Applications of Atomic Force Microscopy in Bone Research

Joseph M Wallace IUPUI Department of Biomedical Engineering ORS Workshop March 15th 2014

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General Seminar Outline Need for nanoscale techniques to analyze bone

bull TEM SEM CryoTEM AFM

Atomic Force Microscopy (AFM) Principles and Imaging Modes

AFM Applications in Bone Research Imaging bull Investigating Bone Cell Activity and Structure bull Processing Bone for Collagen Analysis bull General Analysis of Collagen Structure bull Issues to address when considering AFM for Imaging

AFM Applications in Bone Research Mechanics bull Indentation Calibration assumptions limitations bull Other cool mechanical techniqueshellip

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Bonersquos Hierarchical Structure Hierarchy In Collagen-Based Tissues Most musculoskeletal tissues are hierarchical defined structures can be found at multiple length scales

MineralizedCollagen Fibril

Collagen Mineral Composite

Cortical Bone

Osteon and Lamella

Trabecular Packet and Lamella

TrabecularBone Alpha Chain

Crystal Lattice

Kastelic et al CTR 1978Practical Orthopaedic Sports Medicine amp Arthroscopy 2007

TropocollagenMicrofibrilFibrilFiber FascicleTendonBBML - All R

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Critical Gap in Understanding Collagen Nanoscale collagen fibril primary building block of many collagen-based tissues

bull Critical gap how do nanoscale features influence more clinically-relevant tissue and structural properties

α1

α1

α2

α1

α1

α2

Strain (ε)

Stre

ss (σ

)

Elastic Region Plastic Region

σult

εt

Area under curve Toughness = total energy

Yieldσy

εe

Modulus

Failure

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Methods For Analyzing Collagen Structure bull Bone researchers come from a variety of backgrounds

bull We have borrowed analytical techniques along the way

Spectroscopic bull Fourier Transform Infrared Spectroscopy (FTIR) bull Raman Spectroscopy bull Nuclear magnetic resonance (NMR)

Scattering bull Small Angle X Ray Scattering (SAXS) bull Wide Angle X Ray Diffraction (WAXD) bull Neutron Scattering

Lack the ability to directly answer questions about nanoscale assembly and organization of collagen and mineral in bone BBML - A

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Methods For Imaging Collagen Structure

Transmission andor scanning electron microscopy High resolution imaging of collagen ultrastructure X Harsh sample preparation X Sectioning X Imaging conditions X Reduced ability to draw accurate conclusions

Randall Nature and Structure of Collagen 1953 Quan Methods in Enzymology 2013

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Cryo-TEM

bull Fixation cryosectioing and vitrification

bull Samples maintained in a hydrated state throughout sectioning and microscopy

bull Heavy-metal staining is not necessary

Can be technically challenging to perform

Quan Methods in Enzymology 2013

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Atomic Force Microscopy (AFM)

bull High spatial resolution

bull Samples can remain intact

bull Image in fluid or air

bull Range of temperatures

bull Minimal preparation

bull Characteristics less likely artifacts of processing or imaging

Can be used to extract nanoscale mechanical properties

Wallace et al Langmuir 2010

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AFM Image of Pentacene (C22H14)

Gross et al Science 2009 325 (5944)1110-1114 BBML - All R

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AFM Image of Hexabenzocoronene

Gross et al Science 2012 2371326-1329 BBML - All R

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Basics of AFM Imaging The Probe Nanoscale tip mounted on a microscale cantilever

Probe

Feedback System

X-Y-X Piezo Combination

Cantilever

Wallace Bone 2012

Kopycinska-Muller Ultramicroscopy 2006

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Basics of AFM Imaging System Components bull Piezoelectric actuators precise

movement under electric potential

bull Raster-scanned (x-y direction)

bull Force transducer interaction force (typically cantileverrsquos deflection)

bull Cantilever deflection (z-direction) measured by photodiode

bull Control system maintains a desired force between probe and sample and (movement in z-direction)

Probe

Feedback System

X-Y-X Piezo Combination

Cantilever

Wallace Bone 2012

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van der Waals Potential Energy Curve

FOR

CE

DISTANCE

Repulsive Regime

Attractive Regime

Weak atomic attraction

Net attractive force weakens as interatomic separation decreases

Net force = 0 distance between atoms is 2-3 Å (~ length of a chemical bond)

Attraction increases until electron clouds begin to electrostatically repel each other BBML - A

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Contact Mode ndash Monitor Deflection

Control system maintains user-defined deflection by vertically moving scanner using piezoelectric actuator

Tip-Sample Separation

Forc

e

Frictional and adhesive forces can damage sample and distort image

Probe in constant contact cantilever deflects like a spring according to Hookersquos law (F = kx)

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Tapping Mode ndash Monitor Amplitude

Tip-Sample SeparationForc

e

Intermittent Contact

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Tapping Mode ndash Monitor Amplitude

ApproachWithdrawal

Tip-Sample SeparationForc

e

Cantilever oscillates at user-defined amplitude near cantilever resonance frequency

-15

-1

-05

0

05

1

15

0 5 10 15

Osc

illat

ion

Am

plitu

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Time

-15

-1

-05

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05

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15

0 5 10 15

Osc

illat

ion

Am

plitu

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Time

Setpoint

Bump

-15

-1

-05

0

05

1

15

0 5 10 15

Osc

illat

ion

Am

plitu

de

Time

SetpointBumpValley

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Tapping Mode ndash Monitor Amplitude bull Probe in contact for a small fraction of its tapping period

lateral forces are reduced

bull Probe must be tuned at resonance frequency ndash can be challenging for some probes when in fluid

Tuning in Air Single resonance peak Tuning in Fluid Multiple and spurious peaks wwwasylumresearchcom

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Peak Force Tapping Mode ndash Monitor Force

Control system maintains user-defined maximum force

Cantilever oscillates at low frequency (1-2 kHz)

bull No need for tuning

Slow tapping force curve is captured with each tap

ApproachWithdrawal

Tip-Sample SeparationForc

ePeakForce

Direct control of maximum normal force bull Protects tip and sample wear bull Limits indentation depth to a few nm - increases resolution

by limiting contact area bull Mechanical properties mapped pixel-by-pixel at same

resolution as the height image (PeakForce QNM) BBML - All R

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AFM Applications in Bone Research Imaging

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AFM Imaging of Collagen

bull AFMrsquos adoption in bone research has been slowhellip

bull First AFM-based study in collagen came in 1992 (a previous study in 1989 used Scanning Tunneling Microscopy)

bull Observed fibrillar and monomeric collagen

Chernoff J of Vac Sci Tech 1992

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Imaging of Osteoclast Activity

Bozec Ultramicroscopy 2005

Hassenkam Anat Record 2006

Initial observations of cellular activity were qualitative (analyses of collagen morphology were not presented)

Sasaki J Elec Micro 1993

Sasaki CTI 1995

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Mineralized collagen deposition in vitro

Collagen deposition from MLO-A5 cells

Barragan-Adjemian CTI 2006 BBML - All R

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Imaging of Canilicular Structure

Reilly Annals of BME 2001

Lin J of Microscopy 2010

Average canilicular diameter ~400-500 nm larger than values typically reported from TEM studies

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AFM Imaging in Bone In general we are more interested in collagen in bone

3 microm Diamond Suspension

Unpolished

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AFM Imaging in Bone

The surface is completely mineralizedhellip BBML - All R

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Removing Mineral in Bone to Expose Collagen

Dealing with mineral is a challenge to analyzing collagen ndash some mineral must be removed

bull 10 Citric acid (to remove mineral) 65 Sodium Hypochlorite (to remove non-collagenous proteins) Habelitz J Struct Bio 2002

bull 7 or 17 Phosphoric Acid Bozek IEEE 2005 El Feninat J Biomed Mat Res 1998

bull 5 Formic Acid Ge Mat Sci Eng 2007

bull 30 mM EDTA Kindt Nanotechnology 2007

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Removing Mineral in Bone to Expose Collagen

Dealing with mineral is a challenge to analyzing collagen ndash some mineral must be removed

bull 30 mM EDTA Kindt Nanotechnology 2007 BBML - A

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Demineralization with EDTA EDTA - Ethylenediaminetetraacetic Acid

Chelating agent - sequesters divalent cations (notably Ca2+)

bull Treat with 05M EDTA pH 8 (15-20 minutes) bull Vigorously wash with ultrapure water bull Sonicate to release surface-bound mineral ions bull Repeat 3-4 times

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Demineralization with EDTA

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Demineralization with EDTA

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Issues to Consider Effects of Acid

Immature collagen crosslinks are very sensitive to acid bull Using acids to demineralize bone may inadvertently

destroy crosslinks distort structure

Fixation of tissue may be necessary prior to demineralization bull Fixation (glutaraldehyde or paraformadehyde) may

preserve structural integrity

bull Fixation effects on collagen structure are not fully known

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100 microm x 100 microm

50 microm x 50 microm

25 microm x 25 microm

10 microm x 10 microm

35 microm x 35 microm

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35 microm x 35 microm

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Analysis of Collagen Structure in Bone Spatial organization of newly formed bone following a surgically-induced injury (rat tibial hole)

Baranauskas J Vac Sci Tech 2001 Baranauskas App Surface Sci 2005

ldquohellipstatistical diameter of the fibers is 159 nm in good agreement with the literature values for type I collagen fibersrdquo

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Analysis of Collagen in Bone Fibril Diameter Fibril Diameter easiest to measure property first attempts were a bit crude better with increased resolution

Sasaki J Mat Sci Tech 2002 Thalhammer J Arch Sci 2001

Bozec Ultramicroscopy 2005

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Collagen in Bone Fibril Diameter

Caution must be taken when reporting and interpreting diameters measured from fibrils in bone bull Fibril packing - width may not be fully exposed

Wallace et al Langmuir 2010

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Collagen in Bone Fibril Diameter

Caution must be taken when reporting and interpreting diameters measured from fibrils in bone bull Fibril packing - width may not be fully exposed

Rigozzi ndash J Struct Bio - 2011

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Collagen in Bone Fibril D-Spacing

300 nm 15 nm

N C C N C C N C C N C C

H O H2C CH2 O H

CH2

O OH HHC CH2

CH2

HO

H H

HHN C C

O

H2C CH2

CH2

HN C C

OH

HC CH2

HO

N C C N C C N C C

H O H2C CH2 O

CH2

OH HC CH2

HO

H H

H

CH2 CH2

glycine X - proline Y - hydroxyproline

Hodge and Petruska 1963

Collagen Fibril Axial D-Periodicity BBML - All R

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05

101520

0 02 04 06 08 1

Hei

ght (

nm)

Length (microns)

httpremfdartmoutheduimagesindexhtml

Randall Nature and Structure of Collagen 1953

TEM SEM

AFM

D-Spacing = 67 nm

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Collagen in Bone Fibril D-Spacing Many studies have shown observable D-spacing content that near theoretical value

Sasaki 2002 67 plusmn 2 nm Bozek 2005 665 plusmn 14 nm Ge 2007 666 plusmn 38 nm

Habelitz J Structural Bio 2002

A 2002 study in dentin got me interestedhellip

n = 322 wet n = 363 dry

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Collagen in Bone Fibril D-Spacing

bull Internal structure bull Wallace et al Bone 2010 46 1349-1354 bull Wallace et al J Stuct Bio 2011 173 146-152 bull Kemp et al J Stuct Bio 2012 180 428-438 bull Warden et al Endocrinology 2013 154(9)

3178-3187 bull Gallant et al Bone 2014 61 191-200 bull Bart et al Conn Tiss Res 2014 In Press

bull Enzymatic and non-

enzymatic cross-linking bull Hammond et al Bone 2014 60 26-32 bull Diaz Gonzalez et al J Biomech 2014 47(3)

681-686 bull Voytik-Harbin et al In Preparation 2014

D-periodic spacing important structural feature that captures aspects related to

Mouse Femur Mid-diaphysis BBML - All R

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2D Fast Fourier Transform Analysis

Harmonics

Fibril D-Spacing

Erickson B Wallace JM et al Biotechnology Journal 2013

Raw AFM Image 2D FFT Power Spectrum

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2D Fast Fourier Transform Analysis

Harmonics

Fibril D-Spacing

Raw AFM Image 2D FFT Power Spectrum

0

20000

40000

60000

80000

100000

120000

0 001 002 003 004 005 006

Am

plitu

de (

microV2 )

Position (1nm)

D-periodic spacing 0015 (1nm) ~ 67 nm

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D-Spacing Distribution

0

10

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40

50

60

70

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100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Overall Mean 670 plusmn 00 nm

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D-Spacing Distribution

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Overall Mean 670 plusmn 00 nm

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

4 Samples Total of n = 255 fibrils Overall Mean 682 plusmn 12 nm

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Cumulative Distribution Function

0

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60

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100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

This population 255 fibrils - Each fibril 1255 = 0392 - Data range 628-716 nm

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CDF vs Histogram 62 63 64 65 66 67 68 69 70 71 72 73 74

0

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0

5

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50

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

HistoCDF

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Changes in D-Spacing with Disease

0

5

10

15

20

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

0

5

10

15

20

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

Wallace et al Bone 2010 46 1349-1354

KS Test plt0001

Control 680 plusmn 26 nm

Estrogen Dep 659 plusmn 31 nm

t-test on mean p=0048

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Changes in D-Spacing with Disease

Wallace et al Bone 2010 46 1349-1354

0

10

20

30

40

50

60

70

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90

100

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

KS Test plt0001

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Question Model and Tissue Morphology Publication

Tissue Specificity in Mice Male mouse 8 weeks Femur dentin tail tendon

No differences in bone vs dentin both ∆ vs tendon

Wallace et al Langmuir 2011 26(10) 7349-7354

Gender Effects Male and female mice 16 weeks Femur No differences NA

Estrogen Depletion OVX in sheep 5 years + 2 Radius Mean and population shift Wallace et al Bone

2010 46 1349-1354

Estrogen Depletion OVX in female 5 years + 2 Skin (dermis) Significant population shift Erickson et al Biotech

J 2013 8(1) 1174-126

Osteoporosis and Mechanical Loading

OVX in ulnar loading in rats Ulna

No loading effect population shift with OVX

Warden et al Endocrinology 2013 154(9) 3178-3187

Osteogenesis Imperfecta Brtl+ model male mice 2 months Femur

No mean ∆ widening of distribution

Wallace et al J Stuct Bio 2011 173 146-152

Osteogenesis Imperfecta Oimoim model male mice 12 weeks Femur Mean and population shift Bart et al Conn Tiss

Res 2014 In Press

Osteogenesis Imperfecta and Tissue Hydration

Brtl+ model male mice 6 months Tail Tendon Significant population shift Kemp et al J Stuct Bio

2012 180 428-438

Type 2 Diabetes Male ZDSD rats 30 weeks Tibia Significant population shift Hammond et al Bone

2014 60 26-32

Type 2 Diabetes Female ZDSD rats 32 weeks Tail Tendon Significant population shift

Diaz Gonzalez et al J Biomech 2014 47(3)

681-686

Raloxifene (in vivo and in vitro) Female dog treated with Raloxifene Femur Significant population shift Gallant et al Bone

2014 61 191-200 BBML - All R

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Issues to Consider Site Selection and Sampling

KS Test plt0001

0023 of bonersquos length

15 m

m =

150

00 micro

m

Wallace et al Langmuir 2011 26(10) 7349-7354

100 microm x 100 microm

35 microm x 35 microm

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Issues to Consider Site Selection and Sampling

60

62

64

66

68

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72

74

1 (35)

2 (33)

3 (40)

4 (39)

5 (35)

6 (29)

7 (26)

8 (23)

9 (28)

WT (288)

D-P

erio

dic

Gap

Ove

rlap

Spac

ing

(nm

)

Axial Location (Proximal End = 1 Distal End = 9)

( of Fibrils)

91

23

45

87

6

Wallace et al Langmuir 2011 26(10) 7349-7354

288 fibrils for population n=1 for mean comparisons BBML - All R

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Issues to Consider Effects of Hydration

Wet Tendon Dry Tendon Kemp et al J StructBio 2012 180 (3) 428-438

Collagen and bone exist in a hydrated environment in vivo bull Hydration state can impact morphology and mechanics

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Issues to Consider Effects of Hydration

0

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62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

Nonsignificant mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet (n=255) 682 plusmn 12 nm

WT Dry (n=257) 686 plusmn 08 nm

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

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Issues to Consider Effects of Hydration

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62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

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Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

0

10

20

30

40

50

60

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80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

Brtl DryBrtl Wet

KS Test plt0001

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

WT Case 04 nm mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet 682 plusmn 12 nm

WT Dry 686 plusmn 08 nm

Brtl+ Wet 675 plusmn 14 nm

Brtl+ Dry 689 plusmn 10 nm

Brtl+ Case 14 nm increase with drying (plt0001) bull Entire population shifted upward (plt0001)

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

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Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT WetBrtl Wet

KS Test plt0001

Brtl+ distribution shifted downward vs WT when wet

0

10

20

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40

50

60

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90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryBrtl Dry

KS Test plt0001

Brtl+ distribution shifted upward vs WT when dry

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

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Issues to Consider AFM calibration

bull Absolute distance measurements require accurate piezo calibration

bull AFM systems have large scan ranges (150 microm to lt 1 microm)

bull Scanner non-linearities can introduce substantial error over that range

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

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Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

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Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

100 nm 10 80 602 1198 100 nm 7 60 827 1439 100 nm 5 40 814 1427 100 nm 35 20 917 1724 100 nm 2 10 937 1774

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

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AFM calibration ndash Take Away Message

Calibration using a scan size and feature size comparable to features of interest is highly recommended

Calibration addresses absolute accuracy not precision (differential sensitivity between measurements)

bull Does not limit ability to differentiate between groups using the same AFM with the same calibration parameters

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AFM Applications in Bone Research Indentation Mechanics

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AFM-Based Indentation

In addition to high resolution imaging AFM can be used to extract nanoscale mechanical data

ApproachWithdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

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AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Can

tilev

er D

efle

ctio

n (V

olts

)

1 Ramp into a rigid sample ndash measure the slope

Deflection sensitivity x 119899119899119899119899119881119881

2 Calculate cantilever spring constant

3 Hookersquos law takes deflection (volts) and converts to units of force F = kx

Spring constant k 119873119873119899119899

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AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Forc

e (n

N)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

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AFM-Based Indentation Cantilever Calibration Fo

rce

(nN

)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

Tip-Sample Separation (nm)

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AFM-Based Indentation Parameters

Approach Withdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

Peak Force

Area Between Curves Energy Dissipation

Contact Mechanics Fit for Elastic Modulus (E)

Adhesion Force

Indentation Depth (δ)

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Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

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Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

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Hertz Contact Equation

wwwwikipediacom

z

r

δ

R

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119877119877 ∙ 12057512057532

Es Sample modulus νs Poissonrsquos ratio R tip radius of curvature δ load point displacement

Relevant Hertz Assumptions bull Strains are small and within the elastic limit bull Indenter is infinitely stiff (only sample deforms) bull Indentation depth ltlt radius of curvature of indenter bull Surfaces are continuous non-conforming (must be flat) bull Load is normal to surface

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Proper Indentation Model is Essential

δ

r

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119903119903 ∙ 12057512057532

δ

α

119865119865 =2120587120587∙

1198641198641199041199041 minus 1205841205841199041199042

∙ tan120572120572 ∙ 1205751205752

Es Sample modulus νs Poissonrsquos ratio R tip radius α opening angle

Hertz Model Spherical Indenter

depth ltlt radius of curvature

Sneddon Model Conical Indenter

depth ge radius of curvature

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Indentation in OI Tendon Study

x

z

yBrtl+ and WT mice 6 month old males

4 tails per group

2-3 fascicles per tail

25-30 fibrils per tail

4-5 locations per fibril

Indent force 20 nN wet (Sneddon) 50 nN dry (Hertz) BBML - A

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eserved

OI Tendon Differential Effects of Dehydration

Dry 1268 plusmn 718 MPa

Wet 305 plusmn 243 MPa

Dry 1737 plusmn 973 MPa

Wet 301 plusmn 202 MPa

Kemp et al J StructBio 2012 180 (3) 428-438

bull Significant stiffening of fibrils with dehydration

bull More pronounced modulus modulus as a function of disease

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OI Tendon Differential Effects of Dehydration

Kemp et al J StructBio 2012 180 (3) 428-438

Diseased phenotype not present when wet phenotype observed when dried

0

10

20

30

40

50

60

0 20 40 60 80 100 120 140 160 180 200

Gro

up S

ampl

es (

)

Indent Modulus (MPa)

WT WetBrtl Wet

0

2

4

6

8

10

12

14

16

18

20

025

050

075

010

0012

5015

0017

5020

0022

5025

0027

5030

0032

5035

0037

5040

0042

5045

0047

5050

00

Gro

up S

ampl

es (

)Indent Modulus (MPa)

WT DryBrtl Dry

555 WT Indents 305 plusmn 243 MPa

614 Brtl+ Indents 301 plusmn 202 MPa

KS Test p=0129 t Test p=0464 464 WT Indents

1268 plusmn 718 MPa 479 Brtl+ Indents 1737 plusmn 973 MPa

KS Test plt0001 T Test plt0001

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d

Indentation in Diabetic Tendon (Poster 0495)

x

z

y

Control and Diabetic Rat Tail Tendon

4-5 rats per group

2-3 fascicles per rat

~70 fibrilstail

4-5 locationsfibril

20 nN (Sneddon)

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Multiscale Diabetes Study (Poster 0495)

0

5

10

15

20

25

300 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 gt25

Perc

enta

ge o

f Gro

up S

ampl

es

Elastic Modulus (MPa)

CD (1530 indents)ZDSD (913 indents)

CD (n=5) 381 plusmn 149 MPa

ZDSD (n=3) 706 plusmn 263 MPa

0

100

200

300

400

500

600

700

800

900

0 2 4 6 8 10

Mic

rosc

ale

Mod

ulus

(MPa

)

Nanoscale Modulus (MPa)

plt0001

r2=0531 p=0040

bull Increased indentation modulus in diabetic tendon

bull Strong relationship between nanoscale indent modulus and microscale tensile modulus

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Indentation Limitations and Weaknesses

Proper probe selection for intended application bull Cantilever must be stiff relative to sample bull Sharp probes may damage sample easily dull

R or α must be determined for each probe bull Properties provided by manufacturer are unreliable bull Vary from probe to probe can dull during indent process

Spring constant must be determined for each probe

bull Value provided by manufacturer is inaccurate bull Thermal tuning OK for ldquosoftrdquo probes poor performance

for stiffer probes bull Sader method (geometry) added mass reference

cantilevers

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Indentation Limitations and Weaknesses

Veeco Application Technical note Ben Ohler Practical Advice on the Determination of Cantilever Spring Constants

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Indentation Limitations and Weaknesses

bull Sample processing

bull Sample hydration

bull Proper force range

bull Substrate effects for thin samples

bull Proper modulus model and range of data goodness of fit

Bearing in mind these assumptions and limitations AFM can provide true ldquonanoscalerdquo mechanical properties both in terms of forces and deformations

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ights Reserve

d

A Note on Peak Force QNM for Bruker AFMs bull Mechanical properties mapped pixel-by-pixel at same

resolution as the height image (PeakForce QNM)

Height Modulus BBML - All R

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d

A Note on Peak Force QNM

Correct probe stiffness must be used based on sample Pittenger ndash Veeco Product Bulletin

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A Note on Peak Force QNM Extremely sensitive to calibration (details rarely given) Absolute radius and spring constant known

bull especially important from probe to probe bull radius uarr with indent depth ndash must determine R at depth

you expect to image bull Depth may vary as a function of sample (or at different

regions within the sample)

Relative reference material of ldquoknownrdquo modulus used to determine a ratio of 119896119896 119877119877 bull ldquoknownrdquo modulus may come from tension compression

and not indentation bull R is related to depth indentation depth on sample must

be same as on reference BBML - All R

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A Note on Peak Force QNM bull Modulus fit on the fly ndash no ability to monitor fit (or range) bull Local sample topography and sample tilt play a role in

measured parameters bull Assuming all modulus values in an image are correct how to

quantify

512 x 512 pixels = 262144 measurements

n=1 n=262144

n from selected ROIs

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bull Imaging in air on mica ndash substrate effect

bull Same imaging force or indent depth for all

bull For each area were all modulus values pooled

Lamprou et al PLoS ONE 2013 BBML - All R

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d

AFM Applications in Bone Research Other Cool Mechanical Techniques

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d

ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo Bonds within or between collagen fibrils in bone that rupture to dissipate energy as a toughening mechanism

Thompson et al Nature 2001

Fantner et al Nature Mat 2005

Hansma et al JMNI 2005

Fantner et al Nano Letters 2007 BBML - All R

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Other Mechanical Techniques

Jimenez-Palomar J Mech Beh Bio Mat 2012

van der Rijt Macro Bio 2004

Yang J Biomed Mater Res 2007

Yang Biophysical Journal 2008 BBML - All R

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d

Other Cool Techniques bull Piezoresponse Force Microscopy (PFM)

bull Electrochemical AFM (EC-AFM)

bull Force Modulation Microscopy (FMM)

bull Lateral Force Microscopy (LFM)

bull Magnetic Force Microscopy (MFM)

bull Conductive AFM (C-AFM)

bull Force Pulling

bull Cantilever Bending

bull Tensile testing of individual fibrils

bull 3 point bending of fibrils

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Summary Things to rememberhellip

bull Imaging mode imaging medium and probe selection

bull Morphology Measurements bull Sample preparation and hydration state bull Measuring fibril diameter bull Site selection and proper sampling bull Appropriate system calibration

bull Mechanical Measurements

bull Probe selection (stiffness and tip radius) bull Probe calibration (radius angle spring constant) bull Indentation force range and mechanical model bull Hydration in biological samples bull PeakForce QNM

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Acknowledgements BBML

Max Hammond Armando Diaz Gonzalez Zachary Bart Silvia Canelon Creasy Clauser

IU School of Medicine Dr David Burr Dr Matt Allen Dr Stuart Warden Dr Lilian Plotkin

Funding Sources NIHNIDCR 1F32DE018840-01 A1 IUPUI BME Departmental Startup Funds IUPUI Research Support Funds Grant IUPUI Biomechanics and Biomaterials Research Center

NIH NICHD Dr Joan Marini

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Bone Biology and Mechanics Lab (BBML) wwwiupuiedu~bbml Facebook BoneBiologyMechanicsLab

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d

• Slide Number 1
• General Seminar Outline
• Bonersquos Hierarchical Structure
• Critical Gap in Understanding Collagen
• Methods For Analyzing Collagen Structure
• Methods For Imaging Collagen Structure
• Cryo-TEM
• Atomic Force Microscopy (AFM)
• AFM Image of Pentacene (C22H14)
• AFM Image of Hexabenzocoronene
• Basics of AFM Imaging The Probe
• Basics of AFM Imaging System Components
• van der Waals Potential Energy Curve
• Contact Mode ndash Monitor Deflection
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Peak Force Tapping Mode ndash Monitor Force
• AFM Applications in Bone Research Imaging
• AFM Imaging of Collagen
• Imaging of Osteoclast Activity
• Mineralized collagen deposition in vitro
• Imaging of Canilicular Structure
• AFM Imaging in Bone
• AFM Imaging in Bone
• Removing Mineral in Bone to Expose Collagen
• Removing Mineral in Bone to Expose Collagen
• Demineralization with EDTA
• Demineralization with EDTA
• Demineralization with EDTA
• Issues to Consider Effects of Acid
• Slide Number 32
• Slide Number 33
• Analysis of Collagen Structure in Bone
• Analysis of Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril D-Spacing
• Slide Number 39
• Collagen in Bone Fibril D-Spacing
• Collagen in Bone Fibril D-Spacing
• 2D Fast Fourier Transform Analysis
• 2D Fast Fourier Transform Analysis
• D-Spacing Distribution
• D-Spacing Distribution
• Cumulative Distribution Function
• CDF vs Histogram
• Changes in D-Spacing with Disease
• Changes in D-Spacing with Disease
• Slide Number 50
• Issues to Consider Site Selection and Sampling
• Issues to Consider Site Selection and Sampling
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• AFM calibration ndash Take Away Message
• AFM Applications in Bone ResearchIndentation Mechanics
• AFM-Based Indentation
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Parameters
• Hertz Contact Equation
• Hertz Contact Equation
• Hertz Contact Equation
• Proper Indentation Model is Essential
• Indentation in OI Tendon Study
• OI Tendon Differential Effects of Dehydration
• OI Tendon Differential Effects of Dehydration
• Indentation in Diabetic Tendon (Poster 0495)
• Multiscale Diabetes Study (Poster 0495)
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• A Note on Peak Force QNM for Bruker AFMs
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• Slide Number 83
• Slide Number 84
• AFM Applications in Bone ResearchOther Cool Mechanical Techniques
• ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo
• Other Mechanical Techniques
• Other Cool Techniques
• Summary Things to rememberhellip
• Acknowledgements
• Bone Biology and Mechanics Lab (BBML)

General Seminar Outline Need for nanoscale techniques to analyze bone

bull TEM SEM CryoTEM AFM

Atomic Force Microscopy (AFM) Principles and Imaging Modes

AFM Applications in Bone Research Imaging bull Investigating Bone Cell Activity and Structure bull Processing Bone for Collagen Analysis bull General Analysis of Collagen Structure bull Issues to address when considering AFM for Imaging

AFM Applications in Bone Research Mechanics bull Indentation Calibration assumptions limitations bull Other cool mechanical techniqueshellip

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Bonersquos Hierarchical Structure Hierarchy In Collagen-Based Tissues Most musculoskeletal tissues are hierarchical defined structures can be found at multiple length scales

MineralizedCollagen Fibril

Collagen Mineral Composite

Cortical Bone

Osteon and Lamella

Trabecular Packet and Lamella

TrabecularBone Alpha Chain

Crystal Lattice

Kastelic et al CTR 1978Practical Orthopaedic Sports Medicine amp Arthroscopy 2007

TropocollagenMicrofibrilFibrilFiber FascicleTendonBBML - All R

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Critical Gap in Understanding Collagen Nanoscale collagen fibril primary building block of many collagen-based tissues

bull Critical gap how do nanoscale features influence more clinically-relevant tissue and structural properties

α1

α1

α2

α1

α1

α2

Strain (ε)

Stre

ss (σ

)

Elastic Region Plastic Region

σult

εt

Area under curve Toughness = total energy

Yieldσy

εe

Modulus

Failure

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Methods For Analyzing Collagen Structure bull Bone researchers come from a variety of backgrounds

bull We have borrowed analytical techniques along the way

Spectroscopic bull Fourier Transform Infrared Spectroscopy (FTIR) bull Raman Spectroscopy bull Nuclear magnetic resonance (NMR)

Scattering bull Small Angle X Ray Scattering (SAXS) bull Wide Angle X Ray Diffraction (WAXD) bull Neutron Scattering

Lack the ability to directly answer questions about nanoscale assembly and organization of collagen and mineral in bone BBML - A

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Methods For Imaging Collagen Structure

Transmission andor scanning electron microscopy High resolution imaging of collagen ultrastructure X Harsh sample preparation X Sectioning X Imaging conditions X Reduced ability to draw accurate conclusions

Randall Nature and Structure of Collagen 1953 Quan Methods in Enzymology 2013

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Cryo-TEM

bull Fixation cryosectioing and vitrification

bull Samples maintained in a hydrated state throughout sectioning and microscopy

bull Heavy-metal staining is not necessary

Can be technically challenging to perform

Quan Methods in Enzymology 2013

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Atomic Force Microscopy (AFM)

bull High spatial resolution

bull Samples can remain intact

bull Image in fluid or air

bull Range of temperatures

bull Minimal preparation

bull Characteristics less likely artifacts of processing or imaging

Can be used to extract nanoscale mechanical properties

Wallace et al Langmuir 2010

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AFM Image of Pentacene (C22H14)

Gross et al Science 2009 325 (5944)1110-1114 BBML - All R

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AFM Image of Hexabenzocoronene

Gross et al Science 2012 2371326-1329 BBML - All R

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Basics of AFM Imaging The Probe Nanoscale tip mounted on a microscale cantilever

Probe

Feedback System

X-Y-X Piezo Combination

Cantilever

Wallace Bone 2012

Kopycinska-Muller Ultramicroscopy 2006

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Basics of AFM Imaging System Components bull Piezoelectric actuators precise

movement under electric potential

bull Raster-scanned (x-y direction)

bull Force transducer interaction force (typically cantileverrsquos deflection)

bull Cantilever deflection (z-direction) measured by photodiode

bull Control system maintains a desired force between probe and sample and (movement in z-direction)

Probe

Feedback System

X-Y-X Piezo Combination

Cantilever

Wallace Bone 2012

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van der Waals Potential Energy Curve

FOR

CE

DISTANCE

Repulsive Regime

Attractive Regime

Weak atomic attraction

Net attractive force weakens as interatomic separation decreases

Net force = 0 distance between atoms is 2-3 Å (~ length of a chemical bond)

Attraction increases until electron clouds begin to electrostatically repel each other BBML - A

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Contact Mode ndash Monitor Deflection

Control system maintains user-defined deflection by vertically moving scanner using piezoelectric actuator

Tip-Sample Separation

Forc

e

Frictional and adhesive forces can damage sample and distort image

Probe in constant contact cantilever deflects like a spring according to Hookersquos law (F = kx)

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Tapping Mode ndash Monitor Amplitude

Tip-Sample SeparationForc

e

Intermittent Contact

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Tapping Mode ndash Monitor Amplitude

ApproachWithdrawal

Tip-Sample SeparationForc

e

Cantilever oscillates at user-defined amplitude near cantilever resonance frequency

-15

-1

-05

0

05

1

15

0 5 10 15

Osc

illat

ion

Am

plitu

de

Time

-15

-1

-05

0

05

1

15

0 5 10 15

Osc

illat

ion

Am

plitu

de

Time

Setpoint

Bump

-15

-1

-05

0

05

1

15

0 5 10 15

Osc

illat

ion

Am

plitu

de

Time

SetpointBumpValley

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Tapping Mode ndash Monitor Amplitude bull Probe in contact for a small fraction of its tapping period

lateral forces are reduced

bull Probe must be tuned at resonance frequency ndash can be challenging for some probes when in fluid

Tuning in Air Single resonance peak Tuning in Fluid Multiple and spurious peaks wwwasylumresearchcom

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Peak Force Tapping Mode ndash Monitor Force

Control system maintains user-defined maximum force

Cantilever oscillates at low frequency (1-2 kHz)

bull No need for tuning

Slow tapping force curve is captured with each tap

ApproachWithdrawal

Tip-Sample SeparationForc

ePeakForce

Direct control of maximum normal force bull Protects tip and sample wear bull Limits indentation depth to a few nm - increases resolution

by limiting contact area bull Mechanical properties mapped pixel-by-pixel at same

resolution as the height image (PeakForce QNM) BBML - All R

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AFM Applications in Bone Research Imaging

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AFM Imaging of Collagen

bull AFMrsquos adoption in bone research has been slowhellip

bull First AFM-based study in collagen came in 1992 (a previous study in 1989 used Scanning Tunneling Microscopy)

bull Observed fibrillar and monomeric collagen

Chernoff J of Vac Sci Tech 1992

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Imaging of Osteoclast Activity

Bozec Ultramicroscopy 2005

Hassenkam Anat Record 2006

Initial observations of cellular activity were qualitative (analyses of collagen morphology were not presented)

Sasaki J Elec Micro 1993

Sasaki CTI 1995

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Mineralized collagen deposition in vitro

Collagen deposition from MLO-A5 cells

Barragan-Adjemian CTI 2006 BBML - All R

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Imaging of Canilicular Structure

Reilly Annals of BME 2001

Lin J of Microscopy 2010

Average canilicular diameter ~400-500 nm larger than values typically reported from TEM studies

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AFM Imaging in Bone In general we are more interested in collagen in bone

3 microm Diamond Suspension

Unpolished

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AFM Imaging in Bone

The surface is completely mineralizedhellip BBML - All R

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Removing Mineral in Bone to Expose Collagen

Dealing with mineral is a challenge to analyzing collagen ndash some mineral must be removed

bull 10 Citric acid (to remove mineral) 65 Sodium Hypochlorite (to remove non-collagenous proteins) Habelitz J Struct Bio 2002

bull 7 or 17 Phosphoric Acid Bozek IEEE 2005 El Feninat J Biomed Mat Res 1998

bull 5 Formic Acid Ge Mat Sci Eng 2007

bull 30 mM EDTA Kindt Nanotechnology 2007

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Removing Mineral in Bone to Expose Collagen

Dealing with mineral is a challenge to analyzing collagen ndash some mineral must be removed

bull 30 mM EDTA Kindt Nanotechnology 2007 BBML - A

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eserved

Demineralization with EDTA EDTA - Ethylenediaminetetraacetic Acid

Chelating agent - sequesters divalent cations (notably Ca2+)

bull Treat with 05M EDTA pH 8 (15-20 minutes) bull Vigorously wash with ultrapure water bull Sonicate to release surface-bound mineral ions bull Repeat 3-4 times

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Demineralization with EDTA

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Demineralization with EDTA

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Issues to Consider Effects of Acid

Immature collagen crosslinks are very sensitive to acid bull Using acids to demineralize bone may inadvertently

destroy crosslinks distort structure

Fixation of tissue may be necessary prior to demineralization bull Fixation (glutaraldehyde or paraformadehyde) may

preserve structural integrity

bull Fixation effects on collagen structure are not fully known

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100 microm x 100 microm

50 microm x 50 microm

25 microm x 25 microm

10 microm x 10 microm

35 microm x 35 microm

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35 microm x 35 microm

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Analysis of Collagen Structure in Bone Spatial organization of newly formed bone following a surgically-induced injury (rat tibial hole)

Baranauskas J Vac Sci Tech 2001 Baranauskas App Surface Sci 2005

ldquohellipstatistical diameter of the fibers is 159 nm in good agreement with the literature values for type I collagen fibersrdquo

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Analysis of Collagen in Bone Fibril Diameter Fibril Diameter easiest to measure property first attempts were a bit crude better with increased resolution

Sasaki J Mat Sci Tech 2002 Thalhammer J Arch Sci 2001

Bozec Ultramicroscopy 2005

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Collagen in Bone Fibril Diameter

Caution must be taken when reporting and interpreting diameters measured from fibrils in bone bull Fibril packing - width may not be fully exposed

Wallace et al Langmuir 2010

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Collagen in Bone Fibril Diameter

Caution must be taken when reporting and interpreting diameters measured from fibrils in bone bull Fibril packing - width may not be fully exposed

Rigozzi ndash J Struct Bio - 2011

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Collagen in Bone Fibril D-Spacing

300 nm 15 nm

N C C N C C N C C N C C

H O H2C CH2 O H

CH2

O OH HHC CH2

CH2

HO

H H

HHN C C

O

H2C CH2

CH2

HN C C

OH

HC CH2

HO

N C C N C C N C C

H O H2C CH2 O

CH2

OH HC CH2

HO

H H

H

CH2 CH2

glycine X - proline Y - hydroxyproline

Hodge and Petruska 1963

Collagen Fibril Axial D-Periodicity BBML - All R

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d

05

101520

0 02 04 06 08 1

Hei

ght (

nm)

Length (microns)

httpremfdartmoutheduimagesindexhtml

Randall Nature and Structure of Collagen 1953

TEM SEM

AFM

D-Spacing = 67 nm

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Collagen in Bone Fibril D-Spacing Many studies have shown observable D-spacing content that near theoretical value

Sasaki 2002 67 plusmn 2 nm Bozek 2005 665 plusmn 14 nm Ge 2007 666 plusmn 38 nm

Habelitz J Structural Bio 2002

A 2002 study in dentin got me interestedhellip

n = 322 wet n = 363 dry

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Collagen in Bone Fibril D-Spacing

bull Internal structure bull Wallace et al Bone 2010 46 1349-1354 bull Wallace et al J Stuct Bio 2011 173 146-152 bull Kemp et al J Stuct Bio 2012 180 428-438 bull Warden et al Endocrinology 2013 154(9)

3178-3187 bull Gallant et al Bone 2014 61 191-200 bull Bart et al Conn Tiss Res 2014 In Press

bull Enzymatic and non-

enzymatic cross-linking bull Hammond et al Bone 2014 60 26-32 bull Diaz Gonzalez et al J Biomech 2014 47(3)

681-686 bull Voytik-Harbin et al In Preparation 2014

D-periodic spacing important structural feature that captures aspects related to

Mouse Femur Mid-diaphysis BBML - All R

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d

2D Fast Fourier Transform Analysis

Harmonics

Fibril D-Spacing

Erickson B Wallace JM et al Biotechnology Journal 2013

Raw AFM Image 2D FFT Power Spectrum

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2D Fast Fourier Transform Analysis

Harmonics

Fibril D-Spacing

Raw AFM Image 2D FFT Power Spectrum

0

20000

40000

60000

80000

100000

120000

0 001 002 003 004 005 006

Am

plitu

de (

microV2 )

Position (1nm)

D-periodic spacing 0015 (1nm) ~ 67 nm

BBML - All R

ights Reserve

d

D-Spacing Distribution

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Overall Mean 670 plusmn 00 nm

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ights Reserve

d

D-Spacing Distribution

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Overall Mean 670 plusmn 00 nm

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

4 Samples Total of n = 255 fibrils Overall Mean 682 plusmn 12 nm

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ights Reserve

d

Cumulative Distribution Function

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

This population 255 fibrils - Each fibril 1255 = 0392 - Data range 628-716 nm

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ights Reserve

d

CDF vs Histogram 62 63 64 65 66 67 68 69 70 71 72 73 74

0

10

20

30

40

50

60

70

80

90

100

0

5

10

15

20

25

30

35

40

45

50

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

HistoCDF

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ights Reserve

d

Changes in D-Spacing with Disease

0

5

10

15

20

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

0

5

10

15

20

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

Wallace et al Bone 2010 46 1349-1354

KS Test plt0001

Control 680 plusmn 26 nm

Estrogen Dep 659 plusmn 31 nm

t-test on mean p=0048

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ights Reserve

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Changes in D-Spacing with Disease

Wallace et al Bone 2010 46 1349-1354

0

10

20

30

40

50

60

70

80

90

100

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

KS Test plt0001

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d

Question Model and Tissue Morphology Publication

Tissue Specificity in Mice Male mouse 8 weeks Femur dentin tail tendon

No differences in bone vs dentin both ∆ vs tendon

Wallace et al Langmuir 2011 26(10) 7349-7354

Gender Effects Male and female mice 16 weeks Femur No differences NA

Estrogen Depletion OVX in sheep 5 years + 2 Radius Mean and population shift Wallace et al Bone

2010 46 1349-1354

Estrogen Depletion OVX in female 5 years + 2 Skin (dermis) Significant population shift Erickson et al Biotech

J 2013 8(1) 1174-126

Osteoporosis and Mechanical Loading

OVX in ulnar loading in rats Ulna

No loading effect population shift with OVX

Warden et al Endocrinology 2013 154(9) 3178-3187

Osteogenesis Imperfecta Brtl+ model male mice 2 months Femur

No mean ∆ widening of distribution

Wallace et al J Stuct Bio 2011 173 146-152

Osteogenesis Imperfecta Oimoim model male mice 12 weeks Femur Mean and population shift Bart et al Conn Tiss

Res 2014 In Press

Osteogenesis Imperfecta and Tissue Hydration

Brtl+ model male mice 6 months Tail Tendon Significant population shift Kemp et al J Stuct Bio

2012 180 428-438

Type 2 Diabetes Male ZDSD rats 30 weeks Tibia Significant population shift Hammond et al Bone

2014 60 26-32

Type 2 Diabetes Female ZDSD rats 32 weeks Tail Tendon Significant population shift

Diaz Gonzalez et al J Biomech 2014 47(3)

681-686

Raloxifene (in vivo and in vitro) Female dog treated with Raloxifene Femur Significant population shift Gallant et al Bone

2014 61 191-200 BBML - All R

ights Reserve

d

Issues to Consider Site Selection and Sampling

KS Test plt0001

0023 of bonersquos length

15 m

m =

150

00 micro

m

Wallace et al Langmuir 2011 26(10) 7349-7354

100 microm x 100 microm

35 microm x 35 microm

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ights Reserve

d

Issues to Consider Site Selection and Sampling

60

62

64

66

68

70

72

74

1 (35)

2 (33)

3 (40)

4 (39)

5 (35)

6 (29)

7 (26)

8 (23)

9 (28)

WT (288)

D-P

erio

dic

Gap

Ove

rlap

Spac

ing

(nm

)

Axial Location (Proximal End = 1 Distal End = 9)

( of Fibrils)

91

23

45

87

6

Wallace et al Langmuir 2011 26(10) 7349-7354

288 fibrils for population n=1 for mean comparisons BBML - All R

ights Reserve

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Issues to Consider Effects of Hydration

Wet Tendon Dry Tendon Kemp et al J StructBio 2012 180 (3) 428-438

Collagen and bone exist in a hydrated environment in vivo bull Hydration state can impact morphology and mechanics

BBML - All R

ights Reserve

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Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

Nonsignificant mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet (n=255) 682 plusmn 12 nm

WT Dry (n=257) 686 plusmn 08 nm

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

Brtl DryBrtl Wet

KS Test plt0001

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

WT Case 04 nm mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet 682 plusmn 12 nm

WT Dry 686 plusmn 08 nm

Brtl+ Wet 675 plusmn 14 nm

Brtl+ Dry 689 plusmn 10 nm

Brtl+ Case 14 nm increase with drying (plt0001) bull Entire population shifted upward (plt0001)

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

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Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT WetBrtl Wet

KS Test plt0001

Brtl+ distribution shifted downward vs WT when wet

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryBrtl Dry

KS Test plt0001

Brtl+ distribution shifted upward vs WT when dry

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

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Issues to Consider AFM calibration

bull Absolute distance measurements require accurate piezo calibration

bull AFM systems have large scan ranges (150 microm to lt 1 microm)

bull Scanner non-linearities can introduce substantial error over that range

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

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Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

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Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

100 nm 10 80 602 1198 100 nm 7 60 827 1439 100 nm 5 40 814 1427 100 nm 35 20 917 1724 100 nm 2 10 937 1774

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

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AFM calibration ndash Take Away Message

Calibration using a scan size and feature size comparable to features of interest is highly recommended

Calibration addresses absolute accuracy not precision (differential sensitivity between measurements)

bull Does not limit ability to differentiate between groups using the same AFM with the same calibration parameters

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d

AFM Applications in Bone Research Indentation Mechanics

BBML - All R

ights Reserve

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AFM-Based Indentation

In addition to high resolution imaging AFM can be used to extract nanoscale mechanical data

ApproachWithdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Can

tilev

er D

efle

ctio

n (V

olts

)

1 Ramp into a rigid sample ndash measure the slope

Deflection sensitivity x 119899119899119899119899119881119881

2 Calculate cantilever spring constant

3 Hookersquos law takes deflection (volts) and converts to units of force F = kx

Spring constant k 119873119873119899119899

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ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Forc

e (n

N)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

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ights Reserve

d

AFM-Based Indentation Cantilever Calibration Fo

rce

(nN

)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

Tip-Sample Separation (nm)

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ights Reserve

d

AFM-Based Indentation Parameters

Approach Withdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

Peak Force

Area Between Curves Energy Dissipation

Contact Mechanics Fit for Elastic Modulus (E)

Adhesion Force

Indentation Depth (δ)

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

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d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

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ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

z

r

δ

R

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119877119877 ∙ 12057512057532

Es Sample modulus νs Poissonrsquos ratio R tip radius of curvature δ load point displacement

Relevant Hertz Assumptions bull Strains are small and within the elastic limit bull Indenter is infinitely stiff (only sample deforms) bull Indentation depth ltlt radius of curvature of indenter bull Surfaces are continuous non-conforming (must be flat) bull Load is normal to surface

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ights Reserve

d

Proper Indentation Model is Essential

δ

r

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119903119903 ∙ 12057512057532

δ

α

119865119865 =2120587120587∙

1198641198641199041199041 minus 1205841205841199041199042

∙ tan120572120572 ∙ 1205751205752

Es Sample modulus νs Poissonrsquos ratio R tip radius α opening angle

Hertz Model Spherical Indenter

depth ltlt radius of curvature

Sneddon Model Conical Indenter

depth ge radius of curvature

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ights Reserve

d

Indentation in OI Tendon Study

x

z

yBrtl+ and WT mice 6 month old males

4 tails per group

2-3 fascicles per tail

25-30 fibrils per tail

4-5 locations per fibril

Indent force 20 nN wet (Sneddon) 50 nN dry (Hertz) BBML - A

ll Rights R

eserved

OI Tendon Differential Effects of Dehydration

Dry 1268 plusmn 718 MPa

Wet 305 plusmn 243 MPa

Dry 1737 plusmn 973 MPa

Wet 301 plusmn 202 MPa

Kemp et al J StructBio 2012 180 (3) 428-438

bull Significant stiffening of fibrils with dehydration

bull More pronounced modulus modulus as a function of disease

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OI Tendon Differential Effects of Dehydration

Kemp et al J StructBio 2012 180 (3) 428-438

Diseased phenotype not present when wet phenotype observed when dried

0

10

20

30

40

50

60

0 20 40 60 80 100 120 140 160 180 200

Gro

up S

ampl

es (

)

Indent Modulus (MPa)

WT WetBrtl Wet

0

2

4

6

8

10

12

14

16

18

20

025

050

075

010

0012

5015

0017

5020

0022

5025

0027

5030

0032

5035

0037

5040

0042

5045

0047

5050

00

Gro

up S

ampl

es (

)Indent Modulus (MPa)

WT DryBrtl Dry

555 WT Indents 305 plusmn 243 MPa

614 Brtl+ Indents 301 plusmn 202 MPa

KS Test p=0129 t Test p=0464 464 WT Indents

1268 plusmn 718 MPa 479 Brtl+ Indents 1737 plusmn 973 MPa

KS Test plt0001 T Test plt0001

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ights Reserve

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Indentation in Diabetic Tendon (Poster 0495)

x

z

y

Control and Diabetic Rat Tail Tendon

4-5 rats per group

2-3 fascicles per rat

~70 fibrilstail

4-5 locationsfibril

20 nN (Sneddon)

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Multiscale Diabetes Study (Poster 0495)

0

5

10

15

20

25

300 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 gt25

Perc

enta

ge o

f Gro

up S

ampl

es

Elastic Modulus (MPa)

CD (1530 indents)ZDSD (913 indents)

CD (n=5) 381 plusmn 149 MPa

ZDSD (n=3) 706 plusmn 263 MPa

0

100

200

300

400

500

600

700

800

900

0 2 4 6 8 10

Mic

rosc

ale

Mod

ulus

(MPa

)

Nanoscale Modulus (MPa)

plt0001

r2=0531 p=0040

bull Increased indentation modulus in diabetic tendon

bull Strong relationship between nanoscale indent modulus and microscale tensile modulus

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Indentation Limitations and Weaknesses

Proper probe selection for intended application bull Cantilever must be stiff relative to sample bull Sharp probes may damage sample easily dull

R or α must be determined for each probe bull Properties provided by manufacturer are unreliable bull Vary from probe to probe can dull during indent process

Spring constant must be determined for each probe

bull Value provided by manufacturer is inaccurate bull Thermal tuning OK for ldquosoftrdquo probes poor performance

for stiffer probes bull Sader method (geometry) added mass reference

cantilevers

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Indentation Limitations and Weaknesses

Veeco Application Technical note Ben Ohler Practical Advice on the Determination of Cantilever Spring Constants

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Indentation Limitations and Weaknesses

bull Sample processing

bull Sample hydration

bull Proper force range

bull Substrate effects for thin samples

bull Proper modulus model and range of data goodness of fit

Bearing in mind these assumptions and limitations AFM can provide true ldquonanoscalerdquo mechanical properties both in terms of forces and deformations

BBML - All R

ights Reserve

d

A Note on Peak Force QNM for Bruker AFMs bull Mechanical properties mapped pixel-by-pixel at same

resolution as the height image (PeakForce QNM)

Height Modulus BBML - All R

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A Note on Peak Force QNM

Correct probe stiffness must be used based on sample Pittenger ndash Veeco Product Bulletin

BBML - All R

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A Note on Peak Force QNM Extremely sensitive to calibration (details rarely given) Absolute radius and spring constant known

bull especially important from probe to probe bull radius uarr with indent depth ndash must determine R at depth

you expect to image bull Depth may vary as a function of sample (or at different

regions within the sample)

Relative reference material of ldquoknownrdquo modulus used to determine a ratio of 119896119896 119877119877 bull ldquoknownrdquo modulus may come from tension compression

and not indentation bull R is related to depth indentation depth on sample must

be same as on reference BBML - All R

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A Note on Peak Force QNM bull Modulus fit on the fly ndash no ability to monitor fit (or range) bull Local sample topography and sample tilt play a role in

measured parameters bull Assuming all modulus values in an image are correct how to

quantify

512 x 512 pixels = 262144 measurements

n=1 n=262144

n from selected ROIs

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bull Imaging in air on mica ndash substrate effect

bull Same imaging force or indent depth for all

bull For each area were all modulus values pooled

Lamprou et al PLoS ONE 2013 BBML - All R

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d

AFM Applications in Bone Research Other Cool Mechanical Techniques

BBML - All R

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ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo Bonds within or between collagen fibrils in bone that rupture to dissipate energy as a toughening mechanism

Thompson et al Nature 2001

Fantner et al Nature Mat 2005

Hansma et al JMNI 2005

Fantner et al Nano Letters 2007 BBML - All R

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Other Mechanical Techniques

Jimenez-Palomar J Mech Beh Bio Mat 2012

van der Rijt Macro Bio 2004

Yang J Biomed Mater Res 2007

Yang Biophysical Journal 2008 BBML - All R

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Other Cool Techniques bull Piezoresponse Force Microscopy (PFM)

bull Electrochemical AFM (EC-AFM)

bull Force Modulation Microscopy (FMM)

bull Lateral Force Microscopy (LFM)

bull Magnetic Force Microscopy (MFM)

bull Conductive AFM (C-AFM)

bull Force Pulling

bull Cantilever Bending

bull Tensile testing of individual fibrils

bull 3 point bending of fibrils

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Summary Things to rememberhellip

bull Imaging mode imaging medium and probe selection

bull Morphology Measurements bull Sample preparation and hydration state bull Measuring fibril diameter bull Site selection and proper sampling bull Appropriate system calibration

bull Mechanical Measurements

bull Probe selection (stiffness and tip radius) bull Probe calibration (radius angle spring constant) bull Indentation force range and mechanical model bull Hydration in biological samples bull PeakForce QNM

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Acknowledgements BBML

Max Hammond Armando Diaz Gonzalez Zachary Bart Silvia Canelon Creasy Clauser

IU School of Medicine Dr David Burr Dr Matt Allen Dr Stuart Warden Dr Lilian Plotkin

Funding Sources NIHNIDCR 1F32DE018840-01 A1 IUPUI BME Departmental Startup Funds IUPUI Research Support Funds Grant IUPUI Biomechanics and Biomaterials Research Center

NIH NICHD Dr Joan Marini

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Bone Biology and Mechanics Lab (BBML) wwwiupuiedu~bbml Facebook BoneBiologyMechanicsLab

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• Slide Number 1
• General Seminar Outline
• Bonersquos Hierarchical Structure
• Critical Gap in Understanding Collagen
• Methods For Analyzing Collagen Structure
• Methods For Imaging Collagen Structure
• Cryo-TEM
• Atomic Force Microscopy (AFM)
• AFM Image of Pentacene (C22H14)
• AFM Image of Hexabenzocoronene
• Basics of AFM Imaging The Probe
• Basics of AFM Imaging System Components
• van der Waals Potential Energy Curve
• Contact Mode ndash Monitor Deflection
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Peak Force Tapping Mode ndash Monitor Force
• AFM Applications in Bone Research Imaging
• AFM Imaging of Collagen
• Imaging of Osteoclast Activity
• Mineralized collagen deposition in vitro
• Imaging of Canilicular Structure
• AFM Imaging in Bone
• AFM Imaging in Bone
• Removing Mineral in Bone to Expose Collagen
• Removing Mineral in Bone to Expose Collagen
• Demineralization with EDTA
• Demineralization with EDTA
• Demineralization with EDTA
• Issues to Consider Effects of Acid
• Slide Number 32
• Slide Number 33
• Analysis of Collagen Structure in Bone
• Analysis of Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril D-Spacing
• Slide Number 39
• Collagen in Bone Fibril D-Spacing
• Collagen in Bone Fibril D-Spacing
• 2D Fast Fourier Transform Analysis
• 2D Fast Fourier Transform Analysis
• D-Spacing Distribution
• D-Spacing Distribution
• Cumulative Distribution Function
• CDF vs Histogram
• Changes in D-Spacing with Disease
• Changes in D-Spacing with Disease
• Slide Number 50
• Issues to Consider Site Selection and Sampling
• Issues to Consider Site Selection and Sampling
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• AFM calibration ndash Take Away Message
• AFM Applications in Bone ResearchIndentation Mechanics
• AFM-Based Indentation
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Parameters
• Hertz Contact Equation
• Hertz Contact Equation
• Hertz Contact Equation
• Proper Indentation Model is Essential
• Indentation in OI Tendon Study
• OI Tendon Differential Effects of Dehydration
• OI Tendon Differential Effects of Dehydration
• Indentation in Diabetic Tendon (Poster 0495)
• Multiscale Diabetes Study (Poster 0495)
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• A Note on Peak Force QNM for Bruker AFMs
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• Slide Number 83
• Slide Number 84
• AFM Applications in Bone ResearchOther Cool Mechanical Techniques
• ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo
• Other Mechanical Techniques
• Other Cool Techniques
• Summary Things to rememberhellip
• Acknowledgements
• Bone Biology and Mechanics Lab (BBML)

Bonersquos Hierarchical Structure Hierarchy In Collagen-Based Tissues Most musculoskeletal tissues are hierarchical defined structures can be found at multiple length scales

MineralizedCollagen Fibril

Collagen Mineral Composite

Cortical Bone

Osteon and Lamella

Trabecular Packet and Lamella

TrabecularBone Alpha Chain

Crystal Lattice

Kastelic et al CTR 1978Practical Orthopaedic Sports Medicine amp Arthroscopy 2007

TropocollagenMicrofibrilFibrilFiber FascicleTendonBBML - All R

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Critical Gap in Understanding Collagen Nanoscale collagen fibril primary building block of many collagen-based tissues

bull Critical gap how do nanoscale features influence more clinically-relevant tissue and structural properties

α1

α1

α2

α1

α1

α2

Strain (ε)

Stre

ss (σ

)

Elastic Region Plastic Region

σult

εt

Area under curve Toughness = total energy

Yieldσy

εe

Modulus

Failure

BBML - All R

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d

Methods For Analyzing Collagen Structure bull Bone researchers come from a variety of backgrounds

bull We have borrowed analytical techniques along the way

Spectroscopic bull Fourier Transform Infrared Spectroscopy (FTIR) bull Raman Spectroscopy bull Nuclear magnetic resonance (NMR)

Scattering bull Small Angle X Ray Scattering (SAXS) bull Wide Angle X Ray Diffraction (WAXD) bull Neutron Scattering

Lack the ability to directly answer questions about nanoscale assembly and organization of collagen and mineral in bone BBML - A

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Methods For Imaging Collagen Structure

Transmission andor scanning electron microscopy High resolution imaging of collagen ultrastructure X Harsh sample preparation X Sectioning X Imaging conditions X Reduced ability to draw accurate conclusions

Randall Nature and Structure of Collagen 1953 Quan Methods in Enzymology 2013

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Cryo-TEM

bull Fixation cryosectioing and vitrification

bull Samples maintained in a hydrated state throughout sectioning and microscopy

bull Heavy-metal staining is not necessary

Can be technically challenging to perform

Quan Methods in Enzymology 2013

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Atomic Force Microscopy (AFM)

bull High spatial resolution

bull Samples can remain intact

bull Image in fluid or air

bull Range of temperatures

bull Minimal preparation

bull Characteristics less likely artifacts of processing or imaging

Can be used to extract nanoscale mechanical properties

Wallace et al Langmuir 2010

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AFM Image of Pentacene (C22H14)

Gross et al Science 2009 325 (5944)1110-1114 BBML - All R

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AFM Image of Hexabenzocoronene

Gross et al Science 2012 2371326-1329 BBML - All R

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Basics of AFM Imaging The Probe Nanoscale tip mounted on a microscale cantilever

Probe

Feedback System

X-Y-X Piezo Combination

Cantilever

Wallace Bone 2012

Kopycinska-Muller Ultramicroscopy 2006

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Basics of AFM Imaging System Components bull Piezoelectric actuators precise

movement under electric potential

bull Raster-scanned (x-y direction)

bull Force transducer interaction force (typically cantileverrsquos deflection)

bull Cantilever deflection (z-direction) measured by photodiode

bull Control system maintains a desired force between probe and sample and (movement in z-direction)

Probe

Feedback System

X-Y-X Piezo Combination

Cantilever

Wallace Bone 2012

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van der Waals Potential Energy Curve

FOR

CE

DISTANCE

Repulsive Regime

Attractive Regime

Weak atomic attraction

Net attractive force weakens as interatomic separation decreases

Net force = 0 distance between atoms is 2-3 Å (~ length of a chemical bond)

Attraction increases until electron clouds begin to electrostatically repel each other BBML - A

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Contact Mode ndash Monitor Deflection

Control system maintains user-defined deflection by vertically moving scanner using piezoelectric actuator

Tip-Sample Separation

Forc

e

Frictional and adhesive forces can damage sample and distort image

Probe in constant contact cantilever deflects like a spring according to Hookersquos law (F = kx)

BBML - All R

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Tapping Mode ndash Monitor Amplitude

Tip-Sample SeparationForc

e

Intermittent Contact

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Tapping Mode ndash Monitor Amplitude

ApproachWithdrawal

Tip-Sample SeparationForc

e

Cantilever oscillates at user-defined amplitude near cantilever resonance frequency

-15

-1

-05

0

05

1

15

0 5 10 15

Osc

illat

ion

Am

plitu

de

Time

-15

-1

-05

0

05

1

15

0 5 10 15

Osc

illat

ion

Am

plitu

de

Time

Setpoint

Bump

-15

-1

-05

0

05

1

15

0 5 10 15

Osc

illat

ion

Am

plitu

de

Time

SetpointBumpValley

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Tapping Mode ndash Monitor Amplitude bull Probe in contact for a small fraction of its tapping period

lateral forces are reduced

bull Probe must be tuned at resonance frequency ndash can be challenging for some probes when in fluid

Tuning in Air Single resonance peak Tuning in Fluid Multiple and spurious peaks wwwasylumresearchcom

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Peak Force Tapping Mode ndash Monitor Force

Control system maintains user-defined maximum force

Cantilever oscillates at low frequency (1-2 kHz)

bull No need for tuning

Slow tapping force curve is captured with each tap

ApproachWithdrawal

Tip-Sample SeparationForc

ePeakForce

Direct control of maximum normal force bull Protects tip and sample wear bull Limits indentation depth to a few nm - increases resolution

by limiting contact area bull Mechanical properties mapped pixel-by-pixel at same

resolution as the height image (PeakForce QNM) BBML - All R

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AFM Applications in Bone Research Imaging

BBML - All R

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AFM Imaging of Collagen

bull AFMrsquos adoption in bone research has been slowhellip

bull First AFM-based study in collagen came in 1992 (a previous study in 1989 used Scanning Tunneling Microscopy)

bull Observed fibrillar and monomeric collagen

Chernoff J of Vac Sci Tech 1992

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Imaging of Osteoclast Activity

Bozec Ultramicroscopy 2005

Hassenkam Anat Record 2006

Initial observations of cellular activity were qualitative (analyses of collagen morphology were not presented)

Sasaki J Elec Micro 1993

Sasaki CTI 1995

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Mineralized collagen deposition in vitro

Collagen deposition from MLO-A5 cells

Barragan-Adjemian CTI 2006 BBML - All R

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Imaging of Canilicular Structure

Reilly Annals of BME 2001

Lin J of Microscopy 2010

Average canilicular diameter ~400-500 nm larger than values typically reported from TEM studies

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AFM Imaging in Bone In general we are more interested in collagen in bone

3 microm Diamond Suspension

Unpolished

BBML - All R

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d

AFM Imaging in Bone

The surface is completely mineralizedhellip BBML - All R

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Removing Mineral in Bone to Expose Collagen

Dealing with mineral is a challenge to analyzing collagen ndash some mineral must be removed

bull 10 Citric acid (to remove mineral) 65 Sodium Hypochlorite (to remove non-collagenous proteins) Habelitz J Struct Bio 2002

bull 7 or 17 Phosphoric Acid Bozek IEEE 2005 El Feninat J Biomed Mat Res 1998

bull 5 Formic Acid Ge Mat Sci Eng 2007

bull 30 mM EDTA Kindt Nanotechnology 2007

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Removing Mineral in Bone to Expose Collagen

Dealing with mineral is a challenge to analyzing collagen ndash some mineral must be removed

bull 30 mM EDTA Kindt Nanotechnology 2007 BBML - A

ll Rights R

eserved

Demineralization with EDTA EDTA - Ethylenediaminetetraacetic Acid

Chelating agent - sequesters divalent cations (notably Ca2+)

bull Treat with 05M EDTA pH 8 (15-20 minutes) bull Vigorously wash with ultrapure water bull Sonicate to release surface-bound mineral ions bull Repeat 3-4 times

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Demineralization with EDTA

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Demineralization with EDTA

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ights Reserve

d

Issues to Consider Effects of Acid

Immature collagen crosslinks are very sensitive to acid bull Using acids to demineralize bone may inadvertently

destroy crosslinks distort structure

Fixation of tissue may be necessary prior to demineralization bull Fixation (glutaraldehyde or paraformadehyde) may

preserve structural integrity

bull Fixation effects on collagen structure are not fully known

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100 microm x 100 microm

50 microm x 50 microm

25 microm x 25 microm

10 microm x 10 microm

35 microm x 35 microm

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35 microm x 35 microm

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ights Reserve

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Analysis of Collagen Structure in Bone Spatial organization of newly formed bone following a surgically-induced injury (rat tibial hole)

Baranauskas J Vac Sci Tech 2001 Baranauskas App Surface Sci 2005

ldquohellipstatistical diameter of the fibers is 159 nm in good agreement with the literature values for type I collagen fibersrdquo

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ights Reserve

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Analysis of Collagen in Bone Fibril Diameter Fibril Diameter easiest to measure property first attempts were a bit crude better with increased resolution

Sasaki J Mat Sci Tech 2002 Thalhammer J Arch Sci 2001

Bozec Ultramicroscopy 2005

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Collagen in Bone Fibril Diameter

Caution must be taken when reporting and interpreting diameters measured from fibrils in bone bull Fibril packing - width may not be fully exposed

Wallace et al Langmuir 2010

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Collagen in Bone Fibril Diameter

Caution must be taken when reporting and interpreting diameters measured from fibrils in bone bull Fibril packing - width may not be fully exposed

Rigozzi ndash J Struct Bio - 2011

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Collagen in Bone Fibril D-Spacing

300 nm 15 nm

N C C N C C N C C N C C

H O H2C CH2 O H

CH2

O OH HHC CH2

CH2

HO

H H

HHN C C

O

H2C CH2

CH2

HN C C

OH

HC CH2

HO

N C C N C C N C C

H O H2C CH2 O

CH2

OH HC CH2

HO

H H

H

CH2 CH2

glycine X - proline Y - hydroxyproline

Hodge and Petruska 1963

Collagen Fibril Axial D-Periodicity BBML - All R

ights Reserve

d

05

101520

0 02 04 06 08 1

Hei

ght (

nm)

Length (microns)

httpremfdartmoutheduimagesindexhtml

Randall Nature and Structure of Collagen 1953

TEM SEM

AFM

D-Spacing = 67 nm

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ights Reserve

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Collagen in Bone Fibril D-Spacing Many studies have shown observable D-spacing content that near theoretical value

Sasaki 2002 67 plusmn 2 nm Bozek 2005 665 plusmn 14 nm Ge 2007 666 plusmn 38 nm

Habelitz J Structural Bio 2002

A 2002 study in dentin got me interestedhellip

n = 322 wet n = 363 dry

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ights Reserve

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Collagen in Bone Fibril D-Spacing

bull Internal structure bull Wallace et al Bone 2010 46 1349-1354 bull Wallace et al J Stuct Bio 2011 173 146-152 bull Kemp et al J Stuct Bio 2012 180 428-438 bull Warden et al Endocrinology 2013 154(9)

3178-3187 bull Gallant et al Bone 2014 61 191-200 bull Bart et al Conn Tiss Res 2014 In Press

bull Enzymatic and non-

enzymatic cross-linking bull Hammond et al Bone 2014 60 26-32 bull Diaz Gonzalez et al J Biomech 2014 47(3)

681-686 bull Voytik-Harbin et al In Preparation 2014

D-periodic spacing important structural feature that captures aspects related to

Mouse Femur Mid-diaphysis BBML - All R

ights Reserve

d

2D Fast Fourier Transform Analysis

Harmonics

Fibril D-Spacing

Erickson B Wallace JM et al Biotechnology Journal 2013

Raw AFM Image 2D FFT Power Spectrum

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ights Reserve

d

2D Fast Fourier Transform Analysis

Harmonics

Fibril D-Spacing

Raw AFM Image 2D FFT Power Spectrum

0

20000

40000

60000

80000

100000

120000

0 001 002 003 004 005 006

Am

plitu

de (

microV2 )

Position (1nm)

D-periodic spacing 0015 (1nm) ~ 67 nm

BBML - All R

ights Reserve

d

D-Spacing Distribution

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Overall Mean 670 plusmn 00 nm

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ights Reserve

d

D-Spacing Distribution

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Overall Mean 670 plusmn 00 nm

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

4 Samples Total of n = 255 fibrils Overall Mean 682 plusmn 12 nm

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ights Reserve

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Cumulative Distribution Function

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

This population 255 fibrils - Each fibril 1255 = 0392 - Data range 628-716 nm

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ights Reserve

d

CDF vs Histogram 62 63 64 65 66 67 68 69 70 71 72 73 74

0

10

20

30

40

50

60

70

80

90

100

0

5

10

15

20

25

30

35

40

45

50

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

HistoCDF

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ights Reserve

d

Changes in D-Spacing with Disease

0

5

10

15

20

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

0

5

10

15

20

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

Wallace et al Bone 2010 46 1349-1354

KS Test plt0001

Control 680 plusmn 26 nm

Estrogen Dep 659 plusmn 31 nm

t-test on mean p=0048

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ights Reserve

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Changes in D-Spacing with Disease

Wallace et al Bone 2010 46 1349-1354

0

10

20

30

40

50

60

70

80

90

100

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

KS Test plt0001

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ights Reserve

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Question Model and Tissue Morphology Publication

Tissue Specificity in Mice Male mouse 8 weeks Femur dentin tail tendon

No differences in bone vs dentin both ∆ vs tendon

Wallace et al Langmuir 2011 26(10) 7349-7354

Gender Effects Male and female mice 16 weeks Femur No differences NA

Estrogen Depletion OVX in sheep 5 years + 2 Radius Mean and population shift Wallace et al Bone

2010 46 1349-1354

Estrogen Depletion OVX in female 5 years + 2 Skin (dermis) Significant population shift Erickson et al Biotech

J 2013 8(1) 1174-126

Osteoporosis and Mechanical Loading

OVX in ulnar loading in rats Ulna

No loading effect population shift with OVX

Warden et al Endocrinology 2013 154(9) 3178-3187

Osteogenesis Imperfecta Brtl+ model male mice 2 months Femur

No mean ∆ widening of distribution

Wallace et al J Stuct Bio 2011 173 146-152

Osteogenesis Imperfecta Oimoim model male mice 12 weeks Femur Mean and population shift Bart et al Conn Tiss

Res 2014 In Press

Osteogenesis Imperfecta and Tissue Hydration

Brtl+ model male mice 6 months Tail Tendon Significant population shift Kemp et al J Stuct Bio

2012 180 428-438

Type 2 Diabetes Male ZDSD rats 30 weeks Tibia Significant population shift Hammond et al Bone

2014 60 26-32

Type 2 Diabetes Female ZDSD rats 32 weeks Tail Tendon Significant population shift

Diaz Gonzalez et al J Biomech 2014 47(3)

681-686

Raloxifene (in vivo and in vitro) Female dog treated with Raloxifene Femur Significant population shift Gallant et al Bone

2014 61 191-200 BBML - All R

ights Reserve

d

Issues to Consider Site Selection and Sampling

KS Test plt0001

0023 of bonersquos length

15 m

m =

150

00 micro

m

Wallace et al Langmuir 2011 26(10) 7349-7354

100 microm x 100 microm

35 microm x 35 microm

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ights Reserve

d

Issues to Consider Site Selection and Sampling

60

62

64

66

68

70

72

74

1 (35)

2 (33)

3 (40)

4 (39)

5 (35)

6 (29)

7 (26)

8 (23)

9 (28)

WT (288)

D-P

erio

dic

Gap

Ove

rlap

Spac

ing

(nm

)

Axial Location (Proximal End = 1 Distal End = 9)

( of Fibrils)

91

23

45

87

6

Wallace et al Langmuir 2011 26(10) 7349-7354

288 fibrils for population n=1 for mean comparisons BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

Wet Tendon Dry Tendon Kemp et al J StructBio 2012 180 (3) 428-438

Collagen and bone exist in a hydrated environment in vivo bull Hydration state can impact morphology and mechanics

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ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

Nonsignificant mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet (n=255) 682 plusmn 12 nm

WT Dry (n=257) 686 plusmn 08 nm

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

Brtl DryBrtl Wet

KS Test plt0001

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

WT Case 04 nm mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet 682 plusmn 12 nm

WT Dry 686 plusmn 08 nm

Brtl+ Wet 675 plusmn 14 nm

Brtl+ Dry 689 plusmn 10 nm

Brtl+ Case 14 nm increase with drying (plt0001) bull Entire population shifted upward (plt0001)

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

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d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT WetBrtl Wet

KS Test plt0001

Brtl+ distribution shifted downward vs WT when wet

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryBrtl Dry

KS Test plt0001

Brtl+ distribution shifted upward vs WT when dry

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider AFM calibration

bull Absolute distance measurements require accurate piezo calibration

bull AFM systems have large scan ranges (150 microm to lt 1 microm)

bull Scanner non-linearities can introduce substantial error over that range

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

ights Reserve

d

Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

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ights Reserve

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Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

100 nm 10 80 602 1198 100 nm 7 60 827 1439 100 nm 5 40 814 1427 100 nm 35 20 917 1724 100 nm 2 10 937 1774

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

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d

AFM calibration ndash Take Away Message

Calibration using a scan size and feature size comparable to features of interest is highly recommended

Calibration addresses absolute accuracy not precision (differential sensitivity between measurements)

bull Does not limit ability to differentiate between groups using the same AFM with the same calibration parameters

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d

AFM Applications in Bone Research Indentation Mechanics

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AFM-Based Indentation

In addition to high resolution imaging AFM can be used to extract nanoscale mechanical data

ApproachWithdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

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ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Can

tilev

er D

efle

ctio

n (V

olts

)

1 Ramp into a rigid sample ndash measure the slope

Deflection sensitivity x 119899119899119899119899119881119881

2 Calculate cantilever spring constant

3 Hookersquos law takes deflection (volts) and converts to units of force F = kx

Spring constant k 119873119873119899119899

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ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Forc

e (n

N)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

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AFM-Based Indentation Cantilever Calibration Fo

rce

(nN

)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

Tip-Sample Separation (nm)

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ights Reserve

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AFM-Based Indentation Parameters

Approach Withdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

Peak Force

Area Between Curves Energy Dissipation

Contact Mechanics Fit for Elastic Modulus (E)

Adhesion Force

Indentation Depth (δ)

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ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

z

r

δ

R

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119877119877 ∙ 12057512057532

Es Sample modulus νs Poissonrsquos ratio R tip radius of curvature δ load point displacement

Relevant Hertz Assumptions bull Strains are small and within the elastic limit bull Indenter is infinitely stiff (only sample deforms) bull Indentation depth ltlt radius of curvature of indenter bull Surfaces are continuous non-conforming (must be flat) bull Load is normal to surface

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d

Proper Indentation Model is Essential

δ

r

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119903119903 ∙ 12057512057532

δ

α

119865119865 =2120587120587∙

1198641198641199041199041 minus 1205841205841199041199042

∙ tan120572120572 ∙ 1205751205752

Es Sample modulus νs Poissonrsquos ratio R tip radius α opening angle

Hertz Model Spherical Indenter

depth ltlt radius of curvature

Sneddon Model Conical Indenter

depth ge radius of curvature

BBML - All R

ights Reserve

d

Indentation in OI Tendon Study

x

z

yBrtl+ and WT mice 6 month old males

4 tails per group

2-3 fascicles per tail

25-30 fibrils per tail

4-5 locations per fibril

Indent force 20 nN wet (Sneddon) 50 nN dry (Hertz) BBML - A

ll Rights R

eserved

OI Tendon Differential Effects of Dehydration

Dry 1268 plusmn 718 MPa

Wet 305 plusmn 243 MPa

Dry 1737 plusmn 973 MPa

Wet 301 plusmn 202 MPa

Kemp et al J StructBio 2012 180 (3) 428-438

bull Significant stiffening of fibrils with dehydration

bull More pronounced modulus modulus as a function of disease

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OI Tendon Differential Effects of Dehydration

Kemp et al J StructBio 2012 180 (3) 428-438

Diseased phenotype not present when wet phenotype observed when dried

0

10

20

30

40

50

60

0 20 40 60 80 100 120 140 160 180 200

Gro

up S

ampl

es (

)

Indent Modulus (MPa)

WT WetBrtl Wet

0

2

4

6

8

10

12

14

16

18

20

025

050

075

010

0012

5015

0017

5020

0022

5025

0027

5030

0032

5035

0037

5040

0042

5045

0047

5050

00

Gro

up S

ampl

es (

)Indent Modulus (MPa)

WT DryBrtl Dry

555 WT Indents 305 plusmn 243 MPa

614 Brtl+ Indents 301 plusmn 202 MPa

KS Test p=0129 t Test p=0464 464 WT Indents

1268 plusmn 718 MPa 479 Brtl+ Indents 1737 plusmn 973 MPa

KS Test plt0001 T Test plt0001

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d

Indentation in Diabetic Tendon (Poster 0495)

x

z

y

Control and Diabetic Rat Tail Tendon

4-5 rats per group

2-3 fascicles per rat

~70 fibrilstail

4-5 locationsfibril

20 nN (Sneddon)

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d

Multiscale Diabetes Study (Poster 0495)

0

5

10

15

20

25

300 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 gt25

Perc

enta

ge o

f Gro

up S

ampl

es

Elastic Modulus (MPa)

CD (1530 indents)ZDSD (913 indents)

CD (n=5) 381 plusmn 149 MPa

ZDSD (n=3) 706 plusmn 263 MPa

0

100

200

300

400

500

600

700

800

900

0 2 4 6 8 10

Mic

rosc

ale

Mod

ulus

(MPa

)

Nanoscale Modulus (MPa)

plt0001

r2=0531 p=0040

bull Increased indentation modulus in diabetic tendon

bull Strong relationship between nanoscale indent modulus and microscale tensile modulus

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Indentation Limitations and Weaknesses

Proper probe selection for intended application bull Cantilever must be stiff relative to sample bull Sharp probes may damage sample easily dull

R or α must be determined for each probe bull Properties provided by manufacturer are unreliable bull Vary from probe to probe can dull during indent process

Spring constant must be determined for each probe

bull Value provided by manufacturer is inaccurate bull Thermal tuning OK for ldquosoftrdquo probes poor performance

for stiffer probes bull Sader method (geometry) added mass reference

cantilevers

BBML - All R

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d

Indentation Limitations and Weaknesses

Veeco Application Technical note Ben Ohler Practical Advice on the Determination of Cantilever Spring Constants

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d

Indentation Limitations and Weaknesses

bull Sample processing

bull Sample hydration

bull Proper force range

bull Substrate effects for thin samples

bull Proper modulus model and range of data goodness of fit

Bearing in mind these assumptions and limitations AFM can provide true ldquonanoscalerdquo mechanical properties both in terms of forces and deformations

BBML - All R

ights Reserve

d

A Note on Peak Force QNM for Bruker AFMs bull Mechanical properties mapped pixel-by-pixel at same

resolution as the height image (PeakForce QNM)

Height Modulus BBML - All R

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d

A Note on Peak Force QNM

Correct probe stiffness must be used based on sample Pittenger ndash Veeco Product Bulletin

BBML - All R

ights Reserve

d

A Note on Peak Force QNM Extremely sensitive to calibration (details rarely given) Absolute radius and spring constant known

bull especially important from probe to probe bull radius uarr with indent depth ndash must determine R at depth

you expect to image bull Depth may vary as a function of sample (or at different

regions within the sample)

Relative reference material of ldquoknownrdquo modulus used to determine a ratio of 119896119896 119877119877 bull ldquoknownrdquo modulus may come from tension compression

and not indentation bull R is related to depth indentation depth on sample must

be same as on reference BBML - All R

ights Reserve

d

A Note on Peak Force QNM bull Modulus fit on the fly ndash no ability to monitor fit (or range) bull Local sample topography and sample tilt play a role in

measured parameters bull Assuming all modulus values in an image are correct how to

quantify

512 x 512 pixels = 262144 measurements

n=1 n=262144

n from selected ROIs

BBML - All R

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d

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bull Imaging in air on mica ndash substrate effect

bull Same imaging force or indent depth for all

bull For each area were all modulus values pooled

Lamprou et al PLoS ONE 2013 BBML - All R

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d

AFM Applications in Bone Research Other Cool Mechanical Techniques

BBML - All R

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d

ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo Bonds within or between collagen fibrils in bone that rupture to dissipate energy as a toughening mechanism

Thompson et al Nature 2001

Fantner et al Nature Mat 2005

Hansma et al JMNI 2005

Fantner et al Nano Letters 2007 BBML - All R

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d

Other Mechanical Techniques

Jimenez-Palomar J Mech Beh Bio Mat 2012

van der Rijt Macro Bio 2004

Yang J Biomed Mater Res 2007

Yang Biophysical Journal 2008 BBML - All R

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d

Other Cool Techniques bull Piezoresponse Force Microscopy (PFM)

bull Electrochemical AFM (EC-AFM)

bull Force Modulation Microscopy (FMM)

bull Lateral Force Microscopy (LFM)

bull Magnetic Force Microscopy (MFM)

bull Conductive AFM (C-AFM)

bull Force Pulling

bull Cantilever Bending

bull Tensile testing of individual fibrils

bull 3 point bending of fibrils

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d

Summary Things to rememberhellip

bull Imaging mode imaging medium and probe selection

bull Morphology Measurements bull Sample preparation and hydration state bull Measuring fibril diameter bull Site selection and proper sampling bull Appropriate system calibration

bull Mechanical Measurements

bull Probe selection (stiffness and tip radius) bull Probe calibration (radius angle spring constant) bull Indentation force range and mechanical model bull Hydration in biological samples bull PeakForce QNM

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Acknowledgements BBML

Max Hammond Armando Diaz Gonzalez Zachary Bart Silvia Canelon Creasy Clauser

IU School of Medicine Dr David Burr Dr Matt Allen Dr Stuart Warden Dr Lilian Plotkin

Funding Sources NIHNIDCR 1F32DE018840-01 A1 IUPUI BME Departmental Startup Funds IUPUI Research Support Funds Grant IUPUI Biomechanics and Biomaterials Research Center

NIH NICHD Dr Joan Marini

BBML - All R

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d

Bone Biology and Mechanics Lab (BBML) wwwiupuiedu~bbml Facebook BoneBiologyMechanicsLab

BBML - All R

ights Reserve

d

• Slide Number 1
• General Seminar Outline
• Bonersquos Hierarchical Structure
• Critical Gap in Understanding Collagen
• Methods For Analyzing Collagen Structure
• Methods For Imaging Collagen Structure
• Cryo-TEM
• Atomic Force Microscopy (AFM)
• AFM Image of Pentacene (C22H14)
• AFM Image of Hexabenzocoronene
• Basics of AFM Imaging The Probe
• Basics of AFM Imaging System Components
• van der Waals Potential Energy Curve
• Contact Mode ndash Monitor Deflection
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Peak Force Tapping Mode ndash Monitor Force
• AFM Applications in Bone Research Imaging
• AFM Imaging of Collagen
• Imaging of Osteoclast Activity
• Mineralized collagen deposition in vitro
• Imaging of Canilicular Structure
• AFM Imaging in Bone
• AFM Imaging in Bone
• Removing Mineral in Bone to Expose Collagen
• Removing Mineral in Bone to Expose Collagen
• Demineralization with EDTA
• Demineralization with EDTA
• Demineralization with EDTA
• Issues to Consider Effects of Acid
• Slide Number 32
• Slide Number 33
• Analysis of Collagen Structure in Bone
• Analysis of Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril D-Spacing
• Slide Number 39
• Collagen in Bone Fibril D-Spacing
• Collagen in Bone Fibril D-Spacing
• 2D Fast Fourier Transform Analysis
• 2D Fast Fourier Transform Analysis
• D-Spacing Distribution
• D-Spacing Distribution
• Cumulative Distribution Function
• CDF vs Histogram
• Changes in D-Spacing with Disease
• Changes in D-Spacing with Disease
• Slide Number 50
• Issues to Consider Site Selection and Sampling
• Issues to Consider Site Selection and Sampling
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• AFM calibration ndash Take Away Message
• AFM Applications in Bone ResearchIndentation Mechanics
• AFM-Based Indentation
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Parameters
• Hertz Contact Equation
• Hertz Contact Equation
• Hertz Contact Equation
• Proper Indentation Model is Essential
• Indentation in OI Tendon Study
• OI Tendon Differential Effects of Dehydration
• OI Tendon Differential Effects of Dehydration
• Indentation in Diabetic Tendon (Poster 0495)
• Multiscale Diabetes Study (Poster 0495)
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• A Note on Peak Force QNM for Bruker AFMs
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• Slide Number 83
• Slide Number 84
• AFM Applications in Bone ResearchOther Cool Mechanical Techniques
• ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo
• Other Mechanical Techniques
• Other Cool Techniques
• Summary Things to rememberhellip
• Acknowledgements
• Bone Biology and Mechanics Lab (BBML)

Critical Gap in Understanding Collagen Nanoscale collagen fibril primary building block of many collagen-based tissues

bull Critical gap how do nanoscale features influence more clinically-relevant tissue and structural properties

α1

α1

α2

α1

α1

α2

Strain (ε)

Stre

ss (σ

)

Elastic Region Plastic Region

σult

εt

Area under curve Toughness = total energy

Yieldσy

εe

Modulus

Failure

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d

Methods For Analyzing Collagen Structure bull Bone researchers come from a variety of backgrounds

bull We have borrowed analytical techniques along the way

Spectroscopic bull Fourier Transform Infrared Spectroscopy (FTIR) bull Raman Spectroscopy bull Nuclear magnetic resonance (NMR)

Scattering bull Small Angle X Ray Scattering (SAXS) bull Wide Angle X Ray Diffraction (WAXD) bull Neutron Scattering

Lack the ability to directly answer questions about nanoscale assembly and organization of collagen and mineral in bone BBML - A

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eserved

Methods For Imaging Collagen Structure

Transmission andor scanning electron microscopy High resolution imaging of collagen ultrastructure X Harsh sample preparation X Sectioning X Imaging conditions X Reduced ability to draw accurate conclusions

Randall Nature and Structure of Collagen 1953 Quan Methods in Enzymology 2013

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Cryo-TEM

bull Fixation cryosectioing and vitrification

bull Samples maintained in a hydrated state throughout sectioning and microscopy

bull Heavy-metal staining is not necessary

Can be technically challenging to perform

Quan Methods in Enzymology 2013

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Atomic Force Microscopy (AFM)

bull High spatial resolution

bull Samples can remain intact

bull Image in fluid or air

bull Range of temperatures

bull Minimal preparation

bull Characteristics less likely artifacts of processing or imaging

Can be used to extract nanoscale mechanical properties

Wallace et al Langmuir 2010

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AFM Image of Pentacene (C22H14)

Gross et al Science 2009 325 (5944)1110-1114 BBML - All R

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AFM Image of Hexabenzocoronene

Gross et al Science 2012 2371326-1329 BBML - All R

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Basics of AFM Imaging The Probe Nanoscale tip mounted on a microscale cantilever

Probe

Feedback System

X-Y-X Piezo Combination

Cantilever

Wallace Bone 2012

Kopycinska-Muller Ultramicroscopy 2006

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Basics of AFM Imaging System Components bull Piezoelectric actuators precise

movement under electric potential

bull Raster-scanned (x-y direction)

bull Force transducer interaction force (typically cantileverrsquos deflection)

bull Cantilever deflection (z-direction) measured by photodiode

bull Control system maintains a desired force between probe and sample and (movement in z-direction)

Probe

Feedback System

X-Y-X Piezo Combination

Cantilever

Wallace Bone 2012

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van der Waals Potential Energy Curve

FOR

CE

DISTANCE

Repulsive Regime

Attractive Regime

Weak atomic attraction

Net attractive force weakens as interatomic separation decreases

Net force = 0 distance between atoms is 2-3 Å (~ length of a chemical bond)

Attraction increases until electron clouds begin to electrostatically repel each other BBML - A

ll Rights R

eserved

Contact Mode ndash Monitor Deflection

Control system maintains user-defined deflection by vertically moving scanner using piezoelectric actuator

Tip-Sample Separation

Forc

e

Frictional and adhesive forces can damage sample and distort image

Probe in constant contact cantilever deflects like a spring according to Hookersquos law (F = kx)

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Tapping Mode ndash Monitor Amplitude

Tip-Sample SeparationForc

e

Intermittent Contact

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Tapping Mode ndash Monitor Amplitude

ApproachWithdrawal

Tip-Sample SeparationForc

e

Cantilever oscillates at user-defined amplitude near cantilever resonance frequency

-15

-1

-05

0

05

1

15

0 5 10 15

Osc

illat

ion

Am

plitu

de

Time

-15

-1

-05

0

05

1

15

0 5 10 15

Osc

illat

ion

Am

plitu

de

Time

Setpoint

Bump

-15

-1

-05

0

05

1

15

0 5 10 15

Osc

illat

ion

Am

plitu

de

Time

SetpointBumpValley

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Tapping Mode ndash Monitor Amplitude bull Probe in contact for a small fraction of its tapping period

lateral forces are reduced

bull Probe must be tuned at resonance frequency ndash can be challenging for some probes when in fluid

Tuning in Air Single resonance peak Tuning in Fluid Multiple and spurious peaks wwwasylumresearchcom

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Peak Force Tapping Mode ndash Monitor Force

Control system maintains user-defined maximum force

Cantilever oscillates at low frequency (1-2 kHz)

bull No need for tuning

Slow tapping force curve is captured with each tap

ApproachWithdrawal

Tip-Sample SeparationForc

ePeakForce

Direct control of maximum normal force bull Protects tip and sample wear bull Limits indentation depth to a few nm - increases resolution

by limiting contact area bull Mechanical properties mapped pixel-by-pixel at same

resolution as the height image (PeakForce QNM) BBML - All R

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AFM Applications in Bone Research Imaging

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AFM Imaging of Collagen

bull AFMrsquos adoption in bone research has been slowhellip

bull First AFM-based study in collagen came in 1992 (a previous study in 1989 used Scanning Tunneling Microscopy)

bull Observed fibrillar and monomeric collagen

Chernoff J of Vac Sci Tech 1992

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Imaging of Osteoclast Activity

Bozec Ultramicroscopy 2005

Hassenkam Anat Record 2006

Initial observations of cellular activity were qualitative (analyses of collagen morphology were not presented)

Sasaki J Elec Micro 1993

Sasaki CTI 1995

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Mineralized collagen deposition in vitro

Collagen deposition from MLO-A5 cells

Barragan-Adjemian CTI 2006 BBML - All R

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Imaging of Canilicular Structure

Reilly Annals of BME 2001

Lin J of Microscopy 2010

Average canilicular diameter ~400-500 nm larger than values typically reported from TEM studies

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AFM Imaging in Bone In general we are more interested in collagen in bone

3 microm Diamond Suspension

Unpolished

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AFM Imaging in Bone

The surface is completely mineralizedhellip BBML - All R

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Removing Mineral in Bone to Expose Collagen

Dealing with mineral is a challenge to analyzing collagen ndash some mineral must be removed

bull 10 Citric acid (to remove mineral) 65 Sodium Hypochlorite (to remove non-collagenous proteins) Habelitz J Struct Bio 2002

bull 7 or 17 Phosphoric Acid Bozek IEEE 2005 El Feninat J Biomed Mat Res 1998

bull 5 Formic Acid Ge Mat Sci Eng 2007

bull 30 mM EDTA Kindt Nanotechnology 2007

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Removing Mineral in Bone to Expose Collagen

Dealing with mineral is a challenge to analyzing collagen ndash some mineral must be removed

bull 30 mM EDTA Kindt Nanotechnology 2007 BBML - A

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Demineralization with EDTA EDTA - Ethylenediaminetetraacetic Acid

Chelating agent - sequesters divalent cations (notably Ca2+)

bull Treat with 05M EDTA pH 8 (15-20 minutes) bull Vigorously wash with ultrapure water bull Sonicate to release surface-bound mineral ions bull Repeat 3-4 times

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Demineralization with EDTA

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Demineralization with EDTA

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Issues to Consider Effects of Acid

Immature collagen crosslinks are very sensitive to acid bull Using acids to demineralize bone may inadvertently

destroy crosslinks distort structure

Fixation of tissue may be necessary prior to demineralization bull Fixation (glutaraldehyde or paraformadehyde) may

preserve structural integrity

bull Fixation effects on collagen structure are not fully known

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100 microm x 100 microm

50 microm x 50 microm

25 microm x 25 microm

10 microm x 10 microm

35 microm x 35 microm

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35 microm x 35 microm

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Analysis of Collagen Structure in Bone Spatial organization of newly formed bone following a surgically-induced injury (rat tibial hole)

Baranauskas J Vac Sci Tech 2001 Baranauskas App Surface Sci 2005

ldquohellipstatistical diameter of the fibers is 159 nm in good agreement with the literature values for type I collagen fibersrdquo

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Analysis of Collagen in Bone Fibril Diameter Fibril Diameter easiest to measure property first attempts were a bit crude better with increased resolution

Sasaki J Mat Sci Tech 2002 Thalhammer J Arch Sci 2001

Bozec Ultramicroscopy 2005

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Collagen in Bone Fibril Diameter

Caution must be taken when reporting and interpreting diameters measured from fibrils in bone bull Fibril packing - width may not be fully exposed

Wallace et al Langmuir 2010

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Collagen in Bone Fibril Diameter

Caution must be taken when reporting and interpreting diameters measured from fibrils in bone bull Fibril packing - width may not be fully exposed

Rigozzi ndash J Struct Bio - 2011

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Collagen in Bone Fibril D-Spacing

300 nm 15 nm

N C C N C C N C C N C C

H O H2C CH2 O H

CH2

O OH HHC CH2

CH2

HO

H H

HHN C C

O

H2C CH2

CH2

HN C C

OH

HC CH2

HO

N C C N C C N C C

H O H2C CH2 O

CH2

OH HC CH2

HO

H H

H

CH2 CH2

glycine X - proline Y - hydroxyproline

Hodge and Petruska 1963

Collagen Fibril Axial D-Periodicity BBML - All R

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05

101520

0 02 04 06 08 1

Hei

ght (

nm)

Length (microns)

httpremfdartmoutheduimagesindexhtml

Randall Nature and Structure of Collagen 1953

TEM SEM

AFM

D-Spacing = 67 nm

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Collagen in Bone Fibril D-Spacing Many studies have shown observable D-spacing content that near theoretical value

Sasaki 2002 67 plusmn 2 nm Bozek 2005 665 plusmn 14 nm Ge 2007 666 plusmn 38 nm

Habelitz J Structural Bio 2002

A 2002 study in dentin got me interestedhellip

n = 322 wet n = 363 dry

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Collagen in Bone Fibril D-Spacing

bull Internal structure bull Wallace et al Bone 2010 46 1349-1354 bull Wallace et al J Stuct Bio 2011 173 146-152 bull Kemp et al J Stuct Bio 2012 180 428-438 bull Warden et al Endocrinology 2013 154(9)

3178-3187 bull Gallant et al Bone 2014 61 191-200 bull Bart et al Conn Tiss Res 2014 In Press

bull Enzymatic and non-

enzymatic cross-linking bull Hammond et al Bone 2014 60 26-32 bull Diaz Gonzalez et al J Biomech 2014 47(3)

681-686 bull Voytik-Harbin et al In Preparation 2014

D-periodic spacing important structural feature that captures aspects related to

Mouse Femur Mid-diaphysis BBML - All R

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2D Fast Fourier Transform Analysis

Harmonics

Fibril D-Spacing

Erickson B Wallace JM et al Biotechnology Journal 2013

Raw AFM Image 2D FFT Power Spectrum

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2D Fast Fourier Transform Analysis

Harmonics

Fibril D-Spacing

Raw AFM Image 2D FFT Power Spectrum

0

20000

40000

60000

80000

100000

120000

0 001 002 003 004 005 006

Am

plitu

de (

microV2 )

Position (1nm)

D-periodic spacing 0015 (1nm) ~ 67 nm

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D-Spacing Distribution

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Overall Mean 670 plusmn 00 nm

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D-Spacing Distribution

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Overall Mean 670 plusmn 00 nm

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

4 Samples Total of n = 255 fibrils Overall Mean 682 plusmn 12 nm

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Cumulative Distribution Function

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

This population 255 fibrils - Each fibril 1255 = 0392 - Data range 628-716 nm

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CDF vs Histogram 62 63 64 65 66 67 68 69 70 71 72 73 74

0

10

20

30

40

50

60

70

80

90

100

0

5

10

15

20

25

30

35

40

45

50

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

HistoCDF

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Changes in D-Spacing with Disease

0

5

10

15

20

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

0

5

10

15

20

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

Wallace et al Bone 2010 46 1349-1354

KS Test plt0001

Control 680 plusmn 26 nm

Estrogen Dep 659 plusmn 31 nm

t-test on mean p=0048

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Changes in D-Spacing with Disease

Wallace et al Bone 2010 46 1349-1354

0

10

20

30

40

50

60

70

80

90

100

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

KS Test plt0001

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Question Model and Tissue Morphology Publication

Tissue Specificity in Mice Male mouse 8 weeks Femur dentin tail tendon

No differences in bone vs dentin both ∆ vs tendon

Wallace et al Langmuir 2011 26(10) 7349-7354

Gender Effects Male and female mice 16 weeks Femur No differences NA

Estrogen Depletion OVX in sheep 5 years + 2 Radius Mean and population shift Wallace et al Bone

2010 46 1349-1354

Estrogen Depletion OVX in female 5 years + 2 Skin (dermis) Significant population shift Erickson et al Biotech

J 2013 8(1) 1174-126

Osteoporosis and Mechanical Loading

OVX in ulnar loading in rats Ulna

No loading effect population shift with OVX

Warden et al Endocrinology 2013 154(9) 3178-3187

Osteogenesis Imperfecta Brtl+ model male mice 2 months Femur

No mean ∆ widening of distribution

Wallace et al J Stuct Bio 2011 173 146-152

Osteogenesis Imperfecta Oimoim model male mice 12 weeks Femur Mean and population shift Bart et al Conn Tiss

Res 2014 In Press

Osteogenesis Imperfecta and Tissue Hydration

Brtl+ model male mice 6 months Tail Tendon Significant population shift Kemp et al J Stuct Bio

2012 180 428-438

Type 2 Diabetes Male ZDSD rats 30 weeks Tibia Significant population shift Hammond et al Bone

2014 60 26-32

Type 2 Diabetes Female ZDSD rats 32 weeks Tail Tendon Significant population shift

Diaz Gonzalez et al J Biomech 2014 47(3)

681-686

Raloxifene (in vivo and in vitro) Female dog treated with Raloxifene Femur Significant population shift Gallant et al Bone

2014 61 191-200 BBML - All R

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Issues to Consider Site Selection and Sampling

KS Test plt0001

0023 of bonersquos length

15 m

m =

150

00 micro

m

Wallace et al Langmuir 2011 26(10) 7349-7354

100 microm x 100 microm

35 microm x 35 microm

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Issues to Consider Site Selection and Sampling

60

62

64

66

68

70

72

74

1 (35)

2 (33)

3 (40)

4 (39)

5 (35)

6 (29)

7 (26)

8 (23)

9 (28)

WT (288)

D-P

erio

dic

Gap

Ove

rlap

Spac

ing

(nm

)

Axial Location (Proximal End = 1 Distal End = 9)

( of Fibrils)

91

23

45

87

6

Wallace et al Langmuir 2011 26(10) 7349-7354

288 fibrils for population n=1 for mean comparisons BBML - All R

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Issues to Consider Effects of Hydration

Wet Tendon Dry Tendon Kemp et al J StructBio 2012 180 (3) 428-438

Collagen and bone exist in a hydrated environment in vivo bull Hydration state can impact morphology and mechanics

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Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

Nonsignificant mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet (n=255) 682 plusmn 12 nm

WT Dry (n=257) 686 plusmn 08 nm

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

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Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

Brtl DryBrtl Wet

KS Test plt0001

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

WT Case 04 nm mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet 682 plusmn 12 nm

WT Dry 686 plusmn 08 nm

Brtl+ Wet 675 plusmn 14 nm

Brtl+ Dry 689 plusmn 10 nm

Brtl+ Case 14 nm increase with drying (plt0001) bull Entire population shifted upward (plt0001)

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

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d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT WetBrtl Wet

KS Test plt0001

Brtl+ distribution shifted downward vs WT when wet

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryBrtl Dry

KS Test plt0001

Brtl+ distribution shifted upward vs WT when dry

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

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Issues to Consider AFM calibration

bull Absolute distance measurements require accurate piezo calibration

bull AFM systems have large scan ranges (150 microm to lt 1 microm)

bull Scanner non-linearities can introduce substantial error over that range

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

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Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

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Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

100 nm 10 80 602 1198 100 nm 7 60 827 1439 100 nm 5 40 814 1427 100 nm 35 20 917 1724 100 nm 2 10 937 1774

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

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AFM calibration ndash Take Away Message

Calibration using a scan size and feature size comparable to features of interest is highly recommended

Calibration addresses absolute accuracy not precision (differential sensitivity between measurements)

bull Does not limit ability to differentiate between groups using the same AFM with the same calibration parameters

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AFM Applications in Bone Research Indentation Mechanics

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AFM-Based Indentation

In addition to high resolution imaging AFM can be used to extract nanoscale mechanical data

ApproachWithdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

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AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Can

tilev

er D

efle

ctio

n (V

olts

)

1 Ramp into a rigid sample ndash measure the slope

Deflection sensitivity x 119899119899119899119899119881119881

2 Calculate cantilever spring constant

3 Hookersquos law takes deflection (volts) and converts to units of force F = kx

Spring constant k 119873119873119899119899

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ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Forc

e (n

N)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

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AFM-Based Indentation Cantilever Calibration Fo

rce

(nN

)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

Tip-Sample Separation (nm)

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AFM-Based Indentation Parameters

Approach Withdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

Peak Force

Area Between Curves Energy Dissipation

Contact Mechanics Fit for Elastic Modulus (E)

Adhesion Force

Indentation Depth (δ)

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

z

r

δ

R

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119877119877 ∙ 12057512057532

Es Sample modulus νs Poissonrsquos ratio R tip radius of curvature δ load point displacement

Relevant Hertz Assumptions bull Strains are small and within the elastic limit bull Indenter is infinitely stiff (only sample deforms) bull Indentation depth ltlt radius of curvature of indenter bull Surfaces are continuous non-conforming (must be flat) bull Load is normal to surface

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ights Reserve

d

Proper Indentation Model is Essential

δ

r

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119903119903 ∙ 12057512057532

δ

α

119865119865 =2120587120587∙

1198641198641199041199041 minus 1205841205841199041199042

∙ tan120572120572 ∙ 1205751205752

Es Sample modulus νs Poissonrsquos ratio R tip radius α opening angle

Hertz Model Spherical Indenter

depth ltlt radius of curvature

Sneddon Model Conical Indenter

depth ge radius of curvature

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ights Reserve

d

Indentation in OI Tendon Study

x

z

yBrtl+ and WT mice 6 month old males

4 tails per group

2-3 fascicles per tail

25-30 fibrils per tail

4-5 locations per fibril

Indent force 20 nN wet (Sneddon) 50 nN dry (Hertz) BBML - A

ll Rights R

eserved

OI Tendon Differential Effects of Dehydration

Dry 1268 plusmn 718 MPa

Wet 305 plusmn 243 MPa

Dry 1737 plusmn 973 MPa

Wet 301 plusmn 202 MPa

Kemp et al J StructBio 2012 180 (3) 428-438

bull Significant stiffening of fibrils with dehydration

bull More pronounced modulus modulus as a function of disease

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ights Reserve

d

OI Tendon Differential Effects of Dehydration

Kemp et al J StructBio 2012 180 (3) 428-438

Diseased phenotype not present when wet phenotype observed when dried

0

10

20

30

40

50

60

0 20 40 60 80 100 120 140 160 180 200

Gro

up S

ampl

es (

)

Indent Modulus (MPa)

WT WetBrtl Wet

0

2

4

6

8

10

12

14

16

18

20

025

050

075

010

0012

5015

0017

5020

0022

5025

0027

5030

0032

5035

0037

5040

0042

5045

0047

5050

00

Gro

up S

ampl

es (

)Indent Modulus (MPa)

WT DryBrtl Dry

555 WT Indents 305 plusmn 243 MPa

614 Brtl+ Indents 301 plusmn 202 MPa

KS Test p=0129 t Test p=0464 464 WT Indents

1268 plusmn 718 MPa 479 Brtl+ Indents 1737 plusmn 973 MPa

KS Test plt0001 T Test plt0001

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ights Reserve

d

Indentation in Diabetic Tendon (Poster 0495)

x

z

y

Control and Diabetic Rat Tail Tendon

4-5 rats per group

2-3 fascicles per rat

~70 fibrilstail

4-5 locationsfibril

20 nN (Sneddon)

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ights Reserve

d

Multiscale Diabetes Study (Poster 0495)

0

5

10

15

20

25

300 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 gt25

Perc

enta

ge o

f Gro

up S

ampl

es

Elastic Modulus (MPa)

CD (1530 indents)ZDSD (913 indents)

CD (n=5) 381 plusmn 149 MPa

ZDSD (n=3) 706 plusmn 263 MPa

0

100

200

300

400

500

600

700

800

900

0 2 4 6 8 10

Mic

rosc

ale

Mod

ulus

(MPa

)

Nanoscale Modulus (MPa)

plt0001

r2=0531 p=0040

bull Increased indentation modulus in diabetic tendon

bull Strong relationship between nanoscale indent modulus and microscale tensile modulus

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ights Reserve

d

Indentation Limitations and Weaknesses

Proper probe selection for intended application bull Cantilever must be stiff relative to sample bull Sharp probes may damage sample easily dull

R or α must be determined for each probe bull Properties provided by manufacturer are unreliable bull Vary from probe to probe can dull during indent process

Spring constant must be determined for each probe

bull Value provided by manufacturer is inaccurate bull Thermal tuning OK for ldquosoftrdquo probes poor performance

for stiffer probes bull Sader method (geometry) added mass reference

cantilevers

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ights Reserve

d

Indentation Limitations and Weaknesses

Veeco Application Technical note Ben Ohler Practical Advice on the Determination of Cantilever Spring Constants

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ights Reserve

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Indentation Limitations and Weaknesses

bull Sample processing

bull Sample hydration

bull Proper force range

bull Substrate effects for thin samples

bull Proper modulus model and range of data goodness of fit

Bearing in mind these assumptions and limitations AFM can provide true ldquonanoscalerdquo mechanical properties both in terms of forces and deformations

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ights Reserve

d

A Note on Peak Force QNM for Bruker AFMs bull Mechanical properties mapped pixel-by-pixel at same

resolution as the height image (PeakForce QNM)

Height Modulus BBML - All R

ights Reserve

d

A Note on Peak Force QNM

Correct probe stiffness must be used based on sample Pittenger ndash Veeco Product Bulletin

BBML - All R

ights Reserve

d

A Note on Peak Force QNM Extremely sensitive to calibration (details rarely given) Absolute radius and spring constant known

bull especially important from probe to probe bull radius uarr with indent depth ndash must determine R at depth

you expect to image bull Depth may vary as a function of sample (or at different

regions within the sample)

Relative reference material of ldquoknownrdquo modulus used to determine a ratio of 119896119896 119877119877 bull ldquoknownrdquo modulus may come from tension compression

and not indentation bull R is related to depth indentation depth on sample must

be same as on reference BBML - All R

ights Reserve

d

A Note on Peak Force QNM bull Modulus fit on the fly ndash no ability to monitor fit (or range) bull Local sample topography and sample tilt play a role in

measured parameters bull Assuming all modulus values in an image are correct how to

quantify

512 x 512 pixels = 262144 measurements

n=1 n=262144

n from selected ROIs

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ights Reserve

d

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ights Reserve

d

bull Imaging in air on mica ndash substrate effect

bull Same imaging force or indent depth for all

bull For each area were all modulus values pooled

Lamprou et al PLoS ONE 2013 BBML - All R

ights Reserve

d

AFM Applications in Bone Research Other Cool Mechanical Techniques

BBML - All R

ights Reserve

d

ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo Bonds within or between collagen fibrils in bone that rupture to dissipate energy as a toughening mechanism

Thompson et al Nature 2001

Fantner et al Nature Mat 2005

Hansma et al JMNI 2005

Fantner et al Nano Letters 2007 BBML - All R

ights Reserve

d

Other Mechanical Techniques

Jimenez-Palomar J Mech Beh Bio Mat 2012

van der Rijt Macro Bio 2004

Yang J Biomed Mater Res 2007

Yang Biophysical Journal 2008 BBML - All R

ights Reserve

d

Other Cool Techniques bull Piezoresponse Force Microscopy (PFM)

bull Electrochemical AFM (EC-AFM)

bull Force Modulation Microscopy (FMM)

bull Lateral Force Microscopy (LFM)

bull Magnetic Force Microscopy (MFM)

bull Conductive AFM (C-AFM)

bull Force Pulling

bull Cantilever Bending

bull Tensile testing of individual fibrils

bull 3 point bending of fibrils

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ights Reserve

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Summary Things to rememberhellip

bull Imaging mode imaging medium and probe selection

bull Morphology Measurements bull Sample preparation and hydration state bull Measuring fibril diameter bull Site selection and proper sampling bull Appropriate system calibration

bull Mechanical Measurements

bull Probe selection (stiffness and tip radius) bull Probe calibration (radius angle spring constant) bull Indentation force range and mechanical model bull Hydration in biological samples bull PeakForce QNM

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d

Acknowledgements BBML

Max Hammond Armando Diaz Gonzalez Zachary Bart Silvia Canelon Creasy Clauser

IU School of Medicine Dr David Burr Dr Matt Allen Dr Stuart Warden Dr Lilian Plotkin

Funding Sources NIHNIDCR 1F32DE018840-01 A1 IUPUI BME Departmental Startup Funds IUPUI Research Support Funds Grant IUPUI Biomechanics and Biomaterials Research Center

NIH NICHD Dr Joan Marini

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Bone Biology and Mechanics Lab (BBML) wwwiupuiedu~bbml Facebook BoneBiologyMechanicsLab

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ights Reserve

d

• Slide Number 1
• General Seminar Outline
• Bonersquos Hierarchical Structure
• Critical Gap in Understanding Collagen
• Methods For Analyzing Collagen Structure
• Methods For Imaging Collagen Structure
• Cryo-TEM
• Atomic Force Microscopy (AFM)
• AFM Image of Pentacene (C22H14)
• AFM Image of Hexabenzocoronene
• Basics of AFM Imaging The Probe
• Basics of AFM Imaging System Components
• van der Waals Potential Energy Curve
• Contact Mode ndash Monitor Deflection
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Peak Force Tapping Mode ndash Monitor Force
• AFM Applications in Bone Research Imaging
• AFM Imaging of Collagen
• Imaging of Osteoclast Activity
• Mineralized collagen deposition in vitro
• Imaging of Canilicular Structure
• AFM Imaging in Bone
• AFM Imaging in Bone
• Removing Mineral in Bone to Expose Collagen
• Removing Mineral in Bone to Expose Collagen
• Demineralization with EDTA
• Demineralization with EDTA
• Demineralization with EDTA
• Issues to Consider Effects of Acid
• Slide Number 32
• Slide Number 33
• Analysis of Collagen Structure in Bone
• Analysis of Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril D-Spacing
• Slide Number 39
• Collagen in Bone Fibril D-Spacing
• Collagen in Bone Fibril D-Spacing
• 2D Fast Fourier Transform Analysis
• 2D Fast Fourier Transform Analysis
• D-Spacing Distribution
• D-Spacing Distribution
• Cumulative Distribution Function
• CDF vs Histogram
• Changes in D-Spacing with Disease
• Changes in D-Spacing with Disease
• Slide Number 50
• Issues to Consider Site Selection and Sampling
• Issues to Consider Site Selection and Sampling
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• AFM calibration ndash Take Away Message
• AFM Applications in Bone ResearchIndentation Mechanics
• AFM-Based Indentation
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Parameters
• Hertz Contact Equation
• Hertz Contact Equation
• Hertz Contact Equation
• Proper Indentation Model is Essential
• Indentation in OI Tendon Study
• OI Tendon Differential Effects of Dehydration
• OI Tendon Differential Effects of Dehydration
• Indentation in Diabetic Tendon (Poster 0495)
• Multiscale Diabetes Study (Poster 0495)
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• A Note on Peak Force QNM for Bruker AFMs
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• Slide Number 83
• Slide Number 84
• AFM Applications in Bone ResearchOther Cool Mechanical Techniques
• ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo
• Other Mechanical Techniques
• Other Cool Techniques
• Summary Things to rememberhellip
• Acknowledgements
• Bone Biology and Mechanics Lab (BBML)

Methods For Analyzing Collagen Structure bull Bone researchers come from a variety of backgrounds

bull We have borrowed analytical techniques along the way

Spectroscopic bull Fourier Transform Infrared Spectroscopy (FTIR) bull Raman Spectroscopy bull Nuclear magnetic resonance (NMR)

Scattering bull Small Angle X Ray Scattering (SAXS) bull Wide Angle X Ray Diffraction (WAXD) bull Neutron Scattering

Lack the ability to directly answer questions about nanoscale assembly and organization of collagen and mineral in bone BBML - A

ll Rights R

eserved

Methods For Imaging Collagen Structure

Transmission andor scanning electron microscopy High resolution imaging of collagen ultrastructure X Harsh sample preparation X Sectioning X Imaging conditions X Reduced ability to draw accurate conclusions

Randall Nature and Structure of Collagen 1953 Quan Methods in Enzymology 2013

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Cryo-TEM

bull Fixation cryosectioing and vitrification

bull Samples maintained in a hydrated state throughout sectioning and microscopy

bull Heavy-metal staining is not necessary

Can be technically challenging to perform

Quan Methods in Enzymology 2013

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Atomic Force Microscopy (AFM)

bull High spatial resolution

bull Samples can remain intact

bull Image in fluid or air

bull Range of temperatures

bull Minimal preparation

bull Characteristics less likely artifacts of processing or imaging

Can be used to extract nanoscale mechanical properties

Wallace et al Langmuir 2010

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AFM Image of Pentacene (C22H14)

Gross et al Science 2009 325 (5944)1110-1114 BBML - All R

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AFM Image of Hexabenzocoronene

Gross et al Science 2012 2371326-1329 BBML - All R

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Basics of AFM Imaging The Probe Nanoscale tip mounted on a microscale cantilever

Probe

Feedback System

X-Y-X Piezo Combination

Cantilever

Wallace Bone 2012

Kopycinska-Muller Ultramicroscopy 2006

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ights Reserve

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Basics of AFM Imaging System Components bull Piezoelectric actuators precise

movement under electric potential

bull Raster-scanned (x-y direction)

bull Force transducer interaction force (typically cantileverrsquos deflection)

bull Cantilever deflection (z-direction) measured by photodiode

bull Control system maintains a desired force between probe and sample and (movement in z-direction)

Probe

Feedback System

X-Y-X Piezo Combination

Cantilever

Wallace Bone 2012

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d

van der Waals Potential Energy Curve

FOR

CE

DISTANCE

Repulsive Regime

Attractive Regime

Weak atomic attraction

Net attractive force weakens as interatomic separation decreases

Net force = 0 distance between atoms is 2-3 Å (~ length of a chemical bond)

Attraction increases until electron clouds begin to electrostatically repel each other BBML - A

ll Rights R

eserved

Contact Mode ndash Monitor Deflection

Control system maintains user-defined deflection by vertically moving scanner using piezoelectric actuator

Tip-Sample Separation

Forc

e

Frictional and adhesive forces can damage sample and distort image

Probe in constant contact cantilever deflects like a spring according to Hookersquos law (F = kx)

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ights Reserve

d

Tapping Mode ndash Monitor Amplitude

Tip-Sample SeparationForc

e

Intermittent Contact

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ights Reserve

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Tapping Mode ndash Monitor Amplitude

ApproachWithdrawal

Tip-Sample SeparationForc

e

Cantilever oscillates at user-defined amplitude near cantilever resonance frequency

-15

-1

-05

0

05

1

15

0 5 10 15

Osc

illat

ion

Am

plitu

de

Time

-15

-1

-05

0

05

1

15

0 5 10 15

Osc

illat

ion

Am

plitu

de

Time

Setpoint

Bump

-15

-1

-05

0

05

1

15

0 5 10 15

Osc

illat

ion

Am

plitu

de

Time

SetpointBumpValley

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ights Reserve

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Tapping Mode ndash Monitor Amplitude bull Probe in contact for a small fraction of its tapping period

lateral forces are reduced

bull Probe must be tuned at resonance frequency ndash can be challenging for some probes when in fluid

Tuning in Air Single resonance peak Tuning in Fluid Multiple and spurious peaks wwwasylumresearchcom

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Peak Force Tapping Mode ndash Monitor Force

Control system maintains user-defined maximum force

Cantilever oscillates at low frequency (1-2 kHz)

bull No need for tuning

Slow tapping force curve is captured with each tap

ApproachWithdrawal

Tip-Sample SeparationForc

ePeakForce

Direct control of maximum normal force bull Protects tip and sample wear bull Limits indentation depth to a few nm - increases resolution

by limiting contact area bull Mechanical properties mapped pixel-by-pixel at same

resolution as the height image (PeakForce QNM) BBML - All R

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d

AFM Applications in Bone Research Imaging

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d

AFM Imaging of Collagen

bull AFMrsquos adoption in bone research has been slowhellip

bull First AFM-based study in collagen came in 1992 (a previous study in 1989 used Scanning Tunneling Microscopy)

bull Observed fibrillar and monomeric collagen

Chernoff J of Vac Sci Tech 1992

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Imaging of Osteoclast Activity

Bozec Ultramicroscopy 2005

Hassenkam Anat Record 2006

Initial observations of cellular activity were qualitative (analyses of collagen morphology were not presented)

Sasaki J Elec Micro 1993

Sasaki CTI 1995

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Mineralized collagen deposition in vitro

Collagen deposition from MLO-A5 cells

Barragan-Adjemian CTI 2006 BBML - All R

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Imaging of Canilicular Structure

Reilly Annals of BME 2001

Lin J of Microscopy 2010

Average canilicular diameter ~400-500 nm larger than values typically reported from TEM studies

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AFM Imaging in Bone In general we are more interested in collagen in bone

3 microm Diamond Suspension

Unpolished

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d

AFM Imaging in Bone

The surface is completely mineralizedhellip BBML - All R

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d

Removing Mineral in Bone to Expose Collagen

Dealing with mineral is a challenge to analyzing collagen ndash some mineral must be removed

bull 10 Citric acid (to remove mineral) 65 Sodium Hypochlorite (to remove non-collagenous proteins) Habelitz J Struct Bio 2002

bull 7 or 17 Phosphoric Acid Bozek IEEE 2005 El Feninat J Biomed Mat Res 1998

bull 5 Formic Acid Ge Mat Sci Eng 2007

bull 30 mM EDTA Kindt Nanotechnology 2007

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Removing Mineral in Bone to Expose Collagen

Dealing with mineral is a challenge to analyzing collagen ndash some mineral must be removed

bull 30 mM EDTA Kindt Nanotechnology 2007 BBML - A

ll Rights R

eserved

Demineralization with EDTA EDTA - Ethylenediaminetetraacetic Acid

Chelating agent - sequesters divalent cations (notably Ca2+)

bull Treat with 05M EDTA pH 8 (15-20 minutes) bull Vigorously wash with ultrapure water bull Sonicate to release surface-bound mineral ions bull Repeat 3-4 times

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Demineralization with EDTA

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Demineralization with EDTA

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Issues to Consider Effects of Acid

Immature collagen crosslinks are very sensitive to acid bull Using acids to demineralize bone may inadvertently

destroy crosslinks distort structure

Fixation of tissue may be necessary prior to demineralization bull Fixation (glutaraldehyde or paraformadehyde) may

preserve structural integrity

bull Fixation effects on collagen structure are not fully known

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100 microm x 100 microm

50 microm x 50 microm

25 microm x 25 microm

10 microm x 10 microm

35 microm x 35 microm

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d

35 microm x 35 microm

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Analysis of Collagen Structure in Bone Spatial organization of newly formed bone following a surgically-induced injury (rat tibial hole)

Baranauskas J Vac Sci Tech 2001 Baranauskas App Surface Sci 2005

ldquohellipstatistical diameter of the fibers is 159 nm in good agreement with the literature values for type I collagen fibersrdquo

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Analysis of Collagen in Bone Fibril Diameter Fibril Diameter easiest to measure property first attempts were a bit crude better with increased resolution

Sasaki J Mat Sci Tech 2002 Thalhammer J Arch Sci 2001

Bozec Ultramicroscopy 2005

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Collagen in Bone Fibril Diameter

Caution must be taken when reporting and interpreting diameters measured from fibrils in bone bull Fibril packing - width may not be fully exposed

Wallace et al Langmuir 2010

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Collagen in Bone Fibril Diameter

Caution must be taken when reporting and interpreting diameters measured from fibrils in bone bull Fibril packing - width may not be fully exposed

Rigozzi ndash J Struct Bio - 2011

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Collagen in Bone Fibril D-Spacing

300 nm 15 nm

N C C N C C N C C N C C

H O H2C CH2 O H

CH2

O OH HHC CH2

CH2

HO

H H

HHN C C

O

H2C CH2

CH2

HN C C

OH

HC CH2

HO

N C C N C C N C C

H O H2C CH2 O

CH2

OH HC CH2

HO

H H

H

CH2 CH2

glycine X - proline Y - hydroxyproline

Hodge and Petruska 1963

Collagen Fibril Axial D-Periodicity BBML - All R

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d

05

101520

0 02 04 06 08 1

Hei

ght (

nm)

Length (microns)

httpremfdartmoutheduimagesindexhtml

Randall Nature and Structure of Collagen 1953

TEM SEM

AFM

D-Spacing = 67 nm

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Collagen in Bone Fibril D-Spacing Many studies have shown observable D-spacing content that near theoretical value

Sasaki 2002 67 plusmn 2 nm Bozek 2005 665 plusmn 14 nm Ge 2007 666 plusmn 38 nm

Habelitz J Structural Bio 2002

A 2002 study in dentin got me interestedhellip

n = 322 wet n = 363 dry

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Collagen in Bone Fibril D-Spacing

bull Internal structure bull Wallace et al Bone 2010 46 1349-1354 bull Wallace et al J Stuct Bio 2011 173 146-152 bull Kemp et al J Stuct Bio 2012 180 428-438 bull Warden et al Endocrinology 2013 154(9)

3178-3187 bull Gallant et al Bone 2014 61 191-200 bull Bart et al Conn Tiss Res 2014 In Press

bull Enzymatic and non-

enzymatic cross-linking bull Hammond et al Bone 2014 60 26-32 bull Diaz Gonzalez et al J Biomech 2014 47(3)

681-686 bull Voytik-Harbin et al In Preparation 2014

D-periodic spacing important structural feature that captures aspects related to

Mouse Femur Mid-diaphysis BBML - All R

ights Reserve

d

2D Fast Fourier Transform Analysis

Harmonics

Fibril D-Spacing

Erickson B Wallace JM et al Biotechnology Journal 2013

Raw AFM Image 2D FFT Power Spectrum

BBML - All R

ights Reserve

d

2D Fast Fourier Transform Analysis

Harmonics

Fibril D-Spacing

Raw AFM Image 2D FFT Power Spectrum

0

20000

40000

60000

80000

100000

120000

0 001 002 003 004 005 006

Am

plitu

de (

microV2 )

Position (1nm)

D-periodic spacing 0015 (1nm) ~ 67 nm

BBML - All R

ights Reserve

d

D-Spacing Distribution

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Overall Mean 670 plusmn 00 nm

BBML - All R

ights Reserve

d

D-Spacing Distribution

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Overall Mean 670 plusmn 00 nm

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

4 Samples Total of n = 255 fibrils Overall Mean 682 plusmn 12 nm

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ights Reserve

d

Cumulative Distribution Function

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

This population 255 fibrils - Each fibril 1255 = 0392 - Data range 628-716 nm

BBML - All R

ights Reserve

d

CDF vs Histogram 62 63 64 65 66 67 68 69 70 71 72 73 74

0

10

20

30

40

50

60

70

80

90

100

0

5

10

15

20

25

30

35

40

45

50

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

HistoCDF

BBML - All R

ights Reserve

d

Changes in D-Spacing with Disease

0

5

10

15

20

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

0

5

10

15

20

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

Wallace et al Bone 2010 46 1349-1354

KS Test plt0001

Control 680 plusmn 26 nm

Estrogen Dep 659 plusmn 31 nm

t-test on mean p=0048

BBML - All R

ights Reserve

d

Changes in D-Spacing with Disease

Wallace et al Bone 2010 46 1349-1354

0

10

20

30

40

50

60

70

80

90

100

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

KS Test plt0001

BBML - All R

ights Reserve

d

Question Model and Tissue Morphology Publication

Tissue Specificity in Mice Male mouse 8 weeks Femur dentin tail tendon

No differences in bone vs dentin both ∆ vs tendon

Wallace et al Langmuir 2011 26(10) 7349-7354

Gender Effects Male and female mice 16 weeks Femur No differences NA

Estrogen Depletion OVX in sheep 5 years + 2 Radius Mean and population shift Wallace et al Bone

2010 46 1349-1354

Estrogen Depletion OVX in female 5 years + 2 Skin (dermis) Significant population shift Erickson et al Biotech

J 2013 8(1) 1174-126

Osteoporosis and Mechanical Loading

OVX in ulnar loading in rats Ulna

No loading effect population shift with OVX

Warden et al Endocrinology 2013 154(9) 3178-3187

Osteogenesis Imperfecta Brtl+ model male mice 2 months Femur

No mean ∆ widening of distribution

Wallace et al J Stuct Bio 2011 173 146-152

Osteogenesis Imperfecta Oimoim model male mice 12 weeks Femur Mean and population shift Bart et al Conn Tiss

Res 2014 In Press

Osteogenesis Imperfecta and Tissue Hydration

Brtl+ model male mice 6 months Tail Tendon Significant population shift Kemp et al J Stuct Bio

2012 180 428-438

Type 2 Diabetes Male ZDSD rats 30 weeks Tibia Significant population shift Hammond et al Bone

2014 60 26-32

Type 2 Diabetes Female ZDSD rats 32 weeks Tail Tendon Significant population shift

Diaz Gonzalez et al J Biomech 2014 47(3)

681-686

Raloxifene (in vivo and in vitro) Female dog treated with Raloxifene Femur Significant population shift Gallant et al Bone

2014 61 191-200 BBML - All R

ights Reserve

d

Issues to Consider Site Selection and Sampling

KS Test plt0001

0023 of bonersquos length

15 m

m =

150

00 micro

m

Wallace et al Langmuir 2011 26(10) 7349-7354

100 microm x 100 microm

35 microm x 35 microm

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ights Reserve

d

Issues to Consider Site Selection and Sampling

60

62

64

66

68

70

72

74

1 (35)

2 (33)

3 (40)

4 (39)

5 (35)

6 (29)

7 (26)

8 (23)

9 (28)

WT (288)

D-P

erio

dic

Gap

Ove

rlap

Spac

ing

(nm

)

Axial Location (Proximal End = 1 Distal End = 9)

( of Fibrils)

91

23

45

87

6

Wallace et al Langmuir 2011 26(10) 7349-7354

288 fibrils for population n=1 for mean comparisons BBML - All R

ights Reserve

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Issues to Consider Effects of Hydration

Wet Tendon Dry Tendon Kemp et al J StructBio 2012 180 (3) 428-438

Collagen and bone exist in a hydrated environment in vivo bull Hydration state can impact morphology and mechanics

BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

Nonsignificant mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet (n=255) 682 plusmn 12 nm

WT Dry (n=257) 686 plusmn 08 nm

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

Brtl DryBrtl Wet

KS Test plt0001

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

WT Case 04 nm mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet 682 plusmn 12 nm

WT Dry 686 plusmn 08 nm

Brtl+ Wet 675 plusmn 14 nm

Brtl+ Dry 689 plusmn 10 nm

Brtl+ Case 14 nm increase with drying (plt0001) bull Entire population shifted upward (plt0001)

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

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Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT WetBrtl Wet

KS Test plt0001

Brtl+ distribution shifted downward vs WT when wet

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryBrtl Dry

KS Test plt0001

Brtl+ distribution shifted upward vs WT when dry

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider AFM calibration

bull Absolute distance measurements require accurate piezo calibration

bull AFM systems have large scan ranges (150 microm to lt 1 microm)

bull Scanner non-linearities can introduce substantial error over that range

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

ights Reserve

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Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

BBML - All R

ights Reserve

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Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

100 nm 10 80 602 1198 100 nm 7 60 827 1439 100 nm 5 40 814 1427 100 nm 35 20 917 1724 100 nm 2 10 937 1774

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

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d

AFM calibration ndash Take Away Message

Calibration using a scan size and feature size comparable to features of interest is highly recommended

Calibration addresses absolute accuracy not precision (differential sensitivity between measurements)

bull Does not limit ability to differentiate between groups using the same AFM with the same calibration parameters

BBML - All R

ights Reserve

d

AFM Applications in Bone Research Indentation Mechanics

BBML - All R

ights Reserve

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AFM-Based Indentation

In addition to high resolution imaging AFM can be used to extract nanoscale mechanical data

ApproachWithdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Can

tilev

er D

efle

ctio

n (V

olts

)

1 Ramp into a rigid sample ndash measure the slope

Deflection sensitivity x 119899119899119899119899119881119881

2 Calculate cantilever spring constant

3 Hookersquos law takes deflection (volts) and converts to units of force F = kx

Spring constant k 119873119873119899119899

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ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Forc

e (n

N)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

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ights Reserve

d

AFM-Based Indentation Cantilever Calibration Fo

rce

(nN

)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

Tip-Sample Separation (nm)

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ights Reserve

d

AFM-Based Indentation Parameters

Approach Withdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

Peak Force

Area Between Curves Energy Dissipation

Contact Mechanics Fit for Elastic Modulus (E)

Adhesion Force

Indentation Depth (δ)

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

z

r

δ

R

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119877119877 ∙ 12057512057532

Es Sample modulus νs Poissonrsquos ratio R tip radius of curvature δ load point displacement

Relevant Hertz Assumptions bull Strains are small and within the elastic limit bull Indenter is infinitely stiff (only sample deforms) bull Indentation depth ltlt radius of curvature of indenter bull Surfaces are continuous non-conforming (must be flat) bull Load is normal to surface

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ights Reserve

d

Proper Indentation Model is Essential

δ

r

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119903119903 ∙ 12057512057532

δ

α

119865119865 =2120587120587∙

1198641198641199041199041 minus 1205841205841199041199042

∙ tan120572120572 ∙ 1205751205752

Es Sample modulus νs Poissonrsquos ratio R tip radius α opening angle

Hertz Model Spherical Indenter

depth ltlt radius of curvature

Sneddon Model Conical Indenter

depth ge radius of curvature

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ights Reserve

d

Indentation in OI Tendon Study

x

z

yBrtl+ and WT mice 6 month old males

4 tails per group

2-3 fascicles per tail

25-30 fibrils per tail

4-5 locations per fibril

Indent force 20 nN wet (Sneddon) 50 nN dry (Hertz) BBML - A

ll Rights R

eserved

OI Tendon Differential Effects of Dehydration

Dry 1268 plusmn 718 MPa

Wet 305 plusmn 243 MPa

Dry 1737 plusmn 973 MPa

Wet 301 plusmn 202 MPa

Kemp et al J StructBio 2012 180 (3) 428-438

bull Significant stiffening of fibrils with dehydration

bull More pronounced modulus modulus as a function of disease

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ights Reserve

d

OI Tendon Differential Effects of Dehydration

Kemp et al J StructBio 2012 180 (3) 428-438

Diseased phenotype not present when wet phenotype observed when dried

0

10

20

30

40

50

60

0 20 40 60 80 100 120 140 160 180 200

Gro

up S

ampl

es (

)

Indent Modulus (MPa)

WT WetBrtl Wet

0

2

4

6

8

10

12

14

16

18

20

025

050

075

010

0012

5015

0017

5020

0022

5025

0027

5030

0032

5035

0037

5040

0042

5045

0047

5050

00

Gro

up S

ampl

es (

)Indent Modulus (MPa)

WT DryBrtl Dry

555 WT Indents 305 plusmn 243 MPa

614 Brtl+ Indents 301 plusmn 202 MPa

KS Test p=0129 t Test p=0464 464 WT Indents

1268 plusmn 718 MPa 479 Brtl+ Indents 1737 plusmn 973 MPa

KS Test plt0001 T Test plt0001

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ights Reserve

d

Indentation in Diabetic Tendon (Poster 0495)

x

z

y

Control and Diabetic Rat Tail Tendon

4-5 rats per group

2-3 fascicles per rat

~70 fibrilstail

4-5 locationsfibril

20 nN (Sneddon)

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ights Reserve

d

Multiscale Diabetes Study (Poster 0495)

0

5

10

15

20

25

300 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 gt25

Perc

enta

ge o

f Gro

up S

ampl

es

Elastic Modulus (MPa)

CD (1530 indents)ZDSD (913 indents)

CD (n=5) 381 plusmn 149 MPa

ZDSD (n=3) 706 plusmn 263 MPa

0

100

200

300

400

500

600

700

800

900

0 2 4 6 8 10

Mic

rosc

ale

Mod

ulus

(MPa

)

Nanoscale Modulus (MPa)

plt0001

r2=0531 p=0040

bull Increased indentation modulus in diabetic tendon

bull Strong relationship between nanoscale indent modulus and microscale tensile modulus

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ights Reserve

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Indentation Limitations and Weaknesses

Proper probe selection for intended application bull Cantilever must be stiff relative to sample bull Sharp probes may damage sample easily dull

R or α must be determined for each probe bull Properties provided by manufacturer are unreliable bull Vary from probe to probe can dull during indent process

Spring constant must be determined for each probe

bull Value provided by manufacturer is inaccurate bull Thermal tuning OK for ldquosoftrdquo probes poor performance

for stiffer probes bull Sader method (geometry) added mass reference

cantilevers

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d

Indentation Limitations and Weaknesses

Veeco Application Technical note Ben Ohler Practical Advice on the Determination of Cantilever Spring Constants

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Indentation Limitations and Weaknesses

bull Sample processing

bull Sample hydration

bull Proper force range

bull Substrate effects for thin samples

bull Proper modulus model and range of data goodness of fit

Bearing in mind these assumptions and limitations AFM can provide true ldquonanoscalerdquo mechanical properties both in terms of forces and deformations

BBML - All R

ights Reserve

d

A Note on Peak Force QNM for Bruker AFMs bull Mechanical properties mapped pixel-by-pixel at same

resolution as the height image (PeakForce QNM)

Height Modulus BBML - All R

ights Reserve

d

A Note on Peak Force QNM

Correct probe stiffness must be used based on sample Pittenger ndash Veeco Product Bulletin

BBML - All R

ights Reserve

d

A Note on Peak Force QNM Extremely sensitive to calibration (details rarely given) Absolute radius and spring constant known

bull especially important from probe to probe bull radius uarr with indent depth ndash must determine R at depth

you expect to image bull Depth may vary as a function of sample (or at different

regions within the sample)

Relative reference material of ldquoknownrdquo modulus used to determine a ratio of 119896119896 119877119877 bull ldquoknownrdquo modulus may come from tension compression

and not indentation bull R is related to depth indentation depth on sample must

be same as on reference BBML - All R

ights Reserve

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A Note on Peak Force QNM bull Modulus fit on the fly ndash no ability to monitor fit (or range) bull Local sample topography and sample tilt play a role in

measured parameters bull Assuming all modulus values in an image are correct how to

quantify

512 x 512 pixels = 262144 measurements

n=1 n=262144

n from selected ROIs

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bull Imaging in air on mica ndash substrate effect

bull Same imaging force or indent depth for all

bull For each area were all modulus values pooled

Lamprou et al PLoS ONE 2013 BBML - All R

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d

AFM Applications in Bone Research Other Cool Mechanical Techniques

BBML - All R

ights Reserve

d

ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo Bonds within or between collagen fibrils in bone that rupture to dissipate energy as a toughening mechanism

Thompson et al Nature 2001

Fantner et al Nature Mat 2005

Hansma et al JMNI 2005

Fantner et al Nano Letters 2007 BBML - All R

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Other Mechanical Techniques

Jimenez-Palomar J Mech Beh Bio Mat 2012

van der Rijt Macro Bio 2004

Yang J Biomed Mater Res 2007

Yang Biophysical Journal 2008 BBML - All R

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Other Cool Techniques bull Piezoresponse Force Microscopy (PFM)

bull Electrochemical AFM (EC-AFM)

bull Force Modulation Microscopy (FMM)

bull Lateral Force Microscopy (LFM)

bull Magnetic Force Microscopy (MFM)

bull Conductive AFM (C-AFM)

bull Force Pulling

bull Cantilever Bending

bull Tensile testing of individual fibrils

bull 3 point bending of fibrils

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Summary Things to rememberhellip

bull Imaging mode imaging medium and probe selection

bull Morphology Measurements bull Sample preparation and hydration state bull Measuring fibril diameter bull Site selection and proper sampling bull Appropriate system calibration

bull Mechanical Measurements

bull Probe selection (stiffness and tip radius) bull Probe calibration (radius angle spring constant) bull Indentation force range and mechanical model bull Hydration in biological samples bull PeakForce QNM

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Acknowledgements BBML

Max Hammond Armando Diaz Gonzalez Zachary Bart Silvia Canelon Creasy Clauser

IU School of Medicine Dr David Burr Dr Matt Allen Dr Stuart Warden Dr Lilian Plotkin

Funding Sources NIHNIDCR 1F32DE018840-01 A1 IUPUI BME Departmental Startup Funds IUPUI Research Support Funds Grant IUPUI Biomechanics and Biomaterials Research Center

NIH NICHD Dr Joan Marini

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ights Reserve

d

Bone Biology and Mechanics Lab (BBML) wwwiupuiedu~bbml Facebook BoneBiologyMechanicsLab

BBML - All R

ights Reserve

d

• Slide Number 1
• General Seminar Outline
• Bonersquos Hierarchical Structure
• Critical Gap in Understanding Collagen
• Methods For Analyzing Collagen Structure
• Methods For Imaging Collagen Structure
• Cryo-TEM
• Atomic Force Microscopy (AFM)
• AFM Image of Pentacene (C22H14)
• AFM Image of Hexabenzocoronene
• Basics of AFM Imaging The Probe
• Basics of AFM Imaging System Components
• van der Waals Potential Energy Curve
• Contact Mode ndash Monitor Deflection
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Peak Force Tapping Mode ndash Monitor Force
• AFM Applications in Bone Research Imaging
• AFM Imaging of Collagen
• Imaging of Osteoclast Activity
• Mineralized collagen deposition in vitro
• Imaging of Canilicular Structure
• AFM Imaging in Bone
• AFM Imaging in Bone
• Removing Mineral in Bone to Expose Collagen
• Removing Mineral in Bone to Expose Collagen
• Demineralization with EDTA
• Demineralization with EDTA
• Demineralization with EDTA
• Issues to Consider Effects of Acid
• Slide Number 32
• Slide Number 33
• Analysis of Collagen Structure in Bone
• Analysis of Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril D-Spacing
• Slide Number 39
• Collagen in Bone Fibril D-Spacing
• Collagen in Bone Fibril D-Spacing
• 2D Fast Fourier Transform Analysis
• 2D Fast Fourier Transform Analysis
• D-Spacing Distribution
• D-Spacing Distribution
• Cumulative Distribution Function
• CDF vs Histogram
• Changes in D-Spacing with Disease
• Changes in D-Spacing with Disease
• Slide Number 50
• Issues to Consider Site Selection and Sampling
• Issues to Consider Site Selection and Sampling
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• AFM calibration ndash Take Away Message
• AFM Applications in Bone ResearchIndentation Mechanics
• AFM-Based Indentation
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Parameters
• Hertz Contact Equation
• Hertz Contact Equation
• Hertz Contact Equation
• Proper Indentation Model is Essential
• Indentation in OI Tendon Study
• OI Tendon Differential Effects of Dehydration
• OI Tendon Differential Effects of Dehydration
• Indentation in Diabetic Tendon (Poster 0495)
• Multiscale Diabetes Study (Poster 0495)
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• A Note on Peak Force QNM for Bruker AFMs
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• Slide Number 83
• Slide Number 84
• AFM Applications in Bone ResearchOther Cool Mechanical Techniques
• ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo
• Other Mechanical Techniques
• Other Cool Techniques
• Summary Things to rememberhellip
• Acknowledgements
• Bone Biology and Mechanics Lab (BBML)

Methods For Imaging Collagen Structure

Transmission andor scanning electron microscopy High resolution imaging of collagen ultrastructure X Harsh sample preparation X Sectioning X Imaging conditions X Reduced ability to draw accurate conclusions

Randall Nature and Structure of Collagen 1953 Quan Methods in Enzymology 2013

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Cryo-TEM

bull Fixation cryosectioing and vitrification

bull Samples maintained in a hydrated state throughout sectioning and microscopy

bull Heavy-metal staining is not necessary

Can be technically challenging to perform

Quan Methods in Enzymology 2013

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Atomic Force Microscopy (AFM)

bull High spatial resolution

bull Samples can remain intact

bull Image in fluid or air

bull Range of temperatures

bull Minimal preparation

bull Characteristics less likely artifacts of processing or imaging

Can be used to extract nanoscale mechanical properties

Wallace et al Langmuir 2010

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AFM Image of Pentacene (C22H14)

Gross et al Science 2009 325 (5944)1110-1114 BBML - All R

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AFM Image of Hexabenzocoronene

Gross et al Science 2012 2371326-1329 BBML - All R

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Basics of AFM Imaging The Probe Nanoscale tip mounted on a microscale cantilever

Probe

Feedback System

X-Y-X Piezo Combination

Cantilever

Wallace Bone 2012

Kopycinska-Muller Ultramicroscopy 2006

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Basics of AFM Imaging System Components bull Piezoelectric actuators precise

movement under electric potential

bull Raster-scanned (x-y direction)

bull Force transducer interaction force (typically cantileverrsquos deflection)

bull Cantilever deflection (z-direction) measured by photodiode

bull Control system maintains a desired force between probe and sample and (movement in z-direction)

Probe

Feedback System

X-Y-X Piezo Combination

Cantilever

Wallace Bone 2012

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van der Waals Potential Energy Curve

FOR

CE

DISTANCE

Repulsive Regime

Attractive Regime

Weak atomic attraction

Net attractive force weakens as interatomic separation decreases

Net force = 0 distance between atoms is 2-3 Å (~ length of a chemical bond)

Attraction increases until electron clouds begin to electrostatically repel each other BBML - A

ll Rights R

eserved

Contact Mode ndash Monitor Deflection

Control system maintains user-defined deflection by vertically moving scanner using piezoelectric actuator

Tip-Sample Separation

Forc

e

Frictional and adhesive forces can damage sample and distort image

Probe in constant contact cantilever deflects like a spring according to Hookersquos law (F = kx)

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Tapping Mode ndash Monitor Amplitude

Tip-Sample SeparationForc

e

Intermittent Contact

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d

Tapping Mode ndash Monitor Amplitude

ApproachWithdrawal

Tip-Sample SeparationForc

e

Cantilever oscillates at user-defined amplitude near cantilever resonance frequency

-15

-1

-05

0

05

1

15

0 5 10 15

Osc

illat

ion

Am

plitu

de

Time

-15

-1

-05

0

05

1

15

0 5 10 15

Osc

illat

ion

Am

plitu

de

Time

Setpoint

Bump

-15

-1

-05

0

05

1

15

0 5 10 15

Osc

illat

ion

Am

plitu

de

Time

SetpointBumpValley

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Tapping Mode ndash Monitor Amplitude bull Probe in contact for a small fraction of its tapping period

lateral forces are reduced

bull Probe must be tuned at resonance frequency ndash can be challenging for some probes when in fluid

Tuning in Air Single resonance peak Tuning in Fluid Multiple and spurious peaks wwwasylumresearchcom

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Peak Force Tapping Mode ndash Monitor Force

Control system maintains user-defined maximum force

Cantilever oscillates at low frequency (1-2 kHz)

bull No need for tuning

Slow tapping force curve is captured with each tap

ApproachWithdrawal

Tip-Sample SeparationForc

ePeakForce

Direct control of maximum normal force bull Protects tip and sample wear bull Limits indentation depth to a few nm - increases resolution

by limiting contact area bull Mechanical properties mapped pixel-by-pixel at same

resolution as the height image (PeakForce QNM) BBML - All R

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d

AFM Applications in Bone Research Imaging

BBML - All R

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d

AFM Imaging of Collagen

bull AFMrsquos adoption in bone research has been slowhellip

bull First AFM-based study in collagen came in 1992 (a previous study in 1989 used Scanning Tunneling Microscopy)

bull Observed fibrillar and monomeric collagen

Chernoff J of Vac Sci Tech 1992

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Imaging of Osteoclast Activity

Bozec Ultramicroscopy 2005

Hassenkam Anat Record 2006

Initial observations of cellular activity were qualitative (analyses of collagen morphology were not presented)

Sasaki J Elec Micro 1993

Sasaki CTI 1995

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Mineralized collagen deposition in vitro

Collagen deposition from MLO-A5 cells

Barragan-Adjemian CTI 2006 BBML - All R

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Imaging of Canilicular Structure

Reilly Annals of BME 2001

Lin J of Microscopy 2010

Average canilicular diameter ~400-500 nm larger than values typically reported from TEM studies

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AFM Imaging in Bone In general we are more interested in collagen in bone

3 microm Diamond Suspension

Unpolished

BBML - All R

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d

AFM Imaging in Bone

The surface is completely mineralizedhellip BBML - All R

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Removing Mineral in Bone to Expose Collagen

Dealing with mineral is a challenge to analyzing collagen ndash some mineral must be removed

bull 10 Citric acid (to remove mineral) 65 Sodium Hypochlorite (to remove non-collagenous proteins) Habelitz J Struct Bio 2002

bull 7 or 17 Phosphoric Acid Bozek IEEE 2005 El Feninat J Biomed Mat Res 1998

bull 5 Formic Acid Ge Mat Sci Eng 2007

bull 30 mM EDTA Kindt Nanotechnology 2007

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Removing Mineral in Bone to Expose Collagen

Dealing with mineral is a challenge to analyzing collagen ndash some mineral must be removed

bull 30 mM EDTA Kindt Nanotechnology 2007 BBML - A

ll Rights R

eserved

Demineralization with EDTA EDTA - Ethylenediaminetetraacetic Acid

Chelating agent - sequesters divalent cations (notably Ca2+)

bull Treat with 05M EDTA pH 8 (15-20 minutes) bull Vigorously wash with ultrapure water bull Sonicate to release surface-bound mineral ions bull Repeat 3-4 times

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Demineralization with EDTA

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Demineralization with EDTA

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Issues to Consider Effects of Acid

Immature collagen crosslinks are very sensitive to acid bull Using acids to demineralize bone may inadvertently

destroy crosslinks distort structure

Fixation of tissue may be necessary prior to demineralization bull Fixation (glutaraldehyde or paraformadehyde) may

preserve structural integrity

bull Fixation effects on collagen structure are not fully known

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100 microm x 100 microm

50 microm x 50 microm

25 microm x 25 microm

10 microm x 10 microm

35 microm x 35 microm

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35 microm x 35 microm

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Analysis of Collagen Structure in Bone Spatial organization of newly formed bone following a surgically-induced injury (rat tibial hole)

Baranauskas J Vac Sci Tech 2001 Baranauskas App Surface Sci 2005

ldquohellipstatistical diameter of the fibers is 159 nm in good agreement with the literature values for type I collagen fibersrdquo

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Analysis of Collagen in Bone Fibril Diameter Fibril Diameter easiest to measure property first attempts were a bit crude better with increased resolution

Sasaki J Mat Sci Tech 2002 Thalhammer J Arch Sci 2001

Bozec Ultramicroscopy 2005

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Collagen in Bone Fibril Diameter

Caution must be taken when reporting and interpreting diameters measured from fibrils in bone bull Fibril packing - width may not be fully exposed

Wallace et al Langmuir 2010

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Collagen in Bone Fibril Diameter

Caution must be taken when reporting and interpreting diameters measured from fibrils in bone bull Fibril packing - width may not be fully exposed

Rigozzi ndash J Struct Bio - 2011

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Collagen in Bone Fibril D-Spacing

300 nm 15 nm

N C C N C C N C C N C C

H O H2C CH2 O H

CH2

O OH HHC CH2

CH2

HO

H H

HHN C C

O

H2C CH2

CH2

HN C C

OH

HC CH2

HO

N C C N C C N C C

H O H2C CH2 O

CH2

OH HC CH2

HO

H H

H

CH2 CH2

glycine X - proline Y - hydroxyproline

Hodge and Petruska 1963

Collagen Fibril Axial D-Periodicity BBML - All R

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d

05

101520

0 02 04 06 08 1

Hei

ght (

nm)

Length (microns)

httpremfdartmoutheduimagesindexhtml

Randall Nature and Structure of Collagen 1953

TEM SEM

AFM

D-Spacing = 67 nm

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Collagen in Bone Fibril D-Spacing Many studies have shown observable D-spacing content that near theoretical value

Sasaki 2002 67 plusmn 2 nm Bozek 2005 665 plusmn 14 nm Ge 2007 666 plusmn 38 nm

Habelitz J Structural Bio 2002

A 2002 study in dentin got me interestedhellip

n = 322 wet n = 363 dry

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Collagen in Bone Fibril D-Spacing

bull Internal structure bull Wallace et al Bone 2010 46 1349-1354 bull Wallace et al J Stuct Bio 2011 173 146-152 bull Kemp et al J Stuct Bio 2012 180 428-438 bull Warden et al Endocrinology 2013 154(9)

3178-3187 bull Gallant et al Bone 2014 61 191-200 bull Bart et al Conn Tiss Res 2014 In Press

bull Enzymatic and non-

enzymatic cross-linking bull Hammond et al Bone 2014 60 26-32 bull Diaz Gonzalez et al J Biomech 2014 47(3)

681-686 bull Voytik-Harbin et al In Preparation 2014

D-periodic spacing important structural feature that captures aspects related to

Mouse Femur Mid-diaphysis BBML - All R

ights Reserve

d

2D Fast Fourier Transform Analysis

Harmonics

Fibril D-Spacing

Erickson B Wallace JM et al Biotechnology Journal 2013

Raw AFM Image 2D FFT Power Spectrum

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2D Fast Fourier Transform Analysis

Harmonics

Fibril D-Spacing

Raw AFM Image 2D FFT Power Spectrum

0

20000

40000

60000

80000

100000

120000

0 001 002 003 004 005 006

Am

plitu

de (

microV2 )

Position (1nm)

D-periodic spacing 0015 (1nm) ~ 67 nm

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d

D-Spacing Distribution

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Overall Mean 670 plusmn 00 nm

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ights Reserve

d

D-Spacing Distribution

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Overall Mean 670 plusmn 00 nm

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

4 Samples Total of n = 255 fibrils Overall Mean 682 plusmn 12 nm

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Cumulative Distribution Function

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

This population 255 fibrils - Each fibril 1255 = 0392 - Data range 628-716 nm

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CDF vs Histogram 62 63 64 65 66 67 68 69 70 71 72 73 74

0

10

20

30

40

50

60

70

80

90

100

0

5

10

15

20

25

30

35

40

45

50

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

HistoCDF

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d

Changes in D-Spacing with Disease

0

5

10

15

20

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

0

5

10

15

20

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

Wallace et al Bone 2010 46 1349-1354

KS Test plt0001

Control 680 plusmn 26 nm

Estrogen Dep 659 plusmn 31 nm

t-test on mean p=0048

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Changes in D-Spacing with Disease

Wallace et al Bone 2010 46 1349-1354

0

10

20

30

40

50

60

70

80

90

100

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

KS Test plt0001

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Question Model and Tissue Morphology Publication

Tissue Specificity in Mice Male mouse 8 weeks Femur dentin tail tendon

No differences in bone vs dentin both ∆ vs tendon

Wallace et al Langmuir 2011 26(10) 7349-7354

Gender Effects Male and female mice 16 weeks Femur No differences NA

Estrogen Depletion OVX in sheep 5 years + 2 Radius Mean and population shift Wallace et al Bone

2010 46 1349-1354

Estrogen Depletion OVX in female 5 years + 2 Skin (dermis) Significant population shift Erickson et al Biotech

J 2013 8(1) 1174-126

Osteoporosis and Mechanical Loading

OVX in ulnar loading in rats Ulna

No loading effect population shift with OVX

Warden et al Endocrinology 2013 154(9) 3178-3187

Osteogenesis Imperfecta Brtl+ model male mice 2 months Femur

No mean ∆ widening of distribution

Wallace et al J Stuct Bio 2011 173 146-152

Osteogenesis Imperfecta Oimoim model male mice 12 weeks Femur Mean and population shift Bart et al Conn Tiss

Res 2014 In Press

Osteogenesis Imperfecta and Tissue Hydration

Brtl+ model male mice 6 months Tail Tendon Significant population shift Kemp et al J Stuct Bio

2012 180 428-438

Type 2 Diabetes Male ZDSD rats 30 weeks Tibia Significant population shift Hammond et al Bone

2014 60 26-32

Type 2 Diabetes Female ZDSD rats 32 weeks Tail Tendon Significant population shift

Diaz Gonzalez et al J Biomech 2014 47(3)

681-686

Raloxifene (in vivo and in vitro) Female dog treated with Raloxifene Femur Significant population shift Gallant et al Bone

2014 61 191-200 BBML - All R

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Issues to Consider Site Selection and Sampling

KS Test plt0001

0023 of bonersquos length

15 m

m =

150

00 micro

m

Wallace et al Langmuir 2011 26(10) 7349-7354

100 microm x 100 microm

35 microm x 35 microm

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Issues to Consider Site Selection and Sampling

60

62

64

66

68

70

72

74

1 (35)

2 (33)

3 (40)

4 (39)

5 (35)

6 (29)

7 (26)

8 (23)

9 (28)

WT (288)

D-P

erio

dic

Gap

Ove

rlap

Spac

ing

(nm

)

Axial Location (Proximal End = 1 Distal End = 9)

( of Fibrils)

91

23

45

87

6

Wallace et al Langmuir 2011 26(10) 7349-7354

288 fibrils for population n=1 for mean comparisons BBML - All R

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Issues to Consider Effects of Hydration

Wet Tendon Dry Tendon Kemp et al J StructBio 2012 180 (3) 428-438

Collagen and bone exist in a hydrated environment in vivo bull Hydration state can impact morphology and mechanics

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Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

Nonsignificant mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet (n=255) 682 plusmn 12 nm

WT Dry (n=257) 686 plusmn 08 nm

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

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Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

Brtl DryBrtl Wet

KS Test plt0001

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

WT Case 04 nm mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet 682 plusmn 12 nm

WT Dry 686 plusmn 08 nm

Brtl+ Wet 675 plusmn 14 nm

Brtl+ Dry 689 plusmn 10 nm

Brtl+ Case 14 nm increase with drying (plt0001) bull Entire population shifted upward (plt0001)

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT WetBrtl Wet

KS Test plt0001

Brtl+ distribution shifted downward vs WT when wet

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryBrtl Dry

KS Test plt0001

Brtl+ distribution shifted upward vs WT when dry

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

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d

Issues to Consider AFM calibration

bull Absolute distance measurements require accurate piezo calibration

bull AFM systems have large scan ranges (150 microm to lt 1 microm)

bull Scanner non-linearities can introduce substantial error over that range

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

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Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

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Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

100 nm 10 80 602 1198 100 nm 7 60 827 1439 100 nm 5 40 814 1427 100 nm 35 20 917 1724 100 nm 2 10 937 1774

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

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AFM calibration ndash Take Away Message

Calibration using a scan size and feature size comparable to features of interest is highly recommended

Calibration addresses absolute accuracy not precision (differential sensitivity between measurements)

bull Does not limit ability to differentiate between groups using the same AFM with the same calibration parameters

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AFM Applications in Bone Research Indentation Mechanics

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AFM-Based Indentation

In addition to high resolution imaging AFM can be used to extract nanoscale mechanical data

ApproachWithdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

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ights Reserve

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AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Can

tilev

er D

efle

ctio

n (V

olts

)

1 Ramp into a rigid sample ndash measure the slope

Deflection sensitivity x 119899119899119899119899119881119881

2 Calculate cantilever spring constant

3 Hookersquos law takes deflection (volts) and converts to units of force F = kx

Spring constant k 119873119873119899119899

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ights Reserve

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AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Forc

e (n

N)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

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AFM-Based Indentation Cantilever Calibration Fo

rce

(nN

)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

Tip-Sample Separation (nm)

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ights Reserve

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AFM-Based Indentation Parameters

Approach Withdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

Peak Force

Area Between Curves Energy Dissipation

Contact Mechanics Fit for Elastic Modulus (E)

Adhesion Force

Indentation Depth (δ)

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

z

r

δ

R

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119877119877 ∙ 12057512057532

Es Sample modulus νs Poissonrsquos ratio R tip radius of curvature δ load point displacement

Relevant Hertz Assumptions bull Strains are small and within the elastic limit bull Indenter is infinitely stiff (only sample deforms) bull Indentation depth ltlt radius of curvature of indenter bull Surfaces are continuous non-conforming (must be flat) bull Load is normal to surface

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d

Proper Indentation Model is Essential

δ

r

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119903119903 ∙ 12057512057532

δ

α

119865119865 =2120587120587∙

1198641198641199041199041 minus 1205841205841199041199042

∙ tan120572120572 ∙ 1205751205752

Es Sample modulus νs Poissonrsquos ratio R tip radius α opening angle

Hertz Model Spherical Indenter

depth ltlt radius of curvature

Sneddon Model Conical Indenter

depth ge radius of curvature

BBML - All R

ights Reserve

d

Indentation in OI Tendon Study

x

z

yBrtl+ and WT mice 6 month old males

4 tails per group

2-3 fascicles per tail

25-30 fibrils per tail

4-5 locations per fibril

Indent force 20 nN wet (Sneddon) 50 nN dry (Hertz) BBML - A

ll Rights R

eserved

OI Tendon Differential Effects of Dehydration

Dry 1268 plusmn 718 MPa

Wet 305 plusmn 243 MPa

Dry 1737 plusmn 973 MPa

Wet 301 plusmn 202 MPa

Kemp et al J StructBio 2012 180 (3) 428-438

bull Significant stiffening of fibrils with dehydration

bull More pronounced modulus modulus as a function of disease

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OI Tendon Differential Effects of Dehydration

Kemp et al J StructBio 2012 180 (3) 428-438

Diseased phenotype not present when wet phenotype observed when dried

0

10

20

30

40

50

60

0 20 40 60 80 100 120 140 160 180 200

Gro

up S

ampl

es (

)

Indent Modulus (MPa)

WT WetBrtl Wet

0

2

4

6

8

10

12

14

16

18

20

025

050

075

010

0012

5015

0017

5020

0022

5025

0027

5030

0032

5035

0037

5040

0042

5045

0047

5050

00

Gro

up S

ampl

es (

)Indent Modulus (MPa)

WT DryBrtl Dry

555 WT Indents 305 plusmn 243 MPa

614 Brtl+ Indents 301 plusmn 202 MPa

KS Test p=0129 t Test p=0464 464 WT Indents

1268 plusmn 718 MPa 479 Brtl+ Indents 1737 plusmn 973 MPa

KS Test plt0001 T Test plt0001

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d

Indentation in Diabetic Tendon (Poster 0495)

x

z

y

Control and Diabetic Rat Tail Tendon

4-5 rats per group

2-3 fascicles per rat

~70 fibrilstail

4-5 locationsfibril

20 nN (Sneddon)

BBML - All R

ights Reserve

d

Multiscale Diabetes Study (Poster 0495)

0

5

10

15

20

25

300 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 gt25

Perc

enta

ge o

f Gro

up S

ampl

es

Elastic Modulus (MPa)

CD (1530 indents)ZDSD (913 indents)

CD (n=5) 381 plusmn 149 MPa

ZDSD (n=3) 706 plusmn 263 MPa

0

100

200

300

400

500

600

700

800

900

0 2 4 6 8 10

Mic

rosc

ale

Mod

ulus

(MPa

)

Nanoscale Modulus (MPa)

plt0001

r2=0531 p=0040

bull Increased indentation modulus in diabetic tendon

bull Strong relationship between nanoscale indent modulus and microscale tensile modulus

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Indentation Limitations and Weaknesses

Proper probe selection for intended application bull Cantilever must be stiff relative to sample bull Sharp probes may damage sample easily dull

R or α must be determined for each probe bull Properties provided by manufacturer are unreliable bull Vary from probe to probe can dull during indent process

Spring constant must be determined for each probe

bull Value provided by manufacturer is inaccurate bull Thermal tuning OK for ldquosoftrdquo probes poor performance

for stiffer probes bull Sader method (geometry) added mass reference

cantilevers

BBML - All R

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d

Indentation Limitations and Weaknesses

Veeco Application Technical note Ben Ohler Practical Advice on the Determination of Cantilever Spring Constants

BBML - All R

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d

Indentation Limitations and Weaknesses

bull Sample processing

bull Sample hydration

bull Proper force range

bull Substrate effects for thin samples

bull Proper modulus model and range of data goodness of fit

Bearing in mind these assumptions and limitations AFM can provide true ldquonanoscalerdquo mechanical properties both in terms of forces and deformations

BBML - All R

ights Reserve

d

A Note on Peak Force QNM for Bruker AFMs bull Mechanical properties mapped pixel-by-pixel at same

resolution as the height image (PeakForce QNM)

Height Modulus BBML - All R

ights Reserve

d

A Note on Peak Force QNM

Correct probe stiffness must be used based on sample Pittenger ndash Veeco Product Bulletin

BBML - All R

ights Reserve

d

A Note on Peak Force QNM Extremely sensitive to calibration (details rarely given) Absolute radius and spring constant known

bull especially important from probe to probe bull radius uarr with indent depth ndash must determine R at depth

you expect to image bull Depth may vary as a function of sample (or at different

regions within the sample)

Relative reference material of ldquoknownrdquo modulus used to determine a ratio of 119896119896 119877119877 bull ldquoknownrdquo modulus may come from tension compression

and not indentation bull R is related to depth indentation depth on sample must

be same as on reference BBML - All R

ights Reserve

d

A Note on Peak Force QNM bull Modulus fit on the fly ndash no ability to monitor fit (or range) bull Local sample topography and sample tilt play a role in

measured parameters bull Assuming all modulus values in an image are correct how to

quantify

512 x 512 pixels = 262144 measurements

n=1 n=262144

n from selected ROIs

BBML - All R

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d

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d

bull Imaging in air on mica ndash substrate effect

bull Same imaging force or indent depth for all

bull For each area were all modulus values pooled

Lamprou et al PLoS ONE 2013 BBML - All R

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d

AFM Applications in Bone Research Other Cool Mechanical Techniques

BBML - All R

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d

ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo Bonds within or between collagen fibrils in bone that rupture to dissipate energy as a toughening mechanism

Thompson et al Nature 2001

Fantner et al Nature Mat 2005

Hansma et al JMNI 2005

Fantner et al Nano Letters 2007 BBML - All R

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d

Other Mechanical Techniques

Jimenez-Palomar J Mech Beh Bio Mat 2012

van der Rijt Macro Bio 2004

Yang J Biomed Mater Res 2007

Yang Biophysical Journal 2008 BBML - All R

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d

Other Cool Techniques bull Piezoresponse Force Microscopy (PFM)

bull Electrochemical AFM (EC-AFM)

bull Force Modulation Microscopy (FMM)

bull Lateral Force Microscopy (LFM)

bull Magnetic Force Microscopy (MFM)

bull Conductive AFM (C-AFM)

bull Force Pulling

bull Cantilever Bending

bull Tensile testing of individual fibrils

bull 3 point bending of fibrils

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d

Summary Things to rememberhellip

bull Imaging mode imaging medium and probe selection

bull Morphology Measurements bull Sample preparation and hydration state bull Measuring fibril diameter bull Site selection and proper sampling bull Appropriate system calibration

bull Mechanical Measurements

bull Probe selection (stiffness and tip radius) bull Probe calibration (radius angle spring constant) bull Indentation force range and mechanical model bull Hydration in biological samples bull PeakForce QNM

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Acknowledgements BBML

Max Hammond Armando Diaz Gonzalez Zachary Bart Silvia Canelon Creasy Clauser

IU School of Medicine Dr David Burr Dr Matt Allen Dr Stuart Warden Dr Lilian Plotkin

Funding Sources NIHNIDCR 1F32DE018840-01 A1 IUPUI BME Departmental Startup Funds IUPUI Research Support Funds Grant IUPUI Biomechanics and Biomaterials Research Center

NIH NICHD Dr Joan Marini

BBML - All R

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d

Bone Biology and Mechanics Lab (BBML) wwwiupuiedu~bbml Facebook BoneBiologyMechanicsLab

BBML - All R

ights Reserve

d

• Slide Number 1
• General Seminar Outline
• Bonersquos Hierarchical Structure
• Critical Gap in Understanding Collagen
• Methods For Analyzing Collagen Structure
• Methods For Imaging Collagen Structure
• Cryo-TEM
• Atomic Force Microscopy (AFM)
• AFM Image of Pentacene (C22H14)
• AFM Image of Hexabenzocoronene
• Basics of AFM Imaging The Probe
• Basics of AFM Imaging System Components
• van der Waals Potential Energy Curve
• Contact Mode ndash Monitor Deflection
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Peak Force Tapping Mode ndash Monitor Force
• AFM Applications in Bone Research Imaging
• AFM Imaging of Collagen
• Imaging of Osteoclast Activity
• Mineralized collagen deposition in vitro
• Imaging of Canilicular Structure
• AFM Imaging in Bone
• AFM Imaging in Bone
• Removing Mineral in Bone to Expose Collagen
• Removing Mineral in Bone to Expose Collagen
• Demineralization with EDTA
• Demineralization with EDTA
• Demineralization with EDTA
• Issues to Consider Effects of Acid
• Slide Number 32
• Slide Number 33
• Analysis of Collagen Structure in Bone
• Analysis of Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril D-Spacing
• Slide Number 39
• Collagen in Bone Fibril D-Spacing
• Collagen in Bone Fibril D-Spacing
• 2D Fast Fourier Transform Analysis
• 2D Fast Fourier Transform Analysis
• D-Spacing Distribution
• D-Spacing Distribution
• Cumulative Distribution Function
• CDF vs Histogram
• Changes in D-Spacing with Disease
• Changes in D-Spacing with Disease
• Slide Number 50
• Issues to Consider Site Selection and Sampling
• Issues to Consider Site Selection and Sampling
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• AFM calibration ndash Take Away Message
• AFM Applications in Bone ResearchIndentation Mechanics
• AFM-Based Indentation
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Parameters
• Hertz Contact Equation
• Hertz Contact Equation
• Hertz Contact Equation
• Proper Indentation Model is Essential
• Indentation in OI Tendon Study
• OI Tendon Differential Effects of Dehydration
• OI Tendon Differential Effects of Dehydration
• Indentation in Diabetic Tendon (Poster 0495)
• Multiscale Diabetes Study (Poster 0495)
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• A Note on Peak Force QNM for Bruker AFMs
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• Slide Number 83
• Slide Number 84
• AFM Applications in Bone ResearchOther Cool Mechanical Techniques
• ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo
• Other Mechanical Techniques
• Other Cool Techniques
• Summary Things to rememberhellip
• Acknowledgements
• Bone Biology and Mechanics Lab (BBML)

Cryo-TEM

bull Fixation cryosectioing and vitrification

bull Samples maintained in a hydrated state throughout sectioning and microscopy

bull Heavy-metal staining is not necessary

Can be technically challenging to perform

Quan Methods in Enzymology 2013

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Atomic Force Microscopy (AFM)

bull High spatial resolution

bull Samples can remain intact

bull Image in fluid or air

bull Range of temperatures

bull Minimal preparation

bull Characteristics less likely artifacts of processing or imaging

Can be used to extract nanoscale mechanical properties

Wallace et al Langmuir 2010

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d

AFM Image of Pentacene (C22H14)

Gross et al Science 2009 325 (5944)1110-1114 BBML - All R

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AFM Image of Hexabenzocoronene

Gross et al Science 2012 2371326-1329 BBML - All R

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Basics of AFM Imaging The Probe Nanoscale tip mounted on a microscale cantilever

Probe

Feedback System

X-Y-X Piezo Combination

Cantilever

Wallace Bone 2012

Kopycinska-Muller Ultramicroscopy 2006

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Basics of AFM Imaging System Components bull Piezoelectric actuators precise

movement under electric potential

bull Raster-scanned (x-y direction)

bull Force transducer interaction force (typically cantileverrsquos deflection)

bull Cantilever deflection (z-direction) measured by photodiode

bull Control system maintains a desired force between probe and sample and (movement in z-direction)

Probe

Feedback System

X-Y-X Piezo Combination

Cantilever

Wallace Bone 2012

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van der Waals Potential Energy Curve

FOR

CE

DISTANCE

Repulsive Regime

Attractive Regime

Weak atomic attraction

Net attractive force weakens as interatomic separation decreases

Net force = 0 distance between atoms is 2-3 Å (~ length of a chemical bond)

Attraction increases until electron clouds begin to electrostatically repel each other BBML - A

ll Rights R

eserved

Contact Mode ndash Monitor Deflection

Control system maintains user-defined deflection by vertically moving scanner using piezoelectric actuator

Tip-Sample Separation

Forc

e

Frictional and adhesive forces can damage sample and distort image

Probe in constant contact cantilever deflects like a spring according to Hookersquos law (F = kx)

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ights Reserve

d

Tapping Mode ndash Monitor Amplitude

Tip-Sample SeparationForc

e

Intermittent Contact

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ights Reserve

d

Tapping Mode ndash Monitor Amplitude

ApproachWithdrawal

Tip-Sample SeparationForc

e

Cantilever oscillates at user-defined amplitude near cantilever resonance frequency

-15

-1

-05

0

05

1

15

0 5 10 15

Osc

illat

ion

Am

plitu

de

Time

-15

-1

-05

0

05

1

15

0 5 10 15

Osc

illat

ion

Am

plitu

de

Time

Setpoint

Bump

-15

-1

-05

0

05

1

15

0 5 10 15

Osc

illat

ion

Am

plitu

de

Time

SetpointBumpValley

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ights Reserve

d

Tapping Mode ndash Monitor Amplitude bull Probe in contact for a small fraction of its tapping period

lateral forces are reduced

bull Probe must be tuned at resonance frequency ndash can be challenging for some probes when in fluid

Tuning in Air Single resonance peak Tuning in Fluid Multiple and spurious peaks wwwasylumresearchcom

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Peak Force Tapping Mode ndash Monitor Force

Control system maintains user-defined maximum force

Cantilever oscillates at low frequency (1-2 kHz)

bull No need for tuning

Slow tapping force curve is captured with each tap

ApproachWithdrawal

Tip-Sample SeparationForc

ePeakForce

Direct control of maximum normal force bull Protects tip and sample wear bull Limits indentation depth to a few nm - increases resolution

by limiting contact area bull Mechanical properties mapped pixel-by-pixel at same

resolution as the height image (PeakForce QNM) BBML - All R

ights Reserve

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AFM Applications in Bone Research Imaging

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ights Reserve

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AFM Imaging of Collagen

bull AFMrsquos adoption in bone research has been slowhellip

bull First AFM-based study in collagen came in 1992 (a previous study in 1989 used Scanning Tunneling Microscopy)

bull Observed fibrillar and monomeric collagen

Chernoff J of Vac Sci Tech 1992

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ights Reserve

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Imaging of Osteoclast Activity

Bozec Ultramicroscopy 2005

Hassenkam Anat Record 2006

Initial observations of cellular activity were qualitative (analyses of collagen morphology were not presented)

Sasaki J Elec Micro 1993

Sasaki CTI 1995

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Mineralized collagen deposition in vitro

Collagen deposition from MLO-A5 cells

Barragan-Adjemian CTI 2006 BBML - All R

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Imaging of Canilicular Structure

Reilly Annals of BME 2001

Lin J of Microscopy 2010

Average canilicular diameter ~400-500 nm larger than values typically reported from TEM studies

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AFM Imaging in Bone In general we are more interested in collagen in bone

3 microm Diamond Suspension

Unpolished

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AFM Imaging in Bone

The surface is completely mineralizedhellip BBML - All R

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Removing Mineral in Bone to Expose Collagen

Dealing with mineral is a challenge to analyzing collagen ndash some mineral must be removed

bull 10 Citric acid (to remove mineral) 65 Sodium Hypochlorite (to remove non-collagenous proteins) Habelitz J Struct Bio 2002

bull 7 or 17 Phosphoric Acid Bozek IEEE 2005 El Feninat J Biomed Mat Res 1998

bull 5 Formic Acid Ge Mat Sci Eng 2007

bull 30 mM EDTA Kindt Nanotechnology 2007

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Removing Mineral in Bone to Expose Collagen

Dealing with mineral is a challenge to analyzing collagen ndash some mineral must be removed

bull 30 mM EDTA Kindt Nanotechnology 2007 BBML - A

ll Rights R

eserved

Demineralization with EDTA EDTA - Ethylenediaminetetraacetic Acid

Chelating agent - sequesters divalent cations (notably Ca2+)

bull Treat with 05M EDTA pH 8 (15-20 minutes) bull Vigorously wash with ultrapure water bull Sonicate to release surface-bound mineral ions bull Repeat 3-4 times

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Demineralization with EDTA

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Demineralization with EDTA

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Issues to Consider Effects of Acid

Immature collagen crosslinks are very sensitive to acid bull Using acids to demineralize bone may inadvertently

destroy crosslinks distort structure

Fixation of tissue may be necessary prior to demineralization bull Fixation (glutaraldehyde or paraformadehyde) may

preserve structural integrity

bull Fixation effects on collagen structure are not fully known

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100 microm x 100 microm

50 microm x 50 microm

25 microm x 25 microm

10 microm x 10 microm

35 microm x 35 microm

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ights Reserve

d

35 microm x 35 microm

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ights Reserve

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Analysis of Collagen Structure in Bone Spatial organization of newly formed bone following a surgically-induced injury (rat tibial hole)

Baranauskas J Vac Sci Tech 2001 Baranauskas App Surface Sci 2005

ldquohellipstatistical diameter of the fibers is 159 nm in good agreement with the literature values for type I collagen fibersrdquo

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Analysis of Collagen in Bone Fibril Diameter Fibril Diameter easiest to measure property first attempts were a bit crude better with increased resolution

Sasaki J Mat Sci Tech 2002 Thalhammer J Arch Sci 2001

Bozec Ultramicroscopy 2005

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Collagen in Bone Fibril Diameter

Caution must be taken when reporting and interpreting diameters measured from fibrils in bone bull Fibril packing - width may not be fully exposed

Wallace et al Langmuir 2010

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Collagen in Bone Fibril Diameter

Caution must be taken when reporting and interpreting diameters measured from fibrils in bone bull Fibril packing - width may not be fully exposed

Rigozzi ndash J Struct Bio - 2011

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Collagen in Bone Fibril D-Spacing

300 nm 15 nm

N C C N C C N C C N C C

H O H2C CH2 O H

CH2

O OH HHC CH2

CH2

HO

H H

HHN C C

O

H2C CH2

CH2

HN C C

OH

HC CH2

HO

N C C N C C N C C

H O H2C CH2 O

CH2

OH HC CH2

HO

H H

H

CH2 CH2

glycine X - proline Y - hydroxyproline

Hodge and Petruska 1963

Collagen Fibril Axial D-Periodicity BBML - All R

ights Reserve

d

05

101520

0 02 04 06 08 1

Hei

ght (

nm)

Length (microns)

httpremfdartmoutheduimagesindexhtml

Randall Nature and Structure of Collagen 1953

TEM SEM

AFM

D-Spacing = 67 nm

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Collagen in Bone Fibril D-Spacing Many studies have shown observable D-spacing content that near theoretical value

Sasaki 2002 67 plusmn 2 nm Bozek 2005 665 plusmn 14 nm Ge 2007 666 plusmn 38 nm

Habelitz J Structural Bio 2002

A 2002 study in dentin got me interestedhellip

n = 322 wet n = 363 dry

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Collagen in Bone Fibril D-Spacing

bull Internal structure bull Wallace et al Bone 2010 46 1349-1354 bull Wallace et al J Stuct Bio 2011 173 146-152 bull Kemp et al J Stuct Bio 2012 180 428-438 bull Warden et al Endocrinology 2013 154(9)

3178-3187 bull Gallant et al Bone 2014 61 191-200 bull Bart et al Conn Tiss Res 2014 In Press

bull Enzymatic and non-

enzymatic cross-linking bull Hammond et al Bone 2014 60 26-32 bull Diaz Gonzalez et al J Biomech 2014 47(3)

681-686 bull Voytik-Harbin et al In Preparation 2014

D-periodic spacing important structural feature that captures aspects related to

Mouse Femur Mid-diaphysis BBML - All R

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2D Fast Fourier Transform Analysis

Harmonics

Fibril D-Spacing

Erickson B Wallace JM et al Biotechnology Journal 2013

Raw AFM Image 2D FFT Power Spectrum

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2D Fast Fourier Transform Analysis

Harmonics

Fibril D-Spacing

Raw AFM Image 2D FFT Power Spectrum

0

20000

40000

60000

80000

100000

120000

0 001 002 003 004 005 006

Am

plitu

de (

microV2 )

Position (1nm)

D-periodic spacing 0015 (1nm) ~ 67 nm

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ights Reserve

d

D-Spacing Distribution

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Overall Mean 670 plusmn 00 nm

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ights Reserve

d

D-Spacing Distribution

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Overall Mean 670 plusmn 00 nm

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

4 Samples Total of n = 255 fibrils Overall Mean 682 plusmn 12 nm

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Cumulative Distribution Function

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

This population 255 fibrils - Each fibril 1255 = 0392 - Data range 628-716 nm

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ights Reserve

d

CDF vs Histogram 62 63 64 65 66 67 68 69 70 71 72 73 74

0

10

20

30

40

50

60

70

80

90

100

0

5

10

15

20

25

30

35

40

45

50

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

HistoCDF

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ights Reserve

d

Changes in D-Spacing with Disease

0

5

10

15

20

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

0

5

10

15

20

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

Wallace et al Bone 2010 46 1349-1354

KS Test plt0001

Control 680 plusmn 26 nm

Estrogen Dep 659 plusmn 31 nm

t-test on mean p=0048

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Changes in D-Spacing with Disease

Wallace et al Bone 2010 46 1349-1354

0

10

20

30

40

50

60

70

80

90

100

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

KS Test plt0001

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Question Model and Tissue Morphology Publication

Tissue Specificity in Mice Male mouse 8 weeks Femur dentin tail tendon

No differences in bone vs dentin both ∆ vs tendon

Wallace et al Langmuir 2011 26(10) 7349-7354

Gender Effects Male and female mice 16 weeks Femur No differences NA

Estrogen Depletion OVX in sheep 5 years + 2 Radius Mean and population shift Wallace et al Bone

2010 46 1349-1354

Estrogen Depletion OVX in female 5 years + 2 Skin (dermis) Significant population shift Erickson et al Biotech

J 2013 8(1) 1174-126

Osteoporosis and Mechanical Loading

OVX in ulnar loading in rats Ulna

No loading effect population shift with OVX

Warden et al Endocrinology 2013 154(9) 3178-3187

Osteogenesis Imperfecta Brtl+ model male mice 2 months Femur

No mean ∆ widening of distribution

Wallace et al J Stuct Bio 2011 173 146-152

Osteogenesis Imperfecta Oimoim model male mice 12 weeks Femur Mean and population shift Bart et al Conn Tiss

Res 2014 In Press

Osteogenesis Imperfecta and Tissue Hydration

Brtl+ model male mice 6 months Tail Tendon Significant population shift Kemp et al J Stuct Bio

2012 180 428-438

Type 2 Diabetes Male ZDSD rats 30 weeks Tibia Significant population shift Hammond et al Bone

2014 60 26-32

Type 2 Diabetes Female ZDSD rats 32 weeks Tail Tendon Significant population shift

Diaz Gonzalez et al J Biomech 2014 47(3)

681-686

Raloxifene (in vivo and in vitro) Female dog treated with Raloxifene Femur Significant population shift Gallant et al Bone

2014 61 191-200 BBML - All R

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Issues to Consider Site Selection and Sampling

KS Test plt0001

0023 of bonersquos length

15 m

m =

150

00 micro

m

Wallace et al Langmuir 2011 26(10) 7349-7354

100 microm x 100 microm

35 microm x 35 microm

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Issues to Consider Site Selection and Sampling

60

62

64

66

68

70

72

74

1 (35)

2 (33)

3 (40)

4 (39)

5 (35)

6 (29)

7 (26)

8 (23)

9 (28)

WT (288)

D-P

erio

dic

Gap

Ove

rlap

Spac

ing

(nm

)

Axial Location (Proximal End = 1 Distal End = 9)

( of Fibrils)

91

23

45

87

6

Wallace et al Langmuir 2011 26(10) 7349-7354

288 fibrils for population n=1 for mean comparisons BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

Wet Tendon Dry Tendon Kemp et al J StructBio 2012 180 (3) 428-438

Collagen and bone exist in a hydrated environment in vivo bull Hydration state can impact morphology and mechanics

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ights Reserve

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Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

Nonsignificant mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet (n=255) 682 plusmn 12 nm

WT Dry (n=257) 686 plusmn 08 nm

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

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Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

Brtl DryBrtl Wet

KS Test plt0001

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

WT Case 04 nm mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet 682 plusmn 12 nm

WT Dry 686 plusmn 08 nm

Brtl+ Wet 675 plusmn 14 nm

Brtl+ Dry 689 plusmn 10 nm

Brtl+ Case 14 nm increase with drying (plt0001) bull Entire population shifted upward (plt0001)

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT WetBrtl Wet

KS Test plt0001

Brtl+ distribution shifted downward vs WT when wet

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryBrtl Dry

KS Test plt0001

Brtl+ distribution shifted upward vs WT when dry

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider AFM calibration

bull Absolute distance measurements require accurate piezo calibration

bull AFM systems have large scan ranges (150 microm to lt 1 microm)

bull Scanner non-linearities can introduce substantial error over that range

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

ights Reserve

d

Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

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ights Reserve

d

Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

100 nm 10 80 602 1198 100 nm 7 60 827 1439 100 nm 5 40 814 1427 100 nm 35 20 917 1724 100 nm 2 10 937 1774

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

ights Reserve

d

AFM calibration ndash Take Away Message

Calibration using a scan size and feature size comparable to features of interest is highly recommended

Calibration addresses absolute accuracy not precision (differential sensitivity between measurements)

bull Does not limit ability to differentiate between groups using the same AFM with the same calibration parameters

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AFM Applications in Bone Research Indentation Mechanics

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ights Reserve

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AFM-Based Indentation

In addition to high resolution imaging AFM can be used to extract nanoscale mechanical data

ApproachWithdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Can

tilev

er D

efle

ctio

n (V

olts

)

1 Ramp into a rigid sample ndash measure the slope

Deflection sensitivity x 119899119899119899119899119881119881

2 Calculate cantilever spring constant

3 Hookersquos law takes deflection (volts) and converts to units of force F = kx

Spring constant k 119873119873119899119899

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ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Forc

e (n

N)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

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ights Reserve

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AFM-Based Indentation Cantilever Calibration Fo

rce

(nN

)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

Tip-Sample Separation (nm)

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ights Reserve

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AFM-Based Indentation Parameters

Approach Withdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

Peak Force

Area Between Curves Energy Dissipation

Contact Mechanics Fit for Elastic Modulus (E)

Adhesion Force

Indentation Depth (δ)

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

z

r

δ

R

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119877119877 ∙ 12057512057532

Es Sample modulus νs Poissonrsquos ratio R tip radius of curvature δ load point displacement

Relevant Hertz Assumptions bull Strains are small and within the elastic limit bull Indenter is infinitely stiff (only sample deforms) bull Indentation depth ltlt radius of curvature of indenter bull Surfaces are continuous non-conforming (must be flat) bull Load is normal to surface

BBML - All R

ights Reserve

d

Proper Indentation Model is Essential

δ

r

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119903119903 ∙ 12057512057532

δ

α

119865119865 =2120587120587∙

1198641198641199041199041 minus 1205841205841199041199042

∙ tan120572120572 ∙ 1205751205752

Es Sample modulus νs Poissonrsquos ratio R tip radius α opening angle

Hertz Model Spherical Indenter

depth ltlt radius of curvature

Sneddon Model Conical Indenter

depth ge radius of curvature

BBML - All R

ights Reserve

d

Indentation in OI Tendon Study

x

z

yBrtl+ and WT mice 6 month old males

4 tails per group

2-3 fascicles per tail

25-30 fibrils per tail

4-5 locations per fibril

Indent force 20 nN wet (Sneddon) 50 nN dry (Hertz) BBML - A

ll Rights R

eserved

OI Tendon Differential Effects of Dehydration

Dry 1268 plusmn 718 MPa

Wet 305 plusmn 243 MPa

Dry 1737 plusmn 973 MPa

Wet 301 plusmn 202 MPa

Kemp et al J StructBio 2012 180 (3) 428-438

bull Significant stiffening of fibrils with dehydration

bull More pronounced modulus modulus as a function of disease

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ights Reserve

d

OI Tendon Differential Effects of Dehydration

Kemp et al J StructBio 2012 180 (3) 428-438

Diseased phenotype not present when wet phenotype observed when dried

0

10

20

30

40

50

60

0 20 40 60 80 100 120 140 160 180 200

Gro

up S

ampl

es (

)

Indent Modulus (MPa)

WT WetBrtl Wet

0

2

4

6

8

10

12

14

16

18

20

025

050

075

010

0012

5015

0017

5020

0022

5025

0027

5030

0032

5035

0037

5040

0042

5045

0047

5050

00

Gro

up S

ampl

es (

)Indent Modulus (MPa)

WT DryBrtl Dry

555 WT Indents 305 plusmn 243 MPa

614 Brtl+ Indents 301 plusmn 202 MPa

KS Test p=0129 t Test p=0464 464 WT Indents

1268 plusmn 718 MPa 479 Brtl+ Indents 1737 plusmn 973 MPa

KS Test plt0001 T Test plt0001

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ights Reserve

d

Indentation in Diabetic Tendon (Poster 0495)

x

z

y

Control and Diabetic Rat Tail Tendon

4-5 rats per group

2-3 fascicles per rat

~70 fibrilstail

4-5 locationsfibril

20 nN (Sneddon)

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ights Reserve

d

Multiscale Diabetes Study (Poster 0495)

0

5

10

15

20

25

300 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 gt25

Perc

enta

ge o

f Gro

up S

ampl

es

Elastic Modulus (MPa)

CD (1530 indents)ZDSD (913 indents)

CD (n=5) 381 plusmn 149 MPa

ZDSD (n=3) 706 plusmn 263 MPa

0

100

200

300

400

500

600

700

800

900

0 2 4 6 8 10

Mic

rosc

ale

Mod

ulus

(MPa

)

Nanoscale Modulus (MPa)

plt0001

r2=0531 p=0040

bull Increased indentation modulus in diabetic tendon

bull Strong relationship between nanoscale indent modulus and microscale tensile modulus

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Indentation Limitations and Weaknesses

Proper probe selection for intended application bull Cantilever must be stiff relative to sample bull Sharp probes may damage sample easily dull

R or α must be determined for each probe bull Properties provided by manufacturer are unreliable bull Vary from probe to probe can dull during indent process

Spring constant must be determined for each probe

bull Value provided by manufacturer is inaccurate bull Thermal tuning OK for ldquosoftrdquo probes poor performance

for stiffer probes bull Sader method (geometry) added mass reference

cantilevers

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d

Indentation Limitations and Weaknesses

Veeco Application Technical note Ben Ohler Practical Advice on the Determination of Cantilever Spring Constants

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Indentation Limitations and Weaknesses

bull Sample processing

bull Sample hydration

bull Proper force range

bull Substrate effects for thin samples

bull Proper modulus model and range of data goodness of fit

Bearing in mind these assumptions and limitations AFM can provide true ldquonanoscalerdquo mechanical properties both in terms of forces and deformations

BBML - All R

ights Reserve

d

A Note on Peak Force QNM for Bruker AFMs bull Mechanical properties mapped pixel-by-pixel at same

resolution as the height image (PeakForce QNM)

Height Modulus BBML - All R

ights Reserve

d

A Note on Peak Force QNM

Correct probe stiffness must be used based on sample Pittenger ndash Veeco Product Bulletin

BBML - All R

ights Reserve

d

A Note on Peak Force QNM Extremely sensitive to calibration (details rarely given) Absolute radius and spring constant known

bull especially important from probe to probe bull radius uarr with indent depth ndash must determine R at depth

you expect to image bull Depth may vary as a function of sample (or at different

regions within the sample)

Relative reference material of ldquoknownrdquo modulus used to determine a ratio of 119896119896 119877119877 bull ldquoknownrdquo modulus may come from tension compression

and not indentation bull R is related to depth indentation depth on sample must

be same as on reference BBML - All R

ights Reserve

d

A Note on Peak Force QNM bull Modulus fit on the fly ndash no ability to monitor fit (or range) bull Local sample topography and sample tilt play a role in

measured parameters bull Assuming all modulus values in an image are correct how to

quantify

512 x 512 pixels = 262144 measurements

n=1 n=262144

n from selected ROIs

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d

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ights Reserve

d

bull Imaging in air on mica ndash substrate effect

bull Same imaging force or indent depth for all

bull For each area were all modulus values pooled

Lamprou et al PLoS ONE 2013 BBML - All R

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d

AFM Applications in Bone Research Other Cool Mechanical Techniques

BBML - All R

ights Reserve

d

ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo Bonds within or between collagen fibrils in bone that rupture to dissipate energy as a toughening mechanism

Thompson et al Nature 2001

Fantner et al Nature Mat 2005

Hansma et al JMNI 2005

Fantner et al Nano Letters 2007 BBML - All R

ights Reserve

d

Other Mechanical Techniques

Jimenez-Palomar J Mech Beh Bio Mat 2012

van der Rijt Macro Bio 2004

Yang J Biomed Mater Res 2007

Yang Biophysical Journal 2008 BBML - All R

ights Reserve

d

Other Cool Techniques bull Piezoresponse Force Microscopy (PFM)

bull Electrochemical AFM (EC-AFM)

bull Force Modulation Microscopy (FMM)

bull Lateral Force Microscopy (LFM)

bull Magnetic Force Microscopy (MFM)

bull Conductive AFM (C-AFM)

bull Force Pulling

bull Cantilever Bending

bull Tensile testing of individual fibrils

bull 3 point bending of fibrils

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Summary Things to rememberhellip

bull Imaging mode imaging medium and probe selection

bull Morphology Measurements bull Sample preparation and hydration state bull Measuring fibril diameter bull Site selection and proper sampling bull Appropriate system calibration

bull Mechanical Measurements

bull Probe selection (stiffness and tip radius) bull Probe calibration (radius angle spring constant) bull Indentation force range and mechanical model bull Hydration in biological samples bull PeakForce QNM

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Acknowledgements BBML

Max Hammond Armando Diaz Gonzalez Zachary Bart Silvia Canelon Creasy Clauser

IU School of Medicine Dr David Burr Dr Matt Allen Dr Stuart Warden Dr Lilian Plotkin

Funding Sources NIHNIDCR 1F32DE018840-01 A1 IUPUI BME Departmental Startup Funds IUPUI Research Support Funds Grant IUPUI Biomechanics and Biomaterials Research Center

NIH NICHD Dr Joan Marini

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Bone Biology and Mechanics Lab (BBML) wwwiupuiedu~bbml Facebook BoneBiologyMechanicsLab

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d

• Slide Number 1
• General Seminar Outline
• Bonersquos Hierarchical Structure
• Critical Gap in Understanding Collagen
• Methods For Analyzing Collagen Structure
• Methods For Imaging Collagen Structure
• Cryo-TEM
• Atomic Force Microscopy (AFM)
• AFM Image of Pentacene (C22H14)
• AFM Image of Hexabenzocoronene
• Basics of AFM Imaging The Probe
• Basics of AFM Imaging System Components
• van der Waals Potential Energy Curve
• Contact Mode ndash Monitor Deflection
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Peak Force Tapping Mode ndash Monitor Force
• AFM Applications in Bone Research Imaging
• AFM Imaging of Collagen
• Imaging of Osteoclast Activity
• Mineralized collagen deposition in vitro
• Imaging of Canilicular Structure
• AFM Imaging in Bone
• AFM Imaging in Bone
• Removing Mineral in Bone to Expose Collagen
• Removing Mineral in Bone to Expose Collagen
• Demineralization with EDTA
• Demineralization with EDTA
• Demineralization with EDTA
• Issues to Consider Effects of Acid
• Slide Number 32
• Slide Number 33
• Analysis of Collagen Structure in Bone
• Analysis of Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril D-Spacing
• Slide Number 39
• Collagen in Bone Fibril D-Spacing
• Collagen in Bone Fibril D-Spacing
• 2D Fast Fourier Transform Analysis
• 2D Fast Fourier Transform Analysis
• D-Spacing Distribution
• D-Spacing Distribution
• Cumulative Distribution Function
• CDF vs Histogram
• Changes in D-Spacing with Disease
• Changes in D-Spacing with Disease
• Slide Number 50
• Issues to Consider Site Selection and Sampling
• Issues to Consider Site Selection and Sampling
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• AFM calibration ndash Take Away Message
• AFM Applications in Bone ResearchIndentation Mechanics
• AFM-Based Indentation
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Parameters
• Hertz Contact Equation
• Hertz Contact Equation
• Hertz Contact Equation
• Proper Indentation Model is Essential
• Indentation in OI Tendon Study
• OI Tendon Differential Effects of Dehydration
• OI Tendon Differential Effects of Dehydration
• Indentation in Diabetic Tendon (Poster 0495)
• Multiscale Diabetes Study (Poster 0495)
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• A Note on Peak Force QNM for Bruker AFMs
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• Slide Number 83
• Slide Number 84
• AFM Applications in Bone ResearchOther Cool Mechanical Techniques
• ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo
• Other Mechanical Techniques
• Other Cool Techniques
• Summary Things to rememberhellip
• Acknowledgements
• Bone Biology and Mechanics Lab (BBML)

Atomic Force Microscopy (AFM)

bull High spatial resolution

bull Samples can remain intact

bull Image in fluid or air

bull Range of temperatures

bull Minimal preparation

bull Characteristics less likely artifacts of processing or imaging

Can be used to extract nanoscale mechanical properties

Wallace et al Langmuir 2010

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AFM Image of Pentacene (C22H14)

Gross et al Science 2009 325 (5944)1110-1114 BBML - All R

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AFM Image of Hexabenzocoronene

Gross et al Science 2012 2371326-1329 BBML - All R

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Basics of AFM Imaging The Probe Nanoscale tip mounted on a microscale cantilever

Probe

Feedback System

X-Y-X Piezo Combination

Cantilever

Wallace Bone 2012

Kopycinska-Muller Ultramicroscopy 2006

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Basics of AFM Imaging System Components bull Piezoelectric actuators precise

movement under electric potential

bull Raster-scanned (x-y direction)

bull Force transducer interaction force (typically cantileverrsquos deflection)

bull Cantilever deflection (z-direction) measured by photodiode

bull Control system maintains a desired force between probe and sample and (movement in z-direction)

Probe

Feedback System

X-Y-X Piezo Combination

Cantilever

Wallace Bone 2012

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ights Reserve

d

van der Waals Potential Energy Curve

FOR

CE

DISTANCE

Repulsive Regime

Attractive Regime

Weak atomic attraction

Net attractive force weakens as interatomic separation decreases

Net force = 0 distance between atoms is 2-3 Å (~ length of a chemical bond)

Attraction increases until electron clouds begin to electrostatically repel each other BBML - A

ll Rights R

eserved

Contact Mode ndash Monitor Deflection

Control system maintains user-defined deflection by vertically moving scanner using piezoelectric actuator

Tip-Sample Separation

Forc

e

Frictional and adhesive forces can damage sample and distort image

Probe in constant contact cantilever deflects like a spring according to Hookersquos law (F = kx)

BBML - All R

ights Reserve

d

Tapping Mode ndash Monitor Amplitude

Tip-Sample SeparationForc

e

Intermittent Contact

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ights Reserve

d

Tapping Mode ndash Monitor Amplitude

ApproachWithdrawal

Tip-Sample SeparationForc

e

Cantilever oscillates at user-defined amplitude near cantilever resonance frequency

-15

-1

-05

0

05

1

15

0 5 10 15

Osc

illat

ion

Am

plitu

de

Time

-15

-1

-05

0

05

1

15

0 5 10 15

Osc

illat

ion

Am

plitu

de

Time

Setpoint

Bump

-15

-1

-05

0

05

1

15

0 5 10 15

Osc

illat

ion

Am

plitu

de

Time

SetpointBumpValley

BBML - All R

ights Reserve

d

Tapping Mode ndash Monitor Amplitude bull Probe in contact for a small fraction of its tapping period

lateral forces are reduced

bull Probe must be tuned at resonance frequency ndash can be challenging for some probes when in fluid

Tuning in Air Single resonance peak Tuning in Fluid Multiple and spurious peaks wwwasylumresearchcom

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Peak Force Tapping Mode ndash Monitor Force

Control system maintains user-defined maximum force

Cantilever oscillates at low frequency (1-2 kHz)

bull No need for tuning

Slow tapping force curve is captured with each tap

ApproachWithdrawal

Tip-Sample SeparationForc

ePeakForce

Direct control of maximum normal force bull Protects tip and sample wear bull Limits indentation depth to a few nm - increases resolution

by limiting contact area bull Mechanical properties mapped pixel-by-pixel at same

resolution as the height image (PeakForce QNM) BBML - All R

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d

AFM Applications in Bone Research Imaging

BBML - All R

ights Reserve

d

AFM Imaging of Collagen

bull AFMrsquos adoption in bone research has been slowhellip

bull First AFM-based study in collagen came in 1992 (a previous study in 1989 used Scanning Tunneling Microscopy)

bull Observed fibrillar and monomeric collagen

Chernoff J of Vac Sci Tech 1992

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Imaging of Osteoclast Activity

Bozec Ultramicroscopy 2005

Hassenkam Anat Record 2006

Initial observations of cellular activity were qualitative (analyses of collagen morphology were not presented)

Sasaki J Elec Micro 1993

Sasaki CTI 1995

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Mineralized collagen deposition in vitro

Collagen deposition from MLO-A5 cells

Barragan-Adjemian CTI 2006 BBML - All R

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Imaging of Canilicular Structure

Reilly Annals of BME 2001

Lin J of Microscopy 2010

Average canilicular diameter ~400-500 nm larger than values typically reported from TEM studies

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AFM Imaging in Bone In general we are more interested in collagen in bone

3 microm Diamond Suspension

Unpolished

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AFM Imaging in Bone

The surface is completely mineralizedhellip BBML - All R

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d

Removing Mineral in Bone to Expose Collagen

Dealing with mineral is a challenge to analyzing collagen ndash some mineral must be removed

bull 10 Citric acid (to remove mineral) 65 Sodium Hypochlorite (to remove non-collagenous proteins) Habelitz J Struct Bio 2002

bull 7 or 17 Phosphoric Acid Bozek IEEE 2005 El Feninat J Biomed Mat Res 1998

bull 5 Formic Acid Ge Mat Sci Eng 2007

bull 30 mM EDTA Kindt Nanotechnology 2007

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Removing Mineral in Bone to Expose Collagen

Dealing with mineral is a challenge to analyzing collagen ndash some mineral must be removed

bull 30 mM EDTA Kindt Nanotechnology 2007 BBML - A

ll Rights R

eserved

Demineralization with EDTA EDTA - Ethylenediaminetetraacetic Acid

Chelating agent - sequesters divalent cations (notably Ca2+)

bull Treat with 05M EDTA pH 8 (15-20 minutes) bull Vigorously wash with ultrapure water bull Sonicate to release surface-bound mineral ions bull Repeat 3-4 times

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Demineralization with EDTA

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Demineralization with EDTA

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Issues to Consider Effects of Acid

Immature collagen crosslinks are very sensitive to acid bull Using acids to demineralize bone may inadvertently

destroy crosslinks distort structure

Fixation of tissue may be necessary prior to demineralization bull Fixation (glutaraldehyde or paraformadehyde) may

preserve structural integrity

bull Fixation effects on collagen structure are not fully known

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100 microm x 100 microm

50 microm x 50 microm

25 microm x 25 microm

10 microm x 10 microm

35 microm x 35 microm

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35 microm x 35 microm

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Analysis of Collagen Structure in Bone Spatial organization of newly formed bone following a surgically-induced injury (rat tibial hole)

Baranauskas J Vac Sci Tech 2001 Baranauskas App Surface Sci 2005

ldquohellipstatistical diameter of the fibers is 159 nm in good agreement with the literature values for type I collagen fibersrdquo

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Analysis of Collagen in Bone Fibril Diameter Fibril Diameter easiest to measure property first attempts were a bit crude better with increased resolution

Sasaki J Mat Sci Tech 2002 Thalhammer J Arch Sci 2001

Bozec Ultramicroscopy 2005

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Collagen in Bone Fibril Diameter

Caution must be taken when reporting and interpreting diameters measured from fibrils in bone bull Fibril packing - width may not be fully exposed

Wallace et al Langmuir 2010

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Collagen in Bone Fibril Diameter

Caution must be taken when reporting and interpreting diameters measured from fibrils in bone bull Fibril packing - width may not be fully exposed

Rigozzi ndash J Struct Bio - 2011

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Collagen in Bone Fibril D-Spacing

300 nm 15 nm

N C C N C C N C C N C C

H O H2C CH2 O H

CH2

O OH HHC CH2

CH2

HO

H H

HHN C C

O

H2C CH2

CH2

HN C C

OH

HC CH2

HO

N C C N C C N C C

H O H2C CH2 O

CH2

OH HC CH2

HO

H H

H

CH2 CH2

glycine X - proline Y - hydroxyproline

Hodge and Petruska 1963

Collagen Fibril Axial D-Periodicity BBML - All R

ights Reserve

d

05

101520

0 02 04 06 08 1

Hei

ght (

nm)

Length (microns)

httpremfdartmoutheduimagesindexhtml

Randall Nature and Structure of Collagen 1953

TEM SEM

AFM

D-Spacing = 67 nm

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Collagen in Bone Fibril D-Spacing Many studies have shown observable D-spacing content that near theoretical value

Sasaki 2002 67 plusmn 2 nm Bozek 2005 665 plusmn 14 nm Ge 2007 666 plusmn 38 nm

Habelitz J Structural Bio 2002

A 2002 study in dentin got me interestedhellip

n = 322 wet n = 363 dry

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Collagen in Bone Fibril D-Spacing

bull Internal structure bull Wallace et al Bone 2010 46 1349-1354 bull Wallace et al J Stuct Bio 2011 173 146-152 bull Kemp et al J Stuct Bio 2012 180 428-438 bull Warden et al Endocrinology 2013 154(9)

3178-3187 bull Gallant et al Bone 2014 61 191-200 bull Bart et al Conn Tiss Res 2014 In Press

bull Enzymatic and non-

enzymatic cross-linking bull Hammond et al Bone 2014 60 26-32 bull Diaz Gonzalez et al J Biomech 2014 47(3)

681-686 bull Voytik-Harbin et al In Preparation 2014

D-periodic spacing important structural feature that captures aspects related to

Mouse Femur Mid-diaphysis BBML - All R

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2D Fast Fourier Transform Analysis

Harmonics

Fibril D-Spacing

Erickson B Wallace JM et al Biotechnology Journal 2013

Raw AFM Image 2D FFT Power Spectrum

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2D Fast Fourier Transform Analysis

Harmonics

Fibril D-Spacing

Raw AFM Image 2D FFT Power Spectrum

0

20000

40000

60000

80000

100000

120000

0 001 002 003 004 005 006

Am

plitu

de (

microV2 )

Position (1nm)

D-periodic spacing 0015 (1nm) ~ 67 nm

BBML - All R

ights Reserve

d

D-Spacing Distribution

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Overall Mean 670 plusmn 00 nm

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ights Reserve

d

D-Spacing Distribution

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Overall Mean 670 plusmn 00 nm

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

4 Samples Total of n = 255 fibrils Overall Mean 682 plusmn 12 nm

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ights Reserve

d

Cumulative Distribution Function

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

This population 255 fibrils - Each fibril 1255 = 0392 - Data range 628-716 nm

BBML - All R

ights Reserve

d

CDF vs Histogram 62 63 64 65 66 67 68 69 70 71 72 73 74

0

10

20

30

40

50

60

70

80

90

100

0

5

10

15

20

25

30

35

40

45

50

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

HistoCDF

BBML - All R

ights Reserve

d

Changes in D-Spacing with Disease

0

5

10

15

20

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

0

5

10

15

20

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

Wallace et al Bone 2010 46 1349-1354

KS Test plt0001

Control 680 plusmn 26 nm

Estrogen Dep 659 plusmn 31 nm

t-test on mean p=0048

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ights Reserve

d

Changes in D-Spacing with Disease

Wallace et al Bone 2010 46 1349-1354

0

10

20

30

40

50

60

70

80

90

100

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

KS Test plt0001

BBML - All R

ights Reserve

d

Question Model and Tissue Morphology Publication

Tissue Specificity in Mice Male mouse 8 weeks Femur dentin tail tendon

No differences in bone vs dentin both ∆ vs tendon

Wallace et al Langmuir 2011 26(10) 7349-7354

Gender Effects Male and female mice 16 weeks Femur No differences NA

Estrogen Depletion OVX in sheep 5 years + 2 Radius Mean and population shift Wallace et al Bone

2010 46 1349-1354

Estrogen Depletion OVX in female 5 years + 2 Skin (dermis) Significant population shift Erickson et al Biotech

J 2013 8(1) 1174-126

Osteoporosis and Mechanical Loading

OVX in ulnar loading in rats Ulna

No loading effect population shift with OVX

Warden et al Endocrinology 2013 154(9) 3178-3187

Osteogenesis Imperfecta Brtl+ model male mice 2 months Femur

No mean ∆ widening of distribution

Wallace et al J Stuct Bio 2011 173 146-152

Osteogenesis Imperfecta Oimoim model male mice 12 weeks Femur Mean and population shift Bart et al Conn Tiss

Res 2014 In Press

Osteogenesis Imperfecta and Tissue Hydration

Brtl+ model male mice 6 months Tail Tendon Significant population shift Kemp et al J Stuct Bio

2012 180 428-438

Type 2 Diabetes Male ZDSD rats 30 weeks Tibia Significant population shift Hammond et al Bone

2014 60 26-32

Type 2 Diabetes Female ZDSD rats 32 weeks Tail Tendon Significant population shift

Diaz Gonzalez et al J Biomech 2014 47(3)

681-686

Raloxifene (in vivo and in vitro) Female dog treated with Raloxifene Femur Significant population shift Gallant et al Bone

2014 61 191-200 BBML - All R

ights Reserve

d

Issues to Consider Site Selection and Sampling

KS Test plt0001

0023 of bonersquos length

15 m

m =

150

00 micro

m

Wallace et al Langmuir 2011 26(10) 7349-7354

100 microm x 100 microm

35 microm x 35 microm

BBML - All R

ights Reserve

d

Issues to Consider Site Selection and Sampling

60

62

64

66

68

70

72

74

1 (35)

2 (33)

3 (40)

4 (39)

5 (35)

6 (29)

7 (26)

8 (23)

9 (28)

WT (288)

D-P

erio

dic

Gap

Ove

rlap

Spac

ing

(nm

)

Axial Location (Proximal End = 1 Distal End = 9)

( of Fibrils)

91

23

45

87

6

Wallace et al Langmuir 2011 26(10) 7349-7354

288 fibrils for population n=1 for mean comparisons BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

Wet Tendon Dry Tendon Kemp et al J StructBio 2012 180 (3) 428-438

Collagen and bone exist in a hydrated environment in vivo bull Hydration state can impact morphology and mechanics

BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

Nonsignificant mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet (n=255) 682 plusmn 12 nm

WT Dry (n=257) 686 plusmn 08 nm

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

Brtl DryBrtl Wet

KS Test plt0001

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

WT Case 04 nm mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet 682 plusmn 12 nm

WT Dry 686 plusmn 08 nm

Brtl+ Wet 675 plusmn 14 nm

Brtl+ Dry 689 plusmn 10 nm

Brtl+ Case 14 nm increase with drying (plt0001) bull Entire population shifted upward (plt0001)

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT WetBrtl Wet

KS Test plt0001

Brtl+ distribution shifted downward vs WT when wet

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryBrtl Dry

KS Test plt0001

Brtl+ distribution shifted upward vs WT when dry

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider AFM calibration

bull Absolute distance measurements require accurate piezo calibration

bull AFM systems have large scan ranges (150 microm to lt 1 microm)

bull Scanner non-linearities can introduce substantial error over that range

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

ights Reserve

d

Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

BBML - All R

ights Reserve

d

Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

100 nm 10 80 602 1198 100 nm 7 60 827 1439 100 nm 5 40 814 1427 100 nm 35 20 917 1724 100 nm 2 10 937 1774

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

ights Reserve

d

AFM calibration ndash Take Away Message

Calibration using a scan size and feature size comparable to features of interest is highly recommended

Calibration addresses absolute accuracy not precision (differential sensitivity between measurements)

bull Does not limit ability to differentiate between groups using the same AFM with the same calibration parameters

BBML - All R

ights Reserve

d

AFM Applications in Bone Research Indentation Mechanics

BBML - All R

ights Reserve

d

AFM-Based Indentation

In addition to high resolution imaging AFM can be used to extract nanoscale mechanical data

ApproachWithdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Can

tilev

er D

efle

ctio

n (V

olts

)

1 Ramp into a rigid sample ndash measure the slope

Deflection sensitivity x 119899119899119899119899119881119881

2 Calculate cantilever spring constant

3 Hookersquos law takes deflection (volts) and converts to units of force F = kx

Spring constant k 119873119873119899119899

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Forc

e (n

N)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration Fo

rce

(nN

)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

Tip-Sample Separation (nm)

BBML - All R

ights Reserve

d

AFM-Based Indentation Parameters

Approach Withdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

Peak Force

Area Between Curves Energy Dissipation

Contact Mechanics Fit for Elastic Modulus (E)

Adhesion Force

Indentation Depth (δ)

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

z

r

δ

R

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119877119877 ∙ 12057512057532

Es Sample modulus νs Poissonrsquos ratio R tip radius of curvature δ load point displacement

Relevant Hertz Assumptions bull Strains are small and within the elastic limit bull Indenter is infinitely stiff (only sample deforms) bull Indentation depth ltlt radius of curvature of indenter bull Surfaces are continuous non-conforming (must be flat) bull Load is normal to surface

BBML - All R

ights Reserve

d

Proper Indentation Model is Essential

δ

r

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119903119903 ∙ 12057512057532

δ

α

119865119865 =2120587120587∙

1198641198641199041199041 minus 1205841205841199041199042

∙ tan120572120572 ∙ 1205751205752

Es Sample modulus νs Poissonrsquos ratio R tip radius α opening angle

Hertz Model Spherical Indenter

depth ltlt radius of curvature

Sneddon Model Conical Indenter

depth ge radius of curvature

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ights Reserve

d

Indentation in OI Tendon Study

x

z

yBrtl+ and WT mice 6 month old males

4 tails per group

2-3 fascicles per tail

25-30 fibrils per tail

4-5 locations per fibril

Indent force 20 nN wet (Sneddon) 50 nN dry (Hertz) BBML - A

ll Rights R

eserved

OI Tendon Differential Effects of Dehydration

Dry 1268 plusmn 718 MPa

Wet 305 plusmn 243 MPa

Dry 1737 plusmn 973 MPa

Wet 301 plusmn 202 MPa

Kemp et al J StructBio 2012 180 (3) 428-438

bull Significant stiffening of fibrils with dehydration

bull More pronounced modulus modulus as a function of disease

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ights Reserve

d

OI Tendon Differential Effects of Dehydration

Kemp et al J StructBio 2012 180 (3) 428-438

Diseased phenotype not present when wet phenotype observed when dried

0

10

20

30

40

50

60

0 20 40 60 80 100 120 140 160 180 200

Gro

up S

ampl

es (

)

Indent Modulus (MPa)

WT WetBrtl Wet

0

2

4

6

8

10

12

14

16

18

20

025

050

075

010

0012

5015

0017

5020

0022

5025

0027

5030

0032

5035

0037

5040

0042

5045

0047

5050

00

Gro

up S

ampl

es (

)Indent Modulus (MPa)

WT DryBrtl Dry

555 WT Indents 305 plusmn 243 MPa

614 Brtl+ Indents 301 plusmn 202 MPa

KS Test p=0129 t Test p=0464 464 WT Indents

1268 plusmn 718 MPa 479 Brtl+ Indents 1737 plusmn 973 MPa

KS Test plt0001 T Test plt0001

BBML - All R

ights Reserve

d

Indentation in Diabetic Tendon (Poster 0495)

x

z

y

Control and Diabetic Rat Tail Tendon

4-5 rats per group

2-3 fascicles per rat

~70 fibrilstail

4-5 locationsfibril

20 nN (Sneddon)

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ights Reserve

d

Multiscale Diabetes Study (Poster 0495)

0

5

10

15

20

25

300 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 gt25

Perc

enta

ge o

f Gro

up S

ampl

es

Elastic Modulus (MPa)

CD (1530 indents)ZDSD (913 indents)

CD (n=5) 381 plusmn 149 MPa

ZDSD (n=3) 706 plusmn 263 MPa

0

100

200

300

400

500

600

700

800

900

0 2 4 6 8 10

Mic

rosc

ale

Mod

ulus

(MPa

)

Nanoscale Modulus (MPa)

plt0001

r2=0531 p=0040

bull Increased indentation modulus in diabetic tendon

bull Strong relationship between nanoscale indent modulus and microscale tensile modulus

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ights Reserve

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Indentation Limitations and Weaknesses

Proper probe selection for intended application bull Cantilever must be stiff relative to sample bull Sharp probes may damage sample easily dull

R or α must be determined for each probe bull Properties provided by manufacturer are unreliable bull Vary from probe to probe can dull during indent process

Spring constant must be determined for each probe

bull Value provided by manufacturer is inaccurate bull Thermal tuning OK for ldquosoftrdquo probes poor performance

for stiffer probes bull Sader method (geometry) added mass reference

cantilevers

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

Veeco Application Technical note Ben Ohler Practical Advice on the Determination of Cantilever Spring Constants

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ights Reserve

d

Indentation Limitations and Weaknesses

bull Sample processing

bull Sample hydration

bull Proper force range

bull Substrate effects for thin samples

bull Proper modulus model and range of data goodness of fit

Bearing in mind these assumptions and limitations AFM can provide true ldquonanoscalerdquo mechanical properties both in terms of forces and deformations

BBML - All R

ights Reserve

d

A Note on Peak Force QNM for Bruker AFMs bull Mechanical properties mapped pixel-by-pixel at same

resolution as the height image (PeakForce QNM)

Height Modulus BBML - All R

ights Reserve

d

A Note on Peak Force QNM

Correct probe stiffness must be used based on sample Pittenger ndash Veeco Product Bulletin

BBML - All R

ights Reserve

d

A Note on Peak Force QNM Extremely sensitive to calibration (details rarely given) Absolute radius and spring constant known

bull especially important from probe to probe bull radius uarr with indent depth ndash must determine R at depth

you expect to image bull Depth may vary as a function of sample (or at different

regions within the sample)

Relative reference material of ldquoknownrdquo modulus used to determine a ratio of 119896119896 119877119877 bull ldquoknownrdquo modulus may come from tension compression

and not indentation bull R is related to depth indentation depth on sample must

be same as on reference BBML - All R

ights Reserve

d

A Note on Peak Force QNM bull Modulus fit on the fly ndash no ability to monitor fit (or range) bull Local sample topography and sample tilt play a role in

measured parameters bull Assuming all modulus values in an image are correct how to

quantify

512 x 512 pixels = 262144 measurements

n=1 n=262144

n from selected ROIs

BBML - All R

ights Reserve

d

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ights Reserve

d

bull Imaging in air on mica ndash substrate effect

bull Same imaging force or indent depth for all

bull For each area were all modulus values pooled

Lamprou et al PLoS ONE 2013 BBML - All R

ights Reserve

d

AFM Applications in Bone Research Other Cool Mechanical Techniques

BBML - All R

ights Reserve

d

ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo Bonds within or between collagen fibrils in bone that rupture to dissipate energy as a toughening mechanism

Thompson et al Nature 2001

Fantner et al Nature Mat 2005

Hansma et al JMNI 2005

Fantner et al Nano Letters 2007 BBML - All R

ights Reserve

d

Other Mechanical Techniques

Jimenez-Palomar J Mech Beh Bio Mat 2012

van der Rijt Macro Bio 2004

Yang J Biomed Mater Res 2007

Yang Biophysical Journal 2008 BBML - All R

ights Reserve

d

Other Cool Techniques bull Piezoresponse Force Microscopy (PFM)

bull Electrochemical AFM (EC-AFM)

bull Force Modulation Microscopy (FMM)

bull Lateral Force Microscopy (LFM)

bull Magnetic Force Microscopy (MFM)

bull Conductive AFM (C-AFM)

bull Force Pulling

bull Cantilever Bending

bull Tensile testing of individual fibrils

bull 3 point bending of fibrils

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ights Reserve

d

Summary Things to rememberhellip

bull Imaging mode imaging medium and probe selection

bull Morphology Measurements bull Sample preparation and hydration state bull Measuring fibril diameter bull Site selection and proper sampling bull Appropriate system calibration

bull Mechanical Measurements

bull Probe selection (stiffness and tip radius) bull Probe calibration (radius angle spring constant) bull Indentation force range and mechanical model bull Hydration in biological samples bull PeakForce QNM

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Acknowledgements BBML

Max Hammond Armando Diaz Gonzalez Zachary Bart Silvia Canelon Creasy Clauser

IU School of Medicine Dr David Burr Dr Matt Allen Dr Stuart Warden Dr Lilian Plotkin

Funding Sources NIHNIDCR 1F32DE018840-01 A1 IUPUI BME Departmental Startup Funds IUPUI Research Support Funds Grant IUPUI Biomechanics and Biomaterials Research Center

NIH NICHD Dr Joan Marini

BBML - All R

ights Reserve

d

Bone Biology and Mechanics Lab (BBML) wwwiupuiedu~bbml Facebook BoneBiologyMechanicsLab

BBML - All R

ights Reserve

d

• Slide Number 1
• General Seminar Outline
• Bonersquos Hierarchical Structure
• Critical Gap in Understanding Collagen
• Methods For Analyzing Collagen Structure
• Methods For Imaging Collagen Structure
• Cryo-TEM
• Atomic Force Microscopy (AFM)
• AFM Image of Pentacene (C22H14)
• AFM Image of Hexabenzocoronene
• Basics of AFM Imaging The Probe
• Basics of AFM Imaging System Components
• van der Waals Potential Energy Curve
• Contact Mode ndash Monitor Deflection
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Peak Force Tapping Mode ndash Monitor Force
• AFM Applications in Bone Research Imaging
• AFM Imaging of Collagen
• Imaging of Osteoclast Activity
• Mineralized collagen deposition in vitro
• Imaging of Canilicular Structure
• AFM Imaging in Bone
• AFM Imaging in Bone
• Removing Mineral in Bone to Expose Collagen
• Removing Mineral in Bone to Expose Collagen
• Demineralization with EDTA
• Demineralization with EDTA
• Demineralization with EDTA
• Issues to Consider Effects of Acid
• Slide Number 32
• Slide Number 33
• Analysis of Collagen Structure in Bone
• Analysis of Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril D-Spacing
• Slide Number 39
• Collagen in Bone Fibril D-Spacing
• Collagen in Bone Fibril D-Spacing
• 2D Fast Fourier Transform Analysis
• 2D Fast Fourier Transform Analysis
• D-Spacing Distribution
• D-Spacing Distribution
• Cumulative Distribution Function
• CDF vs Histogram
• Changes in D-Spacing with Disease
• Changes in D-Spacing with Disease
• Slide Number 50
• Issues to Consider Site Selection and Sampling
• Issues to Consider Site Selection and Sampling
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• AFM calibration ndash Take Away Message
• AFM Applications in Bone ResearchIndentation Mechanics
• AFM-Based Indentation
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Parameters
• Hertz Contact Equation
• Hertz Contact Equation
• Hertz Contact Equation
• Proper Indentation Model is Essential
• Indentation in OI Tendon Study
• OI Tendon Differential Effects of Dehydration
• OI Tendon Differential Effects of Dehydration
• Indentation in Diabetic Tendon (Poster 0495)
• Multiscale Diabetes Study (Poster 0495)
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• A Note on Peak Force QNM for Bruker AFMs
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• Slide Number 83
• Slide Number 84
• AFM Applications in Bone ResearchOther Cool Mechanical Techniques
• ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo
• Other Mechanical Techniques
• Other Cool Techniques
• Summary Things to rememberhellip
• Acknowledgements
• Bone Biology and Mechanics Lab (BBML)

AFM Image of Pentacene (C22H14)

Gross et al Science 2009 325 (5944)1110-1114 BBML - All R

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AFM Image of Hexabenzocoronene

Gross et al Science 2012 2371326-1329 BBML - All R

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Basics of AFM Imaging The Probe Nanoscale tip mounted on a microscale cantilever

Probe

Feedback System

X-Y-X Piezo Combination

Cantilever

Wallace Bone 2012

Kopycinska-Muller Ultramicroscopy 2006

BBML - All R

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Basics of AFM Imaging System Components bull Piezoelectric actuators precise

movement under electric potential

bull Raster-scanned (x-y direction)

bull Force transducer interaction force (typically cantileverrsquos deflection)

bull Cantilever deflection (z-direction) measured by photodiode

bull Control system maintains a desired force between probe and sample and (movement in z-direction)

Probe

Feedback System

X-Y-X Piezo Combination

Cantilever

Wallace Bone 2012

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d

van der Waals Potential Energy Curve

FOR

CE

DISTANCE

Repulsive Regime

Attractive Regime

Weak atomic attraction

Net attractive force weakens as interatomic separation decreases

Net force = 0 distance between atoms is 2-3 Å (~ length of a chemical bond)

Attraction increases until electron clouds begin to electrostatically repel each other BBML - A

ll Rights R

eserved

Contact Mode ndash Monitor Deflection

Control system maintains user-defined deflection by vertically moving scanner using piezoelectric actuator

Tip-Sample Separation

Forc

e

Frictional and adhesive forces can damage sample and distort image

Probe in constant contact cantilever deflects like a spring according to Hookersquos law (F = kx)

BBML - All R

ights Reserve

d

Tapping Mode ndash Monitor Amplitude

Tip-Sample SeparationForc

e

Intermittent Contact

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ights Reserve

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Tapping Mode ndash Monitor Amplitude

ApproachWithdrawal

Tip-Sample SeparationForc

e

Cantilever oscillates at user-defined amplitude near cantilever resonance frequency

-15

-1

-05

0

05

1

15

0 5 10 15

Osc

illat

ion

Am

plitu

de

Time

-15

-1

-05

0

05

1

15

0 5 10 15

Osc

illat

ion

Am

plitu

de

Time

Setpoint

Bump

-15

-1

-05

0

05

1

15

0 5 10 15

Osc

illat

ion

Am

plitu

de

Time

SetpointBumpValley

BBML - All R

ights Reserve

d

Tapping Mode ndash Monitor Amplitude bull Probe in contact for a small fraction of its tapping period

lateral forces are reduced

bull Probe must be tuned at resonance frequency ndash can be challenging for some probes when in fluid

Tuning in Air Single resonance peak Tuning in Fluid Multiple and spurious peaks wwwasylumresearchcom

BBML - All R

ights Reserve

d

Peak Force Tapping Mode ndash Monitor Force

Control system maintains user-defined maximum force

Cantilever oscillates at low frequency (1-2 kHz)

bull No need for tuning

Slow tapping force curve is captured with each tap

ApproachWithdrawal

Tip-Sample SeparationForc

ePeakForce

Direct control of maximum normal force bull Protects tip and sample wear bull Limits indentation depth to a few nm - increases resolution

by limiting contact area bull Mechanical properties mapped pixel-by-pixel at same

resolution as the height image (PeakForce QNM) BBML - All R

ights Reserve

d

AFM Applications in Bone Research Imaging

BBML - All R

ights Reserve

d

AFM Imaging of Collagen

bull AFMrsquos adoption in bone research has been slowhellip

bull First AFM-based study in collagen came in 1992 (a previous study in 1989 used Scanning Tunneling Microscopy)

bull Observed fibrillar and monomeric collagen

Chernoff J of Vac Sci Tech 1992

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ights Reserve

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Imaging of Osteoclast Activity

Bozec Ultramicroscopy 2005

Hassenkam Anat Record 2006

Initial observations of cellular activity were qualitative (analyses of collagen morphology were not presented)

Sasaki J Elec Micro 1993

Sasaki CTI 1995

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Mineralized collagen deposition in vitro

Collagen deposition from MLO-A5 cells

Barragan-Adjemian CTI 2006 BBML - All R

ights Reserve

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Imaging of Canilicular Structure

Reilly Annals of BME 2001

Lin J of Microscopy 2010

Average canilicular diameter ~400-500 nm larger than values typically reported from TEM studies

BBML - All R

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d

AFM Imaging in Bone In general we are more interested in collagen in bone

3 microm Diamond Suspension

Unpolished

BBML - All R

ights Reserve

d

AFM Imaging in Bone

The surface is completely mineralizedhellip BBML - All R

ights Reserve

d

Removing Mineral in Bone to Expose Collagen

Dealing with mineral is a challenge to analyzing collagen ndash some mineral must be removed

bull 10 Citric acid (to remove mineral) 65 Sodium Hypochlorite (to remove non-collagenous proteins) Habelitz J Struct Bio 2002

bull 7 or 17 Phosphoric Acid Bozek IEEE 2005 El Feninat J Biomed Mat Res 1998

bull 5 Formic Acid Ge Mat Sci Eng 2007

bull 30 mM EDTA Kindt Nanotechnology 2007

BBML - All R

ights Reserve

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Removing Mineral in Bone to Expose Collagen

Dealing with mineral is a challenge to analyzing collagen ndash some mineral must be removed

bull 30 mM EDTA Kindt Nanotechnology 2007 BBML - A

ll Rights R

eserved

Demineralization with EDTA EDTA - Ethylenediaminetetraacetic Acid

Chelating agent - sequesters divalent cations (notably Ca2+)

bull Treat with 05M EDTA pH 8 (15-20 minutes) bull Vigorously wash with ultrapure water bull Sonicate to release surface-bound mineral ions bull Repeat 3-4 times

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Demineralization with EDTA

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d

Demineralization with EDTA

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Issues to Consider Effects of Acid

Immature collagen crosslinks are very sensitive to acid bull Using acids to demineralize bone may inadvertently

destroy crosslinks distort structure

Fixation of tissue may be necessary prior to demineralization bull Fixation (glutaraldehyde or paraformadehyde) may

preserve structural integrity

bull Fixation effects on collagen structure are not fully known

BBML - All R

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100 microm x 100 microm

50 microm x 50 microm

25 microm x 25 microm

10 microm x 10 microm

35 microm x 35 microm

BBML - All R

ights Reserve

d

35 microm x 35 microm

BBML - All R

ights Reserve

d

Analysis of Collagen Structure in Bone Spatial organization of newly formed bone following a surgically-induced injury (rat tibial hole)

Baranauskas J Vac Sci Tech 2001 Baranauskas App Surface Sci 2005

ldquohellipstatistical diameter of the fibers is 159 nm in good agreement with the literature values for type I collagen fibersrdquo

BBML - All R

ights Reserve

d

Analysis of Collagen in Bone Fibril Diameter Fibril Diameter easiest to measure property first attempts were a bit crude better with increased resolution

Sasaki J Mat Sci Tech 2002 Thalhammer J Arch Sci 2001

Bozec Ultramicroscopy 2005

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ights Reserve

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Collagen in Bone Fibril Diameter

Caution must be taken when reporting and interpreting diameters measured from fibrils in bone bull Fibril packing - width may not be fully exposed

Wallace et al Langmuir 2010

BBML - All R

ights Reserve

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Collagen in Bone Fibril Diameter

Caution must be taken when reporting and interpreting diameters measured from fibrils in bone bull Fibril packing - width may not be fully exposed

Rigozzi ndash J Struct Bio - 2011

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ights Reserve

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Collagen in Bone Fibril D-Spacing

300 nm 15 nm

N C C N C C N C C N C C

H O H2C CH2 O H

CH2

O OH HHC CH2

CH2

HO

H H

HHN C C

O

H2C CH2

CH2

HN C C

OH

HC CH2

HO

N C C N C C N C C

H O H2C CH2 O

CH2

OH HC CH2

HO

H H

H

CH2 CH2

glycine X - proline Y - hydroxyproline

Hodge and Petruska 1963

Collagen Fibril Axial D-Periodicity BBML - All R

ights Reserve

d

05

101520

0 02 04 06 08 1

Hei

ght (

nm)

Length (microns)

httpremfdartmoutheduimagesindexhtml

Randall Nature and Structure of Collagen 1953

TEM SEM

AFM

D-Spacing = 67 nm

BBML - All R

ights Reserve

d

Collagen in Bone Fibril D-Spacing Many studies have shown observable D-spacing content that near theoretical value

Sasaki 2002 67 plusmn 2 nm Bozek 2005 665 plusmn 14 nm Ge 2007 666 plusmn 38 nm

Habelitz J Structural Bio 2002

A 2002 study in dentin got me interestedhellip

n = 322 wet n = 363 dry

BBML - All R

ights Reserve

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Collagen in Bone Fibril D-Spacing

bull Internal structure bull Wallace et al Bone 2010 46 1349-1354 bull Wallace et al J Stuct Bio 2011 173 146-152 bull Kemp et al J Stuct Bio 2012 180 428-438 bull Warden et al Endocrinology 2013 154(9)

3178-3187 bull Gallant et al Bone 2014 61 191-200 bull Bart et al Conn Tiss Res 2014 In Press

bull Enzymatic and non-

enzymatic cross-linking bull Hammond et al Bone 2014 60 26-32 bull Diaz Gonzalez et al J Biomech 2014 47(3)

681-686 bull Voytik-Harbin et al In Preparation 2014

D-periodic spacing important structural feature that captures aspects related to

Mouse Femur Mid-diaphysis BBML - All R

ights Reserve

d

2D Fast Fourier Transform Analysis

Harmonics

Fibril D-Spacing

Erickson B Wallace JM et al Biotechnology Journal 2013

Raw AFM Image 2D FFT Power Spectrum

BBML - All R

ights Reserve

d

2D Fast Fourier Transform Analysis

Harmonics

Fibril D-Spacing

Raw AFM Image 2D FFT Power Spectrum

0

20000

40000

60000

80000

100000

120000

0 001 002 003 004 005 006

Am

plitu

de (

microV2 )

Position (1nm)

D-periodic spacing 0015 (1nm) ~ 67 nm

BBML - All R

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D-Spacing Distribution

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Overall Mean 670 plusmn 00 nm

BBML - All R

ights Reserve

d

D-Spacing Distribution

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Overall Mean 670 plusmn 00 nm

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

4 Samples Total of n = 255 fibrils Overall Mean 682 plusmn 12 nm

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ights Reserve

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Cumulative Distribution Function

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

This population 255 fibrils - Each fibril 1255 = 0392 - Data range 628-716 nm

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CDF vs Histogram 62 63 64 65 66 67 68 69 70 71 72 73 74

0

10

20

30

40

50

60

70

80

90

100

0

5

10

15

20

25

30

35

40

45

50

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

HistoCDF

BBML - All R

ights Reserve

d

Changes in D-Spacing with Disease

0

5

10

15

20

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

0

5

10

15

20

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

Wallace et al Bone 2010 46 1349-1354

KS Test plt0001

Control 680 plusmn 26 nm

Estrogen Dep 659 plusmn 31 nm

t-test on mean p=0048

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ights Reserve

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Changes in D-Spacing with Disease

Wallace et al Bone 2010 46 1349-1354

0

10

20

30

40

50

60

70

80

90

100

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

KS Test plt0001

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ights Reserve

d

Question Model and Tissue Morphology Publication

Tissue Specificity in Mice Male mouse 8 weeks Femur dentin tail tendon

No differences in bone vs dentin both ∆ vs tendon

Wallace et al Langmuir 2011 26(10) 7349-7354

Gender Effects Male and female mice 16 weeks Femur No differences NA

Estrogen Depletion OVX in sheep 5 years + 2 Radius Mean and population shift Wallace et al Bone

2010 46 1349-1354

Estrogen Depletion OVX in female 5 years + 2 Skin (dermis) Significant population shift Erickson et al Biotech

J 2013 8(1) 1174-126

Osteoporosis and Mechanical Loading

OVX in ulnar loading in rats Ulna

No loading effect population shift with OVX

Warden et al Endocrinology 2013 154(9) 3178-3187

Osteogenesis Imperfecta Brtl+ model male mice 2 months Femur

No mean ∆ widening of distribution

Wallace et al J Stuct Bio 2011 173 146-152

Osteogenesis Imperfecta Oimoim model male mice 12 weeks Femur Mean and population shift Bart et al Conn Tiss

Res 2014 In Press

Osteogenesis Imperfecta and Tissue Hydration

Brtl+ model male mice 6 months Tail Tendon Significant population shift Kemp et al J Stuct Bio

2012 180 428-438

Type 2 Diabetes Male ZDSD rats 30 weeks Tibia Significant population shift Hammond et al Bone

2014 60 26-32

Type 2 Diabetes Female ZDSD rats 32 weeks Tail Tendon Significant population shift

Diaz Gonzalez et al J Biomech 2014 47(3)

681-686

Raloxifene (in vivo and in vitro) Female dog treated with Raloxifene Femur Significant population shift Gallant et al Bone

2014 61 191-200 BBML - All R

ights Reserve

d

Issues to Consider Site Selection and Sampling

KS Test plt0001

0023 of bonersquos length

15 m

m =

150

00 micro

m

Wallace et al Langmuir 2011 26(10) 7349-7354

100 microm x 100 microm

35 microm x 35 microm

BBML - All R

ights Reserve

d

Issues to Consider Site Selection and Sampling

60

62

64

66

68

70

72

74

1 (35)

2 (33)

3 (40)

4 (39)

5 (35)

6 (29)

7 (26)

8 (23)

9 (28)

WT (288)

D-P

erio

dic

Gap

Ove

rlap

Spac

ing

(nm

)

Axial Location (Proximal End = 1 Distal End = 9)

( of Fibrils)

91

23

45

87

6

Wallace et al Langmuir 2011 26(10) 7349-7354

288 fibrils for population n=1 for mean comparisons BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

Wet Tendon Dry Tendon Kemp et al J StructBio 2012 180 (3) 428-438

Collagen and bone exist in a hydrated environment in vivo bull Hydration state can impact morphology and mechanics

BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

Nonsignificant mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet (n=255) 682 plusmn 12 nm

WT Dry (n=257) 686 plusmn 08 nm

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

Brtl DryBrtl Wet

KS Test plt0001

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

WT Case 04 nm mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet 682 plusmn 12 nm

WT Dry 686 plusmn 08 nm

Brtl+ Wet 675 plusmn 14 nm

Brtl+ Dry 689 plusmn 10 nm

Brtl+ Case 14 nm increase with drying (plt0001) bull Entire population shifted upward (plt0001)

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT WetBrtl Wet

KS Test plt0001

Brtl+ distribution shifted downward vs WT when wet

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryBrtl Dry

KS Test plt0001

Brtl+ distribution shifted upward vs WT when dry

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider AFM calibration

bull Absolute distance measurements require accurate piezo calibration

bull AFM systems have large scan ranges (150 microm to lt 1 microm)

bull Scanner non-linearities can introduce substantial error over that range

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

ights Reserve

d

Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

BBML - All R

ights Reserve

d

Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

100 nm 10 80 602 1198 100 nm 7 60 827 1439 100 nm 5 40 814 1427 100 nm 35 20 917 1724 100 nm 2 10 937 1774

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

ights Reserve

d

AFM calibration ndash Take Away Message

Calibration using a scan size and feature size comparable to features of interest is highly recommended

Calibration addresses absolute accuracy not precision (differential sensitivity between measurements)

bull Does not limit ability to differentiate between groups using the same AFM with the same calibration parameters

BBML - All R

ights Reserve

d

AFM Applications in Bone Research Indentation Mechanics

BBML - All R

ights Reserve

d

AFM-Based Indentation

In addition to high resolution imaging AFM can be used to extract nanoscale mechanical data

ApproachWithdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Can

tilev

er D

efle

ctio

n (V

olts

)

1 Ramp into a rigid sample ndash measure the slope

Deflection sensitivity x 119899119899119899119899119881119881

2 Calculate cantilever spring constant

3 Hookersquos law takes deflection (volts) and converts to units of force F = kx

Spring constant k 119873119873119899119899

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Forc

e (n

N)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration Fo

rce

(nN

)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

Tip-Sample Separation (nm)

BBML - All R

ights Reserve

d

AFM-Based Indentation Parameters

Approach Withdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

Peak Force

Area Between Curves Energy Dissipation

Contact Mechanics Fit for Elastic Modulus (E)

Adhesion Force

Indentation Depth (δ)

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

z

r

δ

R

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119877119877 ∙ 12057512057532

Es Sample modulus νs Poissonrsquos ratio R tip radius of curvature δ load point displacement

Relevant Hertz Assumptions bull Strains are small and within the elastic limit bull Indenter is infinitely stiff (only sample deforms) bull Indentation depth ltlt radius of curvature of indenter bull Surfaces are continuous non-conforming (must be flat) bull Load is normal to surface

BBML - All R

ights Reserve

d

Proper Indentation Model is Essential

δ

r

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119903119903 ∙ 12057512057532

δ

α

119865119865 =2120587120587∙

1198641198641199041199041 minus 1205841205841199041199042

∙ tan120572120572 ∙ 1205751205752

Es Sample modulus νs Poissonrsquos ratio R tip radius α opening angle

Hertz Model Spherical Indenter

depth ltlt radius of curvature

Sneddon Model Conical Indenter

depth ge radius of curvature

BBML - All R

ights Reserve

d

Indentation in OI Tendon Study

x

z

yBrtl+ and WT mice 6 month old males

4 tails per group

2-3 fascicles per tail

25-30 fibrils per tail

4-5 locations per fibril

Indent force 20 nN wet (Sneddon) 50 nN dry (Hertz) BBML - A

ll Rights R

eserved

OI Tendon Differential Effects of Dehydration

Dry 1268 plusmn 718 MPa

Wet 305 plusmn 243 MPa

Dry 1737 plusmn 973 MPa

Wet 301 plusmn 202 MPa

Kemp et al J StructBio 2012 180 (3) 428-438

bull Significant stiffening of fibrils with dehydration

bull More pronounced modulus modulus as a function of disease

BBML - All R

ights Reserve

d

OI Tendon Differential Effects of Dehydration

Kemp et al J StructBio 2012 180 (3) 428-438

Diseased phenotype not present when wet phenotype observed when dried

0

10

20

30

40

50

60

0 20 40 60 80 100 120 140 160 180 200

Gro

up S

ampl

es (

)

Indent Modulus (MPa)

WT WetBrtl Wet

0

2

4

6

8

10

12

14

16

18

20

025

050

075

010

0012

5015

0017

5020

0022

5025

0027

5030

0032

5035

0037

5040

0042

5045

0047

5050

00

Gro

up S

ampl

es (

)Indent Modulus (MPa)

WT DryBrtl Dry

555 WT Indents 305 plusmn 243 MPa

614 Brtl+ Indents 301 plusmn 202 MPa

KS Test p=0129 t Test p=0464 464 WT Indents

1268 plusmn 718 MPa 479 Brtl+ Indents 1737 plusmn 973 MPa

KS Test plt0001 T Test plt0001

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ights Reserve

d

Indentation in Diabetic Tendon (Poster 0495)

x

z

y

Control and Diabetic Rat Tail Tendon

4-5 rats per group

2-3 fascicles per rat

~70 fibrilstail

4-5 locationsfibril

20 nN (Sneddon)

BBML - All R

ights Reserve

d

Multiscale Diabetes Study (Poster 0495)

0

5

10

15

20

25

300 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 gt25

Perc

enta

ge o

f Gro

up S

ampl

es

Elastic Modulus (MPa)

CD (1530 indents)ZDSD (913 indents)

CD (n=5) 381 plusmn 149 MPa

ZDSD (n=3) 706 plusmn 263 MPa

0

100

200

300

400

500

600

700

800

900

0 2 4 6 8 10

Mic

rosc

ale

Mod

ulus

(MPa

)

Nanoscale Modulus (MPa)

plt0001

r2=0531 p=0040

bull Increased indentation modulus in diabetic tendon

bull Strong relationship between nanoscale indent modulus and microscale tensile modulus

BBML - All R

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Indentation Limitations and Weaknesses

Proper probe selection for intended application bull Cantilever must be stiff relative to sample bull Sharp probes may damage sample easily dull

R or α must be determined for each probe bull Properties provided by manufacturer are unreliable bull Vary from probe to probe can dull during indent process

Spring constant must be determined for each probe

bull Value provided by manufacturer is inaccurate bull Thermal tuning OK for ldquosoftrdquo probes poor performance

for stiffer probes bull Sader method (geometry) added mass reference

cantilevers

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

Veeco Application Technical note Ben Ohler Practical Advice on the Determination of Cantilever Spring Constants

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

bull Sample processing

bull Sample hydration

bull Proper force range

bull Substrate effects for thin samples

bull Proper modulus model and range of data goodness of fit

Bearing in mind these assumptions and limitations AFM can provide true ldquonanoscalerdquo mechanical properties both in terms of forces and deformations

BBML - All R

ights Reserve

d

A Note on Peak Force QNM for Bruker AFMs bull Mechanical properties mapped pixel-by-pixel at same

resolution as the height image (PeakForce QNM)

Height Modulus BBML - All R

ights Reserve

d

A Note on Peak Force QNM

Correct probe stiffness must be used based on sample Pittenger ndash Veeco Product Bulletin

BBML - All R

ights Reserve

d

A Note on Peak Force QNM Extremely sensitive to calibration (details rarely given) Absolute radius and spring constant known

bull especially important from probe to probe bull radius uarr with indent depth ndash must determine R at depth

you expect to image bull Depth may vary as a function of sample (or at different

regions within the sample)

Relative reference material of ldquoknownrdquo modulus used to determine a ratio of 119896119896 119877119877 bull ldquoknownrdquo modulus may come from tension compression

and not indentation bull R is related to depth indentation depth on sample must

be same as on reference BBML - All R

ights Reserve

d

A Note on Peak Force QNM bull Modulus fit on the fly ndash no ability to monitor fit (or range) bull Local sample topography and sample tilt play a role in

measured parameters bull Assuming all modulus values in an image are correct how to

quantify

512 x 512 pixels = 262144 measurements

n=1 n=262144

n from selected ROIs

BBML - All R

ights Reserve

d

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ights Reserve

d

bull Imaging in air on mica ndash substrate effect

bull Same imaging force or indent depth for all

bull For each area were all modulus values pooled

Lamprou et al PLoS ONE 2013 BBML - All R

ights Reserve

d

AFM Applications in Bone Research Other Cool Mechanical Techniques

BBML - All R

ights Reserve

d

ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo Bonds within or between collagen fibrils in bone that rupture to dissipate energy as a toughening mechanism

Thompson et al Nature 2001

Fantner et al Nature Mat 2005

Hansma et al JMNI 2005

Fantner et al Nano Letters 2007 BBML - All R

ights Reserve

d

Other Mechanical Techniques

Jimenez-Palomar J Mech Beh Bio Mat 2012

van der Rijt Macro Bio 2004

Yang J Biomed Mater Res 2007

Yang Biophysical Journal 2008 BBML - All R

ights Reserve

d

Other Cool Techniques bull Piezoresponse Force Microscopy (PFM)

bull Electrochemical AFM (EC-AFM)

bull Force Modulation Microscopy (FMM)

bull Lateral Force Microscopy (LFM)

bull Magnetic Force Microscopy (MFM)

bull Conductive AFM (C-AFM)

bull Force Pulling

bull Cantilever Bending

bull Tensile testing of individual fibrils

bull 3 point bending of fibrils

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ights Reserve

d

Summary Things to rememberhellip

bull Imaging mode imaging medium and probe selection

bull Morphology Measurements bull Sample preparation and hydration state bull Measuring fibril diameter bull Site selection and proper sampling bull Appropriate system calibration

bull Mechanical Measurements

bull Probe selection (stiffness and tip radius) bull Probe calibration (radius angle spring constant) bull Indentation force range and mechanical model bull Hydration in biological samples bull PeakForce QNM

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Acknowledgements BBML

Max Hammond Armando Diaz Gonzalez Zachary Bart Silvia Canelon Creasy Clauser

IU School of Medicine Dr David Burr Dr Matt Allen Dr Stuart Warden Dr Lilian Plotkin

Funding Sources NIHNIDCR 1F32DE018840-01 A1 IUPUI BME Departmental Startup Funds IUPUI Research Support Funds Grant IUPUI Biomechanics and Biomaterials Research Center

NIH NICHD Dr Joan Marini

BBML - All R

ights Reserve

d

Bone Biology and Mechanics Lab (BBML) wwwiupuiedu~bbml Facebook BoneBiologyMechanicsLab

BBML - All R

ights Reserve

d

• Slide Number 1
• General Seminar Outline
• Bonersquos Hierarchical Structure
• Critical Gap in Understanding Collagen
• Methods For Analyzing Collagen Structure
• Methods For Imaging Collagen Structure
• Cryo-TEM
• Atomic Force Microscopy (AFM)
• AFM Image of Pentacene (C22H14)
• AFM Image of Hexabenzocoronene
• Basics of AFM Imaging The Probe
• Basics of AFM Imaging System Components
• van der Waals Potential Energy Curve
• Contact Mode ndash Monitor Deflection
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Peak Force Tapping Mode ndash Monitor Force
• AFM Applications in Bone Research Imaging
• AFM Imaging of Collagen
• Imaging of Osteoclast Activity
• Mineralized collagen deposition in vitro
• Imaging of Canilicular Structure
• AFM Imaging in Bone
• AFM Imaging in Bone
• Removing Mineral in Bone to Expose Collagen
• Removing Mineral in Bone to Expose Collagen
• Demineralization with EDTA
• Demineralization with EDTA
• Demineralization with EDTA
• Issues to Consider Effects of Acid
• Slide Number 32
• Slide Number 33
• Analysis of Collagen Structure in Bone
• Analysis of Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril D-Spacing
• Slide Number 39
• Collagen in Bone Fibril D-Spacing
• Collagen in Bone Fibril D-Spacing
• 2D Fast Fourier Transform Analysis
• 2D Fast Fourier Transform Analysis
• D-Spacing Distribution
• D-Spacing Distribution
• Cumulative Distribution Function
• CDF vs Histogram
• Changes in D-Spacing with Disease
• Changes in D-Spacing with Disease
• Slide Number 50
• Issues to Consider Site Selection and Sampling
• Issues to Consider Site Selection and Sampling
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• AFM calibration ndash Take Away Message
• AFM Applications in Bone ResearchIndentation Mechanics
• AFM-Based Indentation
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Parameters
• Hertz Contact Equation
• Hertz Contact Equation
• Hertz Contact Equation
• Proper Indentation Model is Essential
• Indentation in OI Tendon Study
• OI Tendon Differential Effects of Dehydration
• OI Tendon Differential Effects of Dehydration
• Indentation in Diabetic Tendon (Poster 0495)
• Multiscale Diabetes Study (Poster 0495)
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• A Note on Peak Force QNM for Bruker AFMs
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• Slide Number 83
• Slide Number 84
• AFM Applications in Bone ResearchOther Cool Mechanical Techniques
• ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo
• Other Mechanical Techniques
• Other Cool Techniques
• Summary Things to rememberhellip
• Acknowledgements
• Bone Biology and Mechanics Lab (BBML)

AFM Image of Hexabenzocoronene

Gross et al Science 2012 2371326-1329 BBML - All R

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d

Basics of AFM Imaging The Probe Nanoscale tip mounted on a microscale cantilever

Probe

Feedback System

X-Y-X Piezo Combination

Cantilever

Wallace Bone 2012

Kopycinska-Muller Ultramicroscopy 2006

BBML - All R

ights Reserve

d

Basics of AFM Imaging System Components bull Piezoelectric actuators precise

movement under electric potential

bull Raster-scanned (x-y direction)

bull Force transducer interaction force (typically cantileverrsquos deflection)

bull Cantilever deflection (z-direction) measured by photodiode

bull Control system maintains a desired force between probe and sample and (movement in z-direction)

Probe

Feedback System

X-Y-X Piezo Combination

Cantilever

Wallace Bone 2012

BBML - All R

ights Reserve

d

van der Waals Potential Energy Curve

FOR

CE

DISTANCE

Repulsive Regime

Attractive Regime

Weak atomic attraction

Net attractive force weakens as interatomic separation decreases

Net force = 0 distance between atoms is 2-3 Å (~ length of a chemical bond)

Attraction increases until electron clouds begin to electrostatically repel each other BBML - A

ll Rights R

eserved

Contact Mode ndash Monitor Deflection

Control system maintains user-defined deflection by vertically moving scanner using piezoelectric actuator

Tip-Sample Separation

Forc

e

Frictional and adhesive forces can damage sample and distort image

Probe in constant contact cantilever deflects like a spring according to Hookersquos law (F = kx)

BBML - All R

ights Reserve

d

Tapping Mode ndash Monitor Amplitude

Tip-Sample SeparationForc

e

Intermittent Contact

BBML - All R

ights Reserve

d

Tapping Mode ndash Monitor Amplitude

ApproachWithdrawal

Tip-Sample SeparationForc

e

Cantilever oscillates at user-defined amplitude near cantilever resonance frequency

-15

-1

-05

0

05

1

15

0 5 10 15

Osc

illat

ion

Am

plitu

de

Time

-15

-1

-05

0

05

1

15

0 5 10 15

Osc

illat

ion

Am

plitu

de

Time

Setpoint

Bump

-15

-1

-05

0

05

1

15

0 5 10 15

Osc

illat

ion

Am

plitu

de

Time

SetpointBumpValley

BBML - All R

ights Reserve

d

Tapping Mode ndash Monitor Amplitude bull Probe in contact for a small fraction of its tapping period

lateral forces are reduced

bull Probe must be tuned at resonance frequency ndash can be challenging for some probes when in fluid

Tuning in Air Single resonance peak Tuning in Fluid Multiple and spurious peaks wwwasylumresearchcom

BBML - All R

ights Reserve

d

Peak Force Tapping Mode ndash Monitor Force

Control system maintains user-defined maximum force

Cantilever oscillates at low frequency (1-2 kHz)

bull No need for tuning

Slow tapping force curve is captured with each tap

ApproachWithdrawal

Tip-Sample SeparationForc

ePeakForce

Direct control of maximum normal force bull Protects tip and sample wear bull Limits indentation depth to a few nm - increases resolution

by limiting contact area bull Mechanical properties mapped pixel-by-pixel at same

resolution as the height image (PeakForce QNM) BBML - All R

ights Reserve

d

AFM Applications in Bone Research Imaging

BBML - All R

ights Reserve

d

AFM Imaging of Collagen

bull AFMrsquos adoption in bone research has been slowhellip

bull First AFM-based study in collagen came in 1992 (a previous study in 1989 used Scanning Tunneling Microscopy)

bull Observed fibrillar and monomeric collagen

Chernoff J of Vac Sci Tech 1992

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ights Reserve

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Imaging of Osteoclast Activity

Bozec Ultramicroscopy 2005

Hassenkam Anat Record 2006

Initial observations of cellular activity were qualitative (analyses of collagen morphology were not presented)

Sasaki J Elec Micro 1993

Sasaki CTI 1995

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ights Reserve

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Mineralized collagen deposition in vitro

Collagen deposition from MLO-A5 cells

Barragan-Adjemian CTI 2006 BBML - All R

ights Reserve

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Imaging of Canilicular Structure

Reilly Annals of BME 2001

Lin J of Microscopy 2010

Average canilicular diameter ~400-500 nm larger than values typically reported from TEM studies

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ights Reserve

d

AFM Imaging in Bone In general we are more interested in collagen in bone

3 microm Diamond Suspension

Unpolished

BBML - All R

ights Reserve

d

AFM Imaging in Bone

The surface is completely mineralizedhellip BBML - All R

ights Reserve

d

Removing Mineral in Bone to Expose Collagen

Dealing with mineral is a challenge to analyzing collagen ndash some mineral must be removed

bull 10 Citric acid (to remove mineral) 65 Sodium Hypochlorite (to remove non-collagenous proteins) Habelitz J Struct Bio 2002

bull 7 or 17 Phosphoric Acid Bozek IEEE 2005 El Feninat J Biomed Mat Res 1998

bull 5 Formic Acid Ge Mat Sci Eng 2007

bull 30 mM EDTA Kindt Nanotechnology 2007

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ights Reserve

d

Removing Mineral in Bone to Expose Collagen

Dealing with mineral is a challenge to analyzing collagen ndash some mineral must be removed

bull 30 mM EDTA Kindt Nanotechnology 2007 BBML - A

ll Rights R

eserved

Demineralization with EDTA EDTA - Ethylenediaminetetraacetic Acid

Chelating agent - sequesters divalent cations (notably Ca2+)

bull Treat with 05M EDTA pH 8 (15-20 minutes) bull Vigorously wash with ultrapure water bull Sonicate to release surface-bound mineral ions bull Repeat 3-4 times

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ights Reserve

d

Demineralization with EDTA

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ights Reserve

d

Demineralization with EDTA

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ights Reserve

d

Issues to Consider Effects of Acid

Immature collagen crosslinks are very sensitive to acid bull Using acids to demineralize bone may inadvertently

destroy crosslinks distort structure

Fixation of tissue may be necessary prior to demineralization bull Fixation (glutaraldehyde or paraformadehyde) may

preserve structural integrity

bull Fixation effects on collagen structure are not fully known

BBML - All R

ights Reserve

d

100 microm x 100 microm

50 microm x 50 microm

25 microm x 25 microm

10 microm x 10 microm

35 microm x 35 microm

BBML - All R

ights Reserve

d

35 microm x 35 microm

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ights Reserve

d

Analysis of Collagen Structure in Bone Spatial organization of newly formed bone following a surgically-induced injury (rat tibial hole)

Baranauskas J Vac Sci Tech 2001 Baranauskas App Surface Sci 2005

ldquohellipstatistical diameter of the fibers is 159 nm in good agreement with the literature values for type I collagen fibersrdquo

BBML - All R

ights Reserve

d

Analysis of Collagen in Bone Fibril Diameter Fibril Diameter easiest to measure property first attempts were a bit crude better with increased resolution

Sasaki J Mat Sci Tech 2002 Thalhammer J Arch Sci 2001

Bozec Ultramicroscopy 2005

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ights Reserve

d

Collagen in Bone Fibril Diameter

Caution must be taken when reporting and interpreting diameters measured from fibrils in bone bull Fibril packing - width may not be fully exposed

Wallace et al Langmuir 2010

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ights Reserve

d

Collagen in Bone Fibril Diameter

Caution must be taken when reporting and interpreting diameters measured from fibrils in bone bull Fibril packing - width may not be fully exposed

Rigozzi ndash J Struct Bio - 2011

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d

Collagen in Bone Fibril D-Spacing

300 nm 15 nm

N C C N C C N C C N C C

H O H2C CH2 O H

CH2

O OH HHC CH2

CH2

HO

H H

HHN C C

O

H2C CH2

CH2

HN C C

OH

HC CH2

HO

N C C N C C N C C

H O H2C CH2 O

CH2

OH HC CH2

HO

H H

H

CH2 CH2

glycine X - proline Y - hydroxyproline

Hodge and Petruska 1963

Collagen Fibril Axial D-Periodicity BBML - All R

ights Reserve

d

05

101520

0 02 04 06 08 1

Hei

ght (

nm)

Length (microns)

httpremfdartmoutheduimagesindexhtml

Randall Nature and Structure of Collagen 1953

TEM SEM

AFM

D-Spacing = 67 nm

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ights Reserve

d

Collagen in Bone Fibril D-Spacing Many studies have shown observable D-spacing content that near theoretical value

Sasaki 2002 67 plusmn 2 nm Bozek 2005 665 plusmn 14 nm Ge 2007 666 plusmn 38 nm

Habelitz J Structural Bio 2002

A 2002 study in dentin got me interestedhellip

n = 322 wet n = 363 dry

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ights Reserve

d

Collagen in Bone Fibril D-Spacing

bull Internal structure bull Wallace et al Bone 2010 46 1349-1354 bull Wallace et al J Stuct Bio 2011 173 146-152 bull Kemp et al J Stuct Bio 2012 180 428-438 bull Warden et al Endocrinology 2013 154(9)

3178-3187 bull Gallant et al Bone 2014 61 191-200 bull Bart et al Conn Tiss Res 2014 In Press

bull Enzymatic and non-

enzymatic cross-linking bull Hammond et al Bone 2014 60 26-32 bull Diaz Gonzalez et al J Biomech 2014 47(3)

681-686 bull Voytik-Harbin et al In Preparation 2014

D-periodic spacing important structural feature that captures aspects related to

Mouse Femur Mid-diaphysis BBML - All R

ights Reserve

d

2D Fast Fourier Transform Analysis

Harmonics

Fibril D-Spacing

Erickson B Wallace JM et al Biotechnology Journal 2013

Raw AFM Image 2D FFT Power Spectrum

BBML - All R

ights Reserve

d

2D Fast Fourier Transform Analysis

Harmonics

Fibril D-Spacing

Raw AFM Image 2D FFT Power Spectrum

0

20000

40000

60000

80000

100000

120000

0 001 002 003 004 005 006

Am

plitu

de (

microV2 )

Position (1nm)

D-periodic spacing 0015 (1nm) ~ 67 nm

BBML - All R

ights Reserve

d

D-Spacing Distribution

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Overall Mean 670 plusmn 00 nm

BBML - All R

ights Reserve

d

D-Spacing Distribution

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Overall Mean 670 plusmn 00 nm

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

4 Samples Total of n = 255 fibrils Overall Mean 682 plusmn 12 nm

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ights Reserve

d

Cumulative Distribution Function

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

This population 255 fibrils - Each fibril 1255 = 0392 - Data range 628-716 nm

BBML - All R

ights Reserve

d

CDF vs Histogram 62 63 64 65 66 67 68 69 70 71 72 73 74

0

10

20

30

40

50

60

70

80

90

100

0

5

10

15

20

25

30

35

40

45

50

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

HistoCDF

BBML - All R

ights Reserve

d

Changes in D-Spacing with Disease

0

5

10

15

20

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

0

5

10

15

20

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

Wallace et al Bone 2010 46 1349-1354

KS Test plt0001

Control 680 plusmn 26 nm

Estrogen Dep 659 plusmn 31 nm

t-test on mean p=0048

BBML - All R

ights Reserve

d

Changes in D-Spacing with Disease

Wallace et al Bone 2010 46 1349-1354

0

10

20

30

40

50

60

70

80

90

100

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

KS Test plt0001

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ights Reserve

d

Question Model and Tissue Morphology Publication

Tissue Specificity in Mice Male mouse 8 weeks Femur dentin tail tendon

No differences in bone vs dentin both ∆ vs tendon

Wallace et al Langmuir 2011 26(10) 7349-7354

Gender Effects Male and female mice 16 weeks Femur No differences NA

Estrogen Depletion OVX in sheep 5 years + 2 Radius Mean and population shift Wallace et al Bone

2010 46 1349-1354

Estrogen Depletion OVX in female 5 years + 2 Skin (dermis) Significant population shift Erickson et al Biotech

J 2013 8(1) 1174-126

Osteoporosis and Mechanical Loading

OVX in ulnar loading in rats Ulna

No loading effect population shift with OVX

Warden et al Endocrinology 2013 154(9) 3178-3187

Osteogenesis Imperfecta Brtl+ model male mice 2 months Femur

No mean ∆ widening of distribution

Wallace et al J Stuct Bio 2011 173 146-152

Osteogenesis Imperfecta Oimoim model male mice 12 weeks Femur Mean and population shift Bart et al Conn Tiss

Res 2014 In Press

Osteogenesis Imperfecta and Tissue Hydration

Brtl+ model male mice 6 months Tail Tendon Significant population shift Kemp et al J Stuct Bio

2012 180 428-438

Type 2 Diabetes Male ZDSD rats 30 weeks Tibia Significant population shift Hammond et al Bone

2014 60 26-32

Type 2 Diabetes Female ZDSD rats 32 weeks Tail Tendon Significant population shift

Diaz Gonzalez et al J Biomech 2014 47(3)

681-686

Raloxifene (in vivo and in vitro) Female dog treated with Raloxifene Femur Significant population shift Gallant et al Bone

2014 61 191-200 BBML - All R

ights Reserve

d

Issues to Consider Site Selection and Sampling

KS Test plt0001

0023 of bonersquos length

15 m

m =

150

00 micro

m

Wallace et al Langmuir 2011 26(10) 7349-7354

100 microm x 100 microm

35 microm x 35 microm

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ights Reserve

d

Issues to Consider Site Selection and Sampling

60

62

64

66

68

70

72

74

1 (35)

2 (33)

3 (40)

4 (39)

5 (35)

6 (29)

7 (26)

8 (23)

9 (28)

WT (288)

D-P

erio

dic

Gap

Ove

rlap

Spac

ing

(nm

)

Axial Location (Proximal End = 1 Distal End = 9)

( of Fibrils)

91

23

45

87

6

Wallace et al Langmuir 2011 26(10) 7349-7354

288 fibrils for population n=1 for mean comparisons BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

Wet Tendon Dry Tendon Kemp et al J StructBio 2012 180 (3) 428-438

Collagen and bone exist in a hydrated environment in vivo bull Hydration state can impact morphology and mechanics

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ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

Nonsignificant mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet (n=255) 682 plusmn 12 nm

WT Dry (n=257) 686 plusmn 08 nm

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

Brtl DryBrtl Wet

KS Test plt0001

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

WT Case 04 nm mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet 682 plusmn 12 nm

WT Dry 686 plusmn 08 nm

Brtl+ Wet 675 plusmn 14 nm

Brtl+ Dry 689 plusmn 10 nm

Brtl+ Case 14 nm increase with drying (plt0001) bull Entire population shifted upward (plt0001)

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT WetBrtl Wet

KS Test plt0001

Brtl+ distribution shifted downward vs WT when wet

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryBrtl Dry

KS Test plt0001

Brtl+ distribution shifted upward vs WT when dry

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider AFM calibration

bull Absolute distance measurements require accurate piezo calibration

bull AFM systems have large scan ranges (150 microm to lt 1 microm)

bull Scanner non-linearities can introduce substantial error over that range

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

ights Reserve

d

Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

BBML - All R

ights Reserve

d

Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

100 nm 10 80 602 1198 100 nm 7 60 827 1439 100 nm 5 40 814 1427 100 nm 35 20 917 1724 100 nm 2 10 937 1774

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

ights Reserve

d

AFM calibration ndash Take Away Message

Calibration using a scan size and feature size comparable to features of interest is highly recommended

Calibration addresses absolute accuracy not precision (differential sensitivity between measurements)

bull Does not limit ability to differentiate between groups using the same AFM with the same calibration parameters

BBML - All R

ights Reserve

d

AFM Applications in Bone Research Indentation Mechanics

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ights Reserve

d

AFM-Based Indentation

In addition to high resolution imaging AFM can be used to extract nanoscale mechanical data

ApproachWithdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Can

tilev

er D

efle

ctio

n (V

olts

)

1 Ramp into a rigid sample ndash measure the slope

Deflection sensitivity x 119899119899119899119899119881119881

2 Calculate cantilever spring constant

3 Hookersquos law takes deflection (volts) and converts to units of force F = kx

Spring constant k 119873119873119899119899

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Forc

e (n

N)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration Fo

rce

(nN

)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

Tip-Sample Separation (nm)

BBML - All R

ights Reserve

d

AFM-Based Indentation Parameters

Approach Withdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

Peak Force

Area Between Curves Energy Dissipation

Contact Mechanics Fit for Elastic Modulus (E)

Adhesion Force

Indentation Depth (δ)

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

z

r

δ

R

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119877119877 ∙ 12057512057532

Es Sample modulus νs Poissonrsquos ratio R tip radius of curvature δ load point displacement

Relevant Hertz Assumptions bull Strains are small and within the elastic limit bull Indenter is infinitely stiff (only sample deforms) bull Indentation depth ltlt radius of curvature of indenter bull Surfaces are continuous non-conforming (must be flat) bull Load is normal to surface

BBML - All R

ights Reserve

d

Proper Indentation Model is Essential

δ

r

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119903119903 ∙ 12057512057532

δ

α

119865119865 =2120587120587∙

1198641198641199041199041 minus 1205841205841199041199042

∙ tan120572120572 ∙ 1205751205752

Es Sample modulus νs Poissonrsquos ratio R tip radius α opening angle

Hertz Model Spherical Indenter

depth ltlt radius of curvature

Sneddon Model Conical Indenter

depth ge radius of curvature

BBML - All R

ights Reserve

d

Indentation in OI Tendon Study

x

z

yBrtl+ and WT mice 6 month old males

4 tails per group

2-3 fascicles per tail

25-30 fibrils per tail

4-5 locations per fibril

Indent force 20 nN wet (Sneddon) 50 nN dry (Hertz) BBML - A

ll Rights R

eserved

OI Tendon Differential Effects of Dehydration

Dry 1268 plusmn 718 MPa

Wet 305 plusmn 243 MPa

Dry 1737 plusmn 973 MPa

Wet 301 plusmn 202 MPa

Kemp et al J StructBio 2012 180 (3) 428-438

bull Significant stiffening of fibrils with dehydration

bull More pronounced modulus modulus as a function of disease

BBML - All R

ights Reserve

d

OI Tendon Differential Effects of Dehydration

Kemp et al J StructBio 2012 180 (3) 428-438

Diseased phenotype not present when wet phenotype observed when dried

0

10

20

30

40

50

60

0 20 40 60 80 100 120 140 160 180 200

Gro

up S

ampl

es (

)

Indent Modulus (MPa)

WT WetBrtl Wet

0

2

4

6

8

10

12

14

16

18

20

025

050

075

010

0012

5015

0017

5020

0022

5025

0027

5030

0032

5035

0037

5040

0042

5045

0047

5050

00

Gro

up S

ampl

es (

)Indent Modulus (MPa)

WT DryBrtl Dry

555 WT Indents 305 plusmn 243 MPa

614 Brtl+ Indents 301 plusmn 202 MPa

KS Test p=0129 t Test p=0464 464 WT Indents

1268 plusmn 718 MPa 479 Brtl+ Indents 1737 plusmn 973 MPa

KS Test plt0001 T Test plt0001

BBML - All R

ights Reserve

d

Indentation in Diabetic Tendon (Poster 0495)

x

z

y

Control and Diabetic Rat Tail Tendon

4-5 rats per group

2-3 fascicles per rat

~70 fibrilstail

4-5 locationsfibril

20 nN (Sneddon)

BBML - All R

ights Reserve

d

Multiscale Diabetes Study (Poster 0495)

0

5

10

15

20

25

300 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 gt25

Perc

enta

ge o

f Gro

up S

ampl

es

Elastic Modulus (MPa)

CD (1530 indents)ZDSD (913 indents)

CD (n=5) 381 plusmn 149 MPa

ZDSD (n=3) 706 plusmn 263 MPa

0

100

200

300

400

500

600

700

800

900

0 2 4 6 8 10

Mic

rosc

ale

Mod

ulus

(MPa

)

Nanoscale Modulus (MPa)

plt0001

r2=0531 p=0040

bull Increased indentation modulus in diabetic tendon

bull Strong relationship between nanoscale indent modulus and microscale tensile modulus

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

Proper probe selection for intended application bull Cantilever must be stiff relative to sample bull Sharp probes may damage sample easily dull

R or α must be determined for each probe bull Properties provided by manufacturer are unreliable bull Vary from probe to probe can dull during indent process

Spring constant must be determined for each probe

bull Value provided by manufacturer is inaccurate bull Thermal tuning OK for ldquosoftrdquo probes poor performance

for stiffer probes bull Sader method (geometry) added mass reference

cantilevers

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

Veeco Application Technical note Ben Ohler Practical Advice on the Determination of Cantilever Spring Constants

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

bull Sample processing

bull Sample hydration

bull Proper force range

bull Substrate effects for thin samples

bull Proper modulus model and range of data goodness of fit

Bearing in mind these assumptions and limitations AFM can provide true ldquonanoscalerdquo mechanical properties both in terms of forces and deformations

BBML - All R

ights Reserve

d

A Note on Peak Force QNM for Bruker AFMs bull Mechanical properties mapped pixel-by-pixel at same

resolution as the height image (PeakForce QNM)

Height Modulus BBML - All R

ights Reserve

d

A Note on Peak Force QNM

Correct probe stiffness must be used based on sample Pittenger ndash Veeco Product Bulletin

BBML - All R

ights Reserve

d

A Note on Peak Force QNM Extremely sensitive to calibration (details rarely given) Absolute radius and spring constant known

bull especially important from probe to probe bull radius uarr with indent depth ndash must determine R at depth

you expect to image bull Depth may vary as a function of sample (or at different

regions within the sample)

Relative reference material of ldquoknownrdquo modulus used to determine a ratio of 119896119896 119877119877 bull ldquoknownrdquo modulus may come from tension compression

and not indentation bull R is related to depth indentation depth on sample must

be same as on reference BBML - All R

ights Reserve

d

A Note on Peak Force QNM bull Modulus fit on the fly ndash no ability to monitor fit (or range) bull Local sample topography and sample tilt play a role in

measured parameters bull Assuming all modulus values in an image are correct how to

quantify

512 x 512 pixels = 262144 measurements

n=1 n=262144

n from selected ROIs

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ights Reserve

d

bull Imaging in air on mica ndash substrate effect

bull Same imaging force or indent depth for all

bull For each area were all modulus values pooled

Lamprou et al PLoS ONE 2013 BBML - All R

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d

AFM Applications in Bone Research Other Cool Mechanical Techniques

BBML - All R

ights Reserve

d

ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo Bonds within or between collagen fibrils in bone that rupture to dissipate energy as a toughening mechanism

Thompson et al Nature 2001

Fantner et al Nature Mat 2005

Hansma et al JMNI 2005

Fantner et al Nano Letters 2007 BBML - All R

ights Reserve

d

Other Mechanical Techniques

Jimenez-Palomar J Mech Beh Bio Mat 2012

van der Rijt Macro Bio 2004

Yang J Biomed Mater Res 2007

Yang Biophysical Journal 2008 BBML - All R

ights Reserve

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Other Cool Techniques bull Piezoresponse Force Microscopy (PFM)

bull Electrochemical AFM (EC-AFM)

bull Force Modulation Microscopy (FMM)

bull Lateral Force Microscopy (LFM)

bull Magnetic Force Microscopy (MFM)

bull Conductive AFM (C-AFM)

bull Force Pulling

bull Cantilever Bending

bull Tensile testing of individual fibrils

bull 3 point bending of fibrils

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Summary Things to rememberhellip

bull Imaging mode imaging medium and probe selection

bull Morphology Measurements bull Sample preparation and hydration state bull Measuring fibril diameter bull Site selection and proper sampling bull Appropriate system calibration

bull Mechanical Measurements

bull Probe selection (stiffness and tip radius) bull Probe calibration (radius angle spring constant) bull Indentation force range and mechanical model bull Hydration in biological samples bull PeakForce QNM

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Acknowledgements BBML

Max Hammond Armando Diaz Gonzalez Zachary Bart Silvia Canelon Creasy Clauser

IU School of Medicine Dr David Burr Dr Matt Allen Dr Stuart Warden Dr Lilian Plotkin

Funding Sources NIHNIDCR 1F32DE018840-01 A1 IUPUI BME Departmental Startup Funds IUPUI Research Support Funds Grant IUPUI Biomechanics and Biomaterials Research Center

NIH NICHD Dr Joan Marini

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Bone Biology and Mechanics Lab (BBML) wwwiupuiedu~bbml Facebook BoneBiologyMechanicsLab

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• Slide Number 1
• General Seminar Outline
• Bonersquos Hierarchical Structure
• Critical Gap in Understanding Collagen
• Methods For Analyzing Collagen Structure
• Methods For Imaging Collagen Structure
• Cryo-TEM
• Atomic Force Microscopy (AFM)
• AFM Image of Pentacene (C22H14)
• AFM Image of Hexabenzocoronene
• Basics of AFM Imaging The Probe
• Basics of AFM Imaging System Components
• van der Waals Potential Energy Curve
• Contact Mode ndash Monitor Deflection
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Peak Force Tapping Mode ndash Monitor Force
• AFM Applications in Bone Research Imaging
• AFM Imaging of Collagen
• Imaging of Osteoclast Activity
• Mineralized collagen deposition in vitro
• Imaging of Canilicular Structure
• AFM Imaging in Bone
• AFM Imaging in Bone
• Removing Mineral in Bone to Expose Collagen
• Removing Mineral in Bone to Expose Collagen
• Demineralization with EDTA
• Demineralization with EDTA
• Demineralization with EDTA
• Issues to Consider Effects of Acid
• Slide Number 32
• Slide Number 33
• Analysis of Collagen Structure in Bone
• Analysis of Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril D-Spacing
• Slide Number 39
• Collagen in Bone Fibril D-Spacing
• Collagen in Bone Fibril D-Spacing
• 2D Fast Fourier Transform Analysis
• 2D Fast Fourier Transform Analysis
• D-Spacing Distribution
• D-Spacing Distribution
• Cumulative Distribution Function
• CDF vs Histogram
• Changes in D-Spacing with Disease
• Changes in D-Spacing with Disease
• Slide Number 50
• Issues to Consider Site Selection and Sampling
• Issues to Consider Site Selection and Sampling
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• AFM calibration ndash Take Away Message
• AFM Applications in Bone ResearchIndentation Mechanics
• AFM-Based Indentation
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Parameters
• Hertz Contact Equation
• Hertz Contact Equation
• Hertz Contact Equation
• Proper Indentation Model is Essential
• Indentation in OI Tendon Study
• OI Tendon Differential Effects of Dehydration
• OI Tendon Differential Effects of Dehydration
• Indentation in Diabetic Tendon (Poster 0495)
• Multiscale Diabetes Study (Poster 0495)
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• A Note on Peak Force QNM for Bruker AFMs
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• Slide Number 83
• Slide Number 84
• AFM Applications in Bone ResearchOther Cool Mechanical Techniques
• ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo
• Other Mechanical Techniques
• Other Cool Techniques
• Summary Things to rememberhellip
• Acknowledgements
• Bone Biology and Mechanics Lab (BBML)

Basics of AFM Imaging The Probe Nanoscale tip mounted on a microscale cantilever

Probe

Feedback System

X-Y-X Piezo Combination

Cantilever

Wallace Bone 2012

Kopycinska-Muller Ultramicroscopy 2006

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Basics of AFM Imaging System Components bull Piezoelectric actuators precise

movement under electric potential

bull Raster-scanned (x-y direction)

bull Force transducer interaction force (typically cantileverrsquos deflection)

bull Cantilever deflection (z-direction) measured by photodiode

bull Control system maintains a desired force between probe and sample and (movement in z-direction)

Probe

Feedback System

X-Y-X Piezo Combination

Cantilever

Wallace Bone 2012

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van der Waals Potential Energy Curve

FOR

CE

DISTANCE

Repulsive Regime

Attractive Regime

Weak atomic attraction

Net attractive force weakens as interatomic separation decreases

Net force = 0 distance between atoms is 2-3 Å (~ length of a chemical bond)

Attraction increases until electron clouds begin to electrostatically repel each other BBML - A

ll Rights R

eserved

Contact Mode ndash Monitor Deflection

Control system maintains user-defined deflection by vertically moving scanner using piezoelectric actuator

Tip-Sample Separation

Forc

e

Frictional and adhesive forces can damage sample and distort image

Probe in constant contact cantilever deflects like a spring according to Hookersquos law (F = kx)

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Tapping Mode ndash Monitor Amplitude

Tip-Sample SeparationForc

e

Intermittent Contact

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Tapping Mode ndash Monitor Amplitude

ApproachWithdrawal

Tip-Sample SeparationForc

e

Cantilever oscillates at user-defined amplitude near cantilever resonance frequency

-15

-1

-05

0

05

1

15

0 5 10 15

Osc

illat

ion

Am

plitu

de

Time

-15

-1

-05

0

05

1

15

0 5 10 15

Osc

illat

ion

Am

plitu

de

Time

Setpoint

Bump

-15

-1

-05

0

05

1

15

0 5 10 15

Osc

illat

ion

Am

plitu

de

Time

SetpointBumpValley

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Tapping Mode ndash Monitor Amplitude bull Probe in contact for a small fraction of its tapping period

lateral forces are reduced

bull Probe must be tuned at resonance frequency ndash can be challenging for some probes when in fluid

Tuning in Air Single resonance peak Tuning in Fluid Multiple and spurious peaks wwwasylumresearchcom

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Peak Force Tapping Mode ndash Monitor Force

Control system maintains user-defined maximum force

Cantilever oscillates at low frequency (1-2 kHz)

bull No need for tuning

Slow tapping force curve is captured with each tap

ApproachWithdrawal

Tip-Sample SeparationForc

ePeakForce

Direct control of maximum normal force bull Protects tip and sample wear bull Limits indentation depth to a few nm - increases resolution

by limiting contact area bull Mechanical properties mapped pixel-by-pixel at same

resolution as the height image (PeakForce QNM) BBML - All R

ights Reserve

d

AFM Applications in Bone Research Imaging

BBML - All R

ights Reserve

d

AFM Imaging of Collagen

bull AFMrsquos adoption in bone research has been slowhellip

bull First AFM-based study in collagen came in 1992 (a previous study in 1989 used Scanning Tunneling Microscopy)

bull Observed fibrillar and monomeric collagen

Chernoff J of Vac Sci Tech 1992

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Imaging of Osteoclast Activity

Bozec Ultramicroscopy 2005

Hassenkam Anat Record 2006

Initial observations of cellular activity were qualitative (analyses of collagen morphology were not presented)

Sasaki J Elec Micro 1993

Sasaki CTI 1995

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Mineralized collagen deposition in vitro

Collagen deposition from MLO-A5 cells

Barragan-Adjemian CTI 2006 BBML - All R

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Imaging of Canilicular Structure

Reilly Annals of BME 2001

Lin J of Microscopy 2010

Average canilicular diameter ~400-500 nm larger than values typically reported from TEM studies

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AFM Imaging in Bone In general we are more interested in collagen in bone

3 microm Diamond Suspension

Unpolished

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AFM Imaging in Bone

The surface is completely mineralizedhellip BBML - All R

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Removing Mineral in Bone to Expose Collagen

Dealing with mineral is a challenge to analyzing collagen ndash some mineral must be removed

bull 10 Citric acid (to remove mineral) 65 Sodium Hypochlorite (to remove non-collagenous proteins) Habelitz J Struct Bio 2002

bull 7 or 17 Phosphoric Acid Bozek IEEE 2005 El Feninat J Biomed Mat Res 1998

bull 5 Formic Acid Ge Mat Sci Eng 2007

bull 30 mM EDTA Kindt Nanotechnology 2007

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Removing Mineral in Bone to Expose Collagen

Dealing with mineral is a challenge to analyzing collagen ndash some mineral must be removed

bull 30 mM EDTA Kindt Nanotechnology 2007 BBML - A

ll Rights R

eserved

Demineralization with EDTA EDTA - Ethylenediaminetetraacetic Acid

Chelating agent - sequesters divalent cations (notably Ca2+)

bull Treat with 05M EDTA pH 8 (15-20 minutes) bull Vigorously wash with ultrapure water bull Sonicate to release surface-bound mineral ions bull Repeat 3-4 times

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Demineralization with EDTA

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Demineralization with EDTA

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Issues to Consider Effects of Acid

Immature collagen crosslinks are very sensitive to acid bull Using acids to demineralize bone may inadvertently

destroy crosslinks distort structure

Fixation of tissue may be necessary prior to demineralization bull Fixation (glutaraldehyde or paraformadehyde) may

preserve structural integrity

bull Fixation effects on collagen structure are not fully known

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100 microm x 100 microm

50 microm x 50 microm

25 microm x 25 microm

10 microm x 10 microm

35 microm x 35 microm

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35 microm x 35 microm

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Analysis of Collagen Structure in Bone Spatial organization of newly formed bone following a surgically-induced injury (rat tibial hole)

Baranauskas J Vac Sci Tech 2001 Baranauskas App Surface Sci 2005

ldquohellipstatistical diameter of the fibers is 159 nm in good agreement with the literature values for type I collagen fibersrdquo

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Analysis of Collagen in Bone Fibril Diameter Fibril Diameter easiest to measure property first attempts were a bit crude better with increased resolution

Sasaki J Mat Sci Tech 2002 Thalhammer J Arch Sci 2001

Bozec Ultramicroscopy 2005

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Collagen in Bone Fibril Diameter

Caution must be taken when reporting and interpreting diameters measured from fibrils in bone bull Fibril packing - width may not be fully exposed

Wallace et al Langmuir 2010

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Collagen in Bone Fibril Diameter

Caution must be taken when reporting and interpreting diameters measured from fibrils in bone bull Fibril packing - width may not be fully exposed

Rigozzi ndash J Struct Bio - 2011

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Collagen in Bone Fibril D-Spacing

300 nm 15 nm

N C C N C C N C C N C C

H O H2C CH2 O H

CH2

O OH HHC CH2

CH2

HO

H H

HHN C C

O

H2C CH2

CH2

HN C C

OH

HC CH2

HO

N C C N C C N C C

H O H2C CH2 O

CH2

OH HC CH2

HO

H H

H

CH2 CH2

glycine X - proline Y - hydroxyproline

Hodge and Petruska 1963

Collagen Fibril Axial D-Periodicity BBML - All R

ights Reserve

d

05

101520

0 02 04 06 08 1

Hei

ght (

nm)

Length (microns)

httpremfdartmoutheduimagesindexhtml

Randall Nature and Structure of Collagen 1953

TEM SEM

AFM

D-Spacing = 67 nm

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Collagen in Bone Fibril D-Spacing Many studies have shown observable D-spacing content that near theoretical value

Sasaki 2002 67 plusmn 2 nm Bozek 2005 665 plusmn 14 nm Ge 2007 666 plusmn 38 nm

Habelitz J Structural Bio 2002

A 2002 study in dentin got me interestedhellip

n = 322 wet n = 363 dry

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Collagen in Bone Fibril D-Spacing

bull Internal structure bull Wallace et al Bone 2010 46 1349-1354 bull Wallace et al J Stuct Bio 2011 173 146-152 bull Kemp et al J Stuct Bio 2012 180 428-438 bull Warden et al Endocrinology 2013 154(9)

3178-3187 bull Gallant et al Bone 2014 61 191-200 bull Bart et al Conn Tiss Res 2014 In Press

bull Enzymatic and non-

enzymatic cross-linking bull Hammond et al Bone 2014 60 26-32 bull Diaz Gonzalez et al J Biomech 2014 47(3)

681-686 bull Voytik-Harbin et al In Preparation 2014

D-periodic spacing important structural feature that captures aspects related to

Mouse Femur Mid-diaphysis BBML - All R

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2D Fast Fourier Transform Analysis

Harmonics

Fibril D-Spacing

Erickson B Wallace JM et al Biotechnology Journal 2013

Raw AFM Image 2D FFT Power Spectrum

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2D Fast Fourier Transform Analysis

Harmonics

Fibril D-Spacing

Raw AFM Image 2D FFT Power Spectrum

0

20000

40000

60000

80000

100000

120000

0 001 002 003 004 005 006

Am

plitu

de (

microV2 )

Position (1nm)

D-periodic spacing 0015 (1nm) ~ 67 nm

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D-Spacing Distribution

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Overall Mean 670 plusmn 00 nm

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D-Spacing Distribution

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Overall Mean 670 plusmn 00 nm

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

4 Samples Total of n = 255 fibrils Overall Mean 682 plusmn 12 nm

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Cumulative Distribution Function

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

This population 255 fibrils - Each fibril 1255 = 0392 - Data range 628-716 nm

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CDF vs Histogram 62 63 64 65 66 67 68 69 70 71 72 73 74

0

10

20

30

40

50

60

70

80

90

100

0

5

10

15

20

25

30

35

40

45

50

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

HistoCDF

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Changes in D-Spacing with Disease

0

5

10

15

20

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

0

5

10

15

20

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

Wallace et al Bone 2010 46 1349-1354

KS Test plt0001

Control 680 plusmn 26 nm

Estrogen Dep 659 plusmn 31 nm

t-test on mean p=0048

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Changes in D-Spacing with Disease

Wallace et al Bone 2010 46 1349-1354

0

10

20

30

40

50

60

70

80

90

100

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

KS Test plt0001

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Question Model and Tissue Morphology Publication

Tissue Specificity in Mice Male mouse 8 weeks Femur dentin tail tendon

No differences in bone vs dentin both ∆ vs tendon

Wallace et al Langmuir 2011 26(10) 7349-7354

Gender Effects Male and female mice 16 weeks Femur No differences NA

Estrogen Depletion OVX in sheep 5 years + 2 Radius Mean and population shift Wallace et al Bone

2010 46 1349-1354

Estrogen Depletion OVX in female 5 years + 2 Skin (dermis) Significant population shift Erickson et al Biotech

J 2013 8(1) 1174-126

Osteoporosis and Mechanical Loading

OVX in ulnar loading in rats Ulna

No loading effect population shift with OVX

Warden et al Endocrinology 2013 154(9) 3178-3187

Osteogenesis Imperfecta Brtl+ model male mice 2 months Femur

No mean ∆ widening of distribution

Wallace et al J Stuct Bio 2011 173 146-152

Osteogenesis Imperfecta Oimoim model male mice 12 weeks Femur Mean and population shift Bart et al Conn Tiss

Res 2014 In Press

Osteogenesis Imperfecta and Tissue Hydration

Brtl+ model male mice 6 months Tail Tendon Significant population shift Kemp et al J Stuct Bio

2012 180 428-438

Type 2 Diabetes Male ZDSD rats 30 weeks Tibia Significant population shift Hammond et al Bone

2014 60 26-32

Type 2 Diabetes Female ZDSD rats 32 weeks Tail Tendon Significant population shift

Diaz Gonzalez et al J Biomech 2014 47(3)

681-686

Raloxifene (in vivo and in vitro) Female dog treated with Raloxifene Femur Significant population shift Gallant et al Bone

2014 61 191-200 BBML - All R

ights Reserve

d

Issues to Consider Site Selection and Sampling

KS Test plt0001

0023 of bonersquos length

15 m

m =

150

00 micro

m

Wallace et al Langmuir 2011 26(10) 7349-7354

100 microm x 100 microm

35 microm x 35 microm

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ights Reserve

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Issues to Consider Site Selection and Sampling

60

62

64

66

68

70

72

74

1 (35)

2 (33)

3 (40)

4 (39)

5 (35)

6 (29)

7 (26)

8 (23)

9 (28)

WT (288)

D-P

erio

dic

Gap

Ove

rlap

Spac

ing

(nm

)

Axial Location (Proximal End = 1 Distal End = 9)

( of Fibrils)

91

23

45

87

6

Wallace et al Langmuir 2011 26(10) 7349-7354

288 fibrils for population n=1 for mean comparisons BBML - All R

ights Reserve

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Issues to Consider Effects of Hydration

Wet Tendon Dry Tendon Kemp et al J StructBio 2012 180 (3) 428-438

Collagen and bone exist in a hydrated environment in vivo bull Hydration state can impact morphology and mechanics

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Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

Nonsignificant mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet (n=255) 682 plusmn 12 nm

WT Dry (n=257) 686 plusmn 08 nm

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

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Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

Brtl DryBrtl Wet

KS Test plt0001

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

WT Case 04 nm mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet 682 plusmn 12 nm

WT Dry 686 plusmn 08 nm

Brtl+ Wet 675 plusmn 14 nm

Brtl+ Dry 689 plusmn 10 nm

Brtl+ Case 14 nm increase with drying (plt0001) bull Entire population shifted upward (plt0001)

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

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d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT WetBrtl Wet

KS Test plt0001

Brtl+ distribution shifted downward vs WT when wet

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryBrtl Dry

KS Test plt0001

Brtl+ distribution shifted upward vs WT when dry

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider AFM calibration

bull Absolute distance measurements require accurate piezo calibration

bull AFM systems have large scan ranges (150 microm to lt 1 microm)

bull Scanner non-linearities can introduce substantial error over that range

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

ights Reserve

d

Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

BBML - All R

ights Reserve

d

Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

100 nm 10 80 602 1198 100 nm 7 60 827 1439 100 nm 5 40 814 1427 100 nm 35 20 917 1724 100 nm 2 10 937 1774

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

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d

AFM calibration ndash Take Away Message

Calibration using a scan size and feature size comparable to features of interest is highly recommended

Calibration addresses absolute accuracy not precision (differential sensitivity between measurements)

bull Does not limit ability to differentiate between groups using the same AFM with the same calibration parameters

BBML - All R

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d

AFM Applications in Bone Research Indentation Mechanics

BBML - All R

ights Reserve

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AFM-Based Indentation

In addition to high resolution imaging AFM can be used to extract nanoscale mechanical data

ApproachWithdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Can

tilev

er D

efle

ctio

n (V

olts

)

1 Ramp into a rigid sample ndash measure the slope

Deflection sensitivity x 119899119899119899119899119881119881

2 Calculate cantilever spring constant

3 Hookersquos law takes deflection (volts) and converts to units of force F = kx

Spring constant k 119873119873119899119899

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Forc

e (n

N)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration Fo

rce

(nN

)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

Tip-Sample Separation (nm)

BBML - All R

ights Reserve

d

AFM-Based Indentation Parameters

Approach Withdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

Peak Force

Area Between Curves Energy Dissipation

Contact Mechanics Fit for Elastic Modulus (E)

Adhesion Force

Indentation Depth (δ)

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

z

r

δ

R

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119877119877 ∙ 12057512057532

Es Sample modulus νs Poissonrsquos ratio R tip radius of curvature δ load point displacement

Relevant Hertz Assumptions bull Strains are small and within the elastic limit bull Indenter is infinitely stiff (only sample deforms) bull Indentation depth ltlt radius of curvature of indenter bull Surfaces are continuous non-conforming (must be flat) bull Load is normal to surface

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ights Reserve

d

Proper Indentation Model is Essential

δ

r

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119903119903 ∙ 12057512057532

δ

α

119865119865 =2120587120587∙

1198641198641199041199041 minus 1205841205841199041199042

∙ tan120572120572 ∙ 1205751205752

Es Sample modulus νs Poissonrsquos ratio R tip radius α opening angle

Hertz Model Spherical Indenter

depth ltlt radius of curvature

Sneddon Model Conical Indenter

depth ge radius of curvature

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ights Reserve

d

Indentation in OI Tendon Study

x

z

yBrtl+ and WT mice 6 month old males

4 tails per group

2-3 fascicles per tail

25-30 fibrils per tail

4-5 locations per fibril

Indent force 20 nN wet (Sneddon) 50 nN dry (Hertz) BBML - A

ll Rights R

eserved

OI Tendon Differential Effects of Dehydration

Dry 1268 plusmn 718 MPa

Wet 305 plusmn 243 MPa

Dry 1737 plusmn 973 MPa

Wet 301 plusmn 202 MPa

Kemp et al J StructBio 2012 180 (3) 428-438

bull Significant stiffening of fibrils with dehydration

bull More pronounced modulus modulus as a function of disease

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ights Reserve

d

OI Tendon Differential Effects of Dehydration

Kemp et al J StructBio 2012 180 (3) 428-438

Diseased phenotype not present when wet phenotype observed when dried

0

10

20

30

40

50

60

0 20 40 60 80 100 120 140 160 180 200

Gro

up S

ampl

es (

)

Indent Modulus (MPa)

WT WetBrtl Wet

0

2

4

6

8

10

12

14

16

18

20

025

050

075

010

0012

5015

0017

5020

0022

5025

0027

5030

0032

5035

0037

5040

0042

5045

0047

5050

00

Gro

up S

ampl

es (

)Indent Modulus (MPa)

WT DryBrtl Dry

555 WT Indents 305 plusmn 243 MPa

614 Brtl+ Indents 301 plusmn 202 MPa

KS Test p=0129 t Test p=0464 464 WT Indents

1268 plusmn 718 MPa 479 Brtl+ Indents 1737 plusmn 973 MPa

KS Test plt0001 T Test plt0001

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ights Reserve

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Indentation in Diabetic Tendon (Poster 0495)

x

z

y

Control and Diabetic Rat Tail Tendon

4-5 rats per group

2-3 fascicles per rat

~70 fibrilstail

4-5 locationsfibril

20 nN (Sneddon)

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ights Reserve

d

Multiscale Diabetes Study (Poster 0495)

0

5

10

15

20

25

300 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 gt25

Perc

enta

ge o

f Gro

up S

ampl

es

Elastic Modulus (MPa)

CD (1530 indents)ZDSD (913 indents)

CD (n=5) 381 plusmn 149 MPa

ZDSD (n=3) 706 plusmn 263 MPa

0

100

200

300

400

500

600

700

800

900

0 2 4 6 8 10

Mic

rosc

ale

Mod

ulus

(MPa

)

Nanoscale Modulus (MPa)

plt0001

r2=0531 p=0040

bull Increased indentation modulus in diabetic tendon

bull Strong relationship between nanoscale indent modulus and microscale tensile modulus

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ights Reserve

d

Indentation Limitations and Weaknesses

Proper probe selection for intended application bull Cantilever must be stiff relative to sample bull Sharp probes may damage sample easily dull

R or α must be determined for each probe bull Properties provided by manufacturer are unreliable bull Vary from probe to probe can dull during indent process

Spring constant must be determined for each probe

bull Value provided by manufacturer is inaccurate bull Thermal tuning OK for ldquosoftrdquo probes poor performance

for stiffer probes bull Sader method (geometry) added mass reference

cantilevers

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ights Reserve

d

Indentation Limitations and Weaknesses

Veeco Application Technical note Ben Ohler Practical Advice on the Determination of Cantilever Spring Constants

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ights Reserve

d

Indentation Limitations and Weaknesses

bull Sample processing

bull Sample hydration

bull Proper force range

bull Substrate effects for thin samples

bull Proper modulus model and range of data goodness of fit

Bearing in mind these assumptions and limitations AFM can provide true ldquonanoscalerdquo mechanical properties both in terms of forces and deformations

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ights Reserve

d

A Note on Peak Force QNM for Bruker AFMs bull Mechanical properties mapped pixel-by-pixel at same

resolution as the height image (PeakForce QNM)

Height Modulus BBML - All R

ights Reserve

d

A Note on Peak Force QNM

Correct probe stiffness must be used based on sample Pittenger ndash Veeco Product Bulletin

BBML - All R

ights Reserve

d

A Note on Peak Force QNM Extremely sensitive to calibration (details rarely given) Absolute radius and spring constant known

bull especially important from probe to probe bull radius uarr with indent depth ndash must determine R at depth

you expect to image bull Depth may vary as a function of sample (or at different

regions within the sample)

Relative reference material of ldquoknownrdquo modulus used to determine a ratio of 119896119896 119877119877 bull ldquoknownrdquo modulus may come from tension compression

and not indentation bull R is related to depth indentation depth on sample must

be same as on reference BBML - All R

ights Reserve

d

A Note on Peak Force QNM bull Modulus fit on the fly ndash no ability to monitor fit (or range) bull Local sample topography and sample tilt play a role in

measured parameters bull Assuming all modulus values in an image are correct how to

quantify

512 x 512 pixels = 262144 measurements

n=1 n=262144

n from selected ROIs

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d

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ights Reserve

d

bull Imaging in air on mica ndash substrate effect

bull Same imaging force or indent depth for all

bull For each area were all modulus values pooled

Lamprou et al PLoS ONE 2013 BBML - All R

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d

AFM Applications in Bone Research Other Cool Mechanical Techniques

BBML - All R

ights Reserve

d

ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo Bonds within or between collagen fibrils in bone that rupture to dissipate energy as a toughening mechanism

Thompson et al Nature 2001

Fantner et al Nature Mat 2005

Hansma et al JMNI 2005

Fantner et al Nano Letters 2007 BBML - All R

ights Reserve

d

Other Mechanical Techniques

Jimenez-Palomar J Mech Beh Bio Mat 2012

van der Rijt Macro Bio 2004

Yang J Biomed Mater Res 2007

Yang Biophysical Journal 2008 BBML - All R

ights Reserve

d

Other Cool Techniques bull Piezoresponse Force Microscopy (PFM)

bull Electrochemical AFM (EC-AFM)

bull Force Modulation Microscopy (FMM)

bull Lateral Force Microscopy (LFM)

bull Magnetic Force Microscopy (MFM)

bull Conductive AFM (C-AFM)

bull Force Pulling

bull Cantilever Bending

bull Tensile testing of individual fibrils

bull 3 point bending of fibrils

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Summary Things to rememberhellip

bull Imaging mode imaging medium and probe selection

bull Morphology Measurements bull Sample preparation and hydration state bull Measuring fibril diameter bull Site selection and proper sampling bull Appropriate system calibration

bull Mechanical Measurements

bull Probe selection (stiffness and tip radius) bull Probe calibration (radius angle spring constant) bull Indentation force range and mechanical model bull Hydration in biological samples bull PeakForce QNM

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Acknowledgements BBML

Max Hammond Armando Diaz Gonzalez Zachary Bart Silvia Canelon Creasy Clauser

IU School of Medicine Dr David Burr Dr Matt Allen Dr Stuart Warden Dr Lilian Plotkin

Funding Sources NIHNIDCR 1F32DE018840-01 A1 IUPUI BME Departmental Startup Funds IUPUI Research Support Funds Grant IUPUI Biomechanics and Biomaterials Research Center

NIH NICHD Dr Joan Marini

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d

Bone Biology and Mechanics Lab (BBML) wwwiupuiedu~bbml Facebook BoneBiologyMechanicsLab

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ights Reserve

d

• Slide Number 1
• General Seminar Outline
• Bonersquos Hierarchical Structure
• Critical Gap in Understanding Collagen
• Methods For Analyzing Collagen Structure
• Methods For Imaging Collagen Structure
• Cryo-TEM
• Atomic Force Microscopy (AFM)
• AFM Image of Pentacene (C22H14)
• AFM Image of Hexabenzocoronene
• Basics of AFM Imaging The Probe
• Basics of AFM Imaging System Components
• van der Waals Potential Energy Curve
• Contact Mode ndash Monitor Deflection
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Peak Force Tapping Mode ndash Monitor Force
• AFM Applications in Bone Research Imaging
• AFM Imaging of Collagen
• Imaging of Osteoclast Activity
• Mineralized collagen deposition in vitro
• Imaging of Canilicular Structure
• AFM Imaging in Bone
• AFM Imaging in Bone
• Removing Mineral in Bone to Expose Collagen
• Removing Mineral in Bone to Expose Collagen
• Demineralization with EDTA
• Demineralization with EDTA
• Demineralization with EDTA
• Issues to Consider Effects of Acid
• Slide Number 32
• Slide Number 33
• Analysis of Collagen Structure in Bone
• Analysis of Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril D-Spacing
• Slide Number 39
• Collagen in Bone Fibril D-Spacing
• Collagen in Bone Fibril D-Spacing
• 2D Fast Fourier Transform Analysis
• 2D Fast Fourier Transform Analysis
• D-Spacing Distribution
• D-Spacing Distribution
• Cumulative Distribution Function
• CDF vs Histogram
• Changes in D-Spacing with Disease
• Changes in D-Spacing with Disease
• Slide Number 50
• Issues to Consider Site Selection and Sampling
• Issues to Consider Site Selection and Sampling
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• AFM calibration ndash Take Away Message
• AFM Applications in Bone ResearchIndentation Mechanics
• AFM-Based Indentation
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Parameters
• Hertz Contact Equation
• Hertz Contact Equation
• Hertz Contact Equation
• Proper Indentation Model is Essential
• Indentation in OI Tendon Study
• OI Tendon Differential Effects of Dehydration
• OI Tendon Differential Effects of Dehydration
• Indentation in Diabetic Tendon (Poster 0495)
• Multiscale Diabetes Study (Poster 0495)
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• A Note on Peak Force QNM for Bruker AFMs
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• Slide Number 83
• Slide Number 84
• AFM Applications in Bone ResearchOther Cool Mechanical Techniques
• ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo
• Other Mechanical Techniques
• Other Cool Techniques
• Summary Things to rememberhellip
• Acknowledgements
• Bone Biology and Mechanics Lab (BBML)

Basics of AFM Imaging System Components bull Piezoelectric actuators precise

movement under electric potential

bull Raster-scanned (x-y direction)

bull Force transducer interaction force (typically cantileverrsquos deflection)

bull Cantilever deflection (z-direction) measured by photodiode

bull Control system maintains a desired force between probe and sample and (movement in z-direction)

Probe

Feedback System

X-Y-X Piezo Combination

Cantilever

Wallace Bone 2012

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d

van der Waals Potential Energy Curve

FOR

CE

DISTANCE

Repulsive Regime

Attractive Regime

Weak atomic attraction

Net attractive force weakens as interatomic separation decreases

Net force = 0 distance between atoms is 2-3 Å (~ length of a chemical bond)

Attraction increases until electron clouds begin to electrostatically repel each other BBML - A

ll Rights R

eserved

Contact Mode ndash Monitor Deflection

Control system maintains user-defined deflection by vertically moving scanner using piezoelectric actuator

Tip-Sample Separation

Forc

e

Frictional and adhesive forces can damage sample and distort image

Probe in constant contact cantilever deflects like a spring according to Hookersquos law (F = kx)

BBML - All R

ights Reserve

d

Tapping Mode ndash Monitor Amplitude

Tip-Sample SeparationForc

e

Intermittent Contact

BBML - All R

ights Reserve

d

Tapping Mode ndash Monitor Amplitude

ApproachWithdrawal

Tip-Sample SeparationForc

e

Cantilever oscillates at user-defined amplitude near cantilever resonance frequency

-15

-1

-05

0

05

1

15

0 5 10 15

Osc

illat

ion

Am

plitu

de

Time

-15

-1

-05

0

05

1

15

0 5 10 15

Osc

illat

ion

Am

plitu

de

Time

Setpoint

Bump

-15

-1

-05

0

05

1

15

0 5 10 15

Osc

illat

ion

Am

plitu

de

Time

SetpointBumpValley

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ights Reserve

d

Tapping Mode ndash Monitor Amplitude bull Probe in contact for a small fraction of its tapping period

lateral forces are reduced

bull Probe must be tuned at resonance frequency ndash can be challenging for some probes when in fluid

Tuning in Air Single resonance peak Tuning in Fluid Multiple and spurious peaks wwwasylumresearchcom

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ights Reserve

d

Peak Force Tapping Mode ndash Monitor Force

Control system maintains user-defined maximum force

Cantilever oscillates at low frequency (1-2 kHz)

bull No need for tuning

Slow tapping force curve is captured with each tap

ApproachWithdrawal

Tip-Sample SeparationForc

ePeakForce

Direct control of maximum normal force bull Protects tip and sample wear bull Limits indentation depth to a few nm - increases resolution

by limiting contact area bull Mechanical properties mapped pixel-by-pixel at same

resolution as the height image (PeakForce QNM) BBML - All R

ights Reserve

d

AFM Applications in Bone Research Imaging

BBML - All R

ights Reserve

d

AFM Imaging of Collagen

bull AFMrsquos adoption in bone research has been slowhellip

bull First AFM-based study in collagen came in 1992 (a previous study in 1989 used Scanning Tunneling Microscopy)

bull Observed fibrillar and monomeric collagen

Chernoff J of Vac Sci Tech 1992

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ights Reserve

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Imaging of Osteoclast Activity

Bozec Ultramicroscopy 2005

Hassenkam Anat Record 2006

Initial observations of cellular activity were qualitative (analyses of collagen morphology were not presented)

Sasaki J Elec Micro 1993

Sasaki CTI 1995

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Mineralized collagen deposition in vitro

Collagen deposition from MLO-A5 cells

Barragan-Adjemian CTI 2006 BBML - All R

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d

Imaging of Canilicular Structure

Reilly Annals of BME 2001

Lin J of Microscopy 2010

Average canilicular diameter ~400-500 nm larger than values typically reported from TEM studies

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ights Reserve

d

AFM Imaging in Bone In general we are more interested in collagen in bone

3 microm Diamond Suspension

Unpolished

BBML - All R

ights Reserve

d

AFM Imaging in Bone

The surface is completely mineralizedhellip BBML - All R

ights Reserve

d

Removing Mineral in Bone to Expose Collagen

Dealing with mineral is a challenge to analyzing collagen ndash some mineral must be removed

bull 10 Citric acid (to remove mineral) 65 Sodium Hypochlorite (to remove non-collagenous proteins) Habelitz J Struct Bio 2002

bull 7 or 17 Phosphoric Acid Bozek IEEE 2005 El Feninat J Biomed Mat Res 1998

bull 5 Formic Acid Ge Mat Sci Eng 2007

bull 30 mM EDTA Kindt Nanotechnology 2007

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ights Reserve

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Removing Mineral in Bone to Expose Collagen

Dealing with mineral is a challenge to analyzing collagen ndash some mineral must be removed

bull 30 mM EDTA Kindt Nanotechnology 2007 BBML - A

ll Rights R

eserved

Demineralization with EDTA EDTA - Ethylenediaminetetraacetic Acid

Chelating agent - sequesters divalent cations (notably Ca2+)

bull Treat with 05M EDTA pH 8 (15-20 minutes) bull Vigorously wash with ultrapure water bull Sonicate to release surface-bound mineral ions bull Repeat 3-4 times

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d

Demineralization with EDTA

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ights Reserve

d

Demineralization with EDTA

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ights Reserve

d

Issues to Consider Effects of Acid

Immature collagen crosslinks are very sensitive to acid bull Using acids to demineralize bone may inadvertently

destroy crosslinks distort structure

Fixation of tissue may be necessary prior to demineralization bull Fixation (glutaraldehyde or paraformadehyde) may

preserve structural integrity

bull Fixation effects on collagen structure are not fully known

BBML - All R

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d

100 microm x 100 microm

50 microm x 50 microm

25 microm x 25 microm

10 microm x 10 microm

35 microm x 35 microm

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ights Reserve

d

35 microm x 35 microm

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d

Analysis of Collagen Structure in Bone Spatial organization of newly formed bone following a surgically-induced injury (rat tibial hole)

Baranauskas J Vac Sci Tech 2001 Baranauskas App Surface Sci 2005

ldquohellipstatistical diameter of the fibers is 159 nm in good agreement with the literature values for type I collagen fibersrdquo

BBML - All R

ights Reserve

d

Analysis of Collagen in Bone Fibril Diameter Fibril Diameter easiest to measure property first attempts were a bit crude better with increased resolution

Sasaki J Mat Sci Tech 2002 Thalhammer J Arch Sci 2001

Bozec Ultramicroscopy 2005

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ights Reserve

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Collagen in Bone Fibril Diameter

Caution must be taken when reporting and interpreting diameters measured from fibrils in bone bull Fibril packing - width may not be fully exposed

Wallace et al Langmuir 2010

BBML - All R

ights Reserve

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Collagen in Bone Fibril Diameter

Caution must be taken when reporting and interpreting diameters measured from fibrils in bone bull Fibril packing - width may not be fully exposed

Rigozzi ndash J Struct Bio - 2011

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ights Reserve

d

Collagen in Bone Fibril D-Spacing

300 nm 15 nm

N C C N C C N C C N C C

H O H2C CH2 O H

CH2

O OH HHC CH2

CH2

HO

H H

HHN C C

O

H2C CH2

CH2

HN C C

OH

HC CH2

HO

N C C N C C N C C

H O H2C CH2 O

CH2

OH HC CH2

HO

H H

H

CH2 CH2

glycine X - proline Y - hydroxyproline

Hodge and Petruska 1963

Collagen Fibril Axial D-Periodicity BBML - All R

ights Reserve

d

05

101520

0 02 04 06 08 1

Hei

ght (

nm)

Length (microns)

httpremfdartmoutheduimagesindexhtml

Randall Nature and Structure of Collagen 1953

TEM SEM

AFM

D-Spacing = 67 nm

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ights Reserve

d

Collagen in Bone Fibril D-Spacing Many studies have shown observable D-spacing content that near theoretical value

Sasaki 2002 67 plusmn 2 nm Bozek 2005 665 plusmn 14 nm Ge 2007 666 plusmn 38 nm

Habelitz J Structural Bio 2002

A 2002 study in dentin got me interestedhellip

n = 322 wet n = 363 dry

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ights Reserve

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Collagen in Bone Fibril D-Spacing

bull Internal structure bull Wallace et al Bone 2010 46 1349-1354 bull Wallace et al J Stuct Bio 2011 173 146-152 bull Kemp et al J Stuct Bio 2012 180 428-438 bull Warden et al Endocrinology 2013 154(9)

3178-3187 bull Gallant et al Bone 2014 61 191-200 bull Bart et al Conn Tiss Res 2014 In Press

bull Enzymatic and non-

enzymatic cross-linking bull Hammond et al Bone 2014 60 26-32 bull Diaz Gonzalez et al J Biomech 2014 47(3)

681-686 bull Voytik-Harbin et al In Preparation 2014

D-periodic spacing important structural feature that captures aspects related to

Mouse Femur Mid-diaphysis BBML - All R

ights Reserve

d

2D Fast Fourier Transform Analysis

Harmonics

Fibril D-Spacing

Erickson B Wallace JM et al Biotechnology Journal 2013

Raw AFM Image 2D FFT Power Spectrum

BBML - All R

ights Reserve

d

2D Fast Fourier Transform Analysis

Harmonics

Fibril D-Spacing

Raw AFM Image 2D FFT Power Spectrum

0

20000

40000

60000

80000

100000

120000

0 001 002 003 004 005 006

Am

plitu

de (

microV2 )

Position (1nm)

D-periodic spacing 0015 (1nm) ~ 67 nm

BBML - All R

ights Reserve

d

D-Spacing Distribution

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Overall Mean 670 plusmn 00 nm

BBML - All R

ights Reserve

d

D-Spacing Distribution

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Overall Mean 670 plusmn 00 nm

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

4 Samples Total of n = 255 fibrils Overall Mean 682 plusmn 12 nm

BBML - All R

ights Reserve

d

Cumulative Distribution Function

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

This population 255 fibrils - Each fibril 1255 = 0392 - Data range 628-716 nm

BBML - All R

ights Reserve

d

CDF vs Histogram 62 63 64 65 66 67 68 69 70 71 72 73 74

0

10

20

30

40

50

60

70

80

90

100

0

5

10

15

20

25

30

35

40

45

50

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

HistoCDF

BBML - All R

ights Reserve

d

Changes in D-Spacing with Disease

0

5

10

15

20

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

0

5

10

15

20

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

Wallace et al Bone 2010 46 1349-1354

KS Test plt0001

Control 680 plusmn 26 nm

Estrogen Dep 659 plusmn 31 nm

t-test on mean p=0048

BBML - All R

ights Reserve

d

Changes in D-Spacing with Disease

Wallace et al Bone 2010 46 1349-1354

0

10

20

30

40

50

60

70

80

90

100

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

KS Test plt0001

BBML - All R

ights Reserve

d

Question Model and Tissue Morphology Publication

Tissue Specificity in Mice Male mouse 8 weeks Femur dentin tail tendon

No differences in bone vs dentin both ∆ vs tendon

Wallace et al Langmuir 2011 26(10) 7349-7354

Gender Effects Male and female mice 16 weeks Femur No differences NA

Estrogen Depletion OVX in sheep 5 years + 2 Radius Mean and population shift Wallace et al Bone

2010 46 1349-1354

Estrogen Depletion OVX in female 5 years + 2 Skin (dermis) Significant population shift Erickson et al Biotech

J 2013 8(1) 1174-126

Osteoporosis and Mechanical Loading

OVX in ulnar loading in rats Ulna

No loading effect population shift with OVX

Warden et al Endocrinology 2013 154(9) 3178-3187

Osteogenesis Imperfecta Brtl+ model male mice 2 months Femur

No mean ∆ widening of distribution

Wallace et al J Stuct Bio 2011 173 146-152

Osteogenesis Imperfecta Oimoim model male mice 12 weeks Femur Mean and population shift Bart et al Conn Tiss

Res 2014 In Press

Osteogenesis Imperfecta and Tissue Hydration

Brtl+ model male mice 6 months Tail Tendon Significant population shift Kemp et al J Stuct Bio

2012 180 428-438

Type 2 Diabetes Male ZDSD rats 30 weeks Tibia Significant population shift Hammond et al Bone

2014 60 26-32

Type 2 Diabetes Female ZDSD rats 32 weeks Tail Tendon Significant population shift

Diaz Gonzalez et al J Biomech 2014 47(3)

681-686

Raloxifene (in vivo and in vitro) Female dog treated with Raloxifene Femur Significant population shift Gallant et al Bone

2014 61 191-200 BBML - All R

ights Reserve

d

Issues to Consider Site Selection and Sampling

KS Test plt0001

0023 of bonersquos length

15 m

m =

150

00 micro

m

Wallace et al Langmuir 2011 26(10) 7349-7354

100 microm x 100 microm

35 microm x 35 microm

BBML - All R

ights Reserve

d

Issues to Consider Site Selection and Sampling

60

62

64

66

68

70

72

74

1 (35)

2 (33)

3 (40)

4 (39)

5 (35)

6 (29)

7 (26)

8 (23)

9 (28)

WT (288)

D-P

erio

dic

Gap

Ove

rlap

Spac

ing

(nm

)

Axial Location (Proximal End = 1 Distal End = 9)

( of Fibrils)

91

23

45

87

6

Wallace et al Langmuir 2011 26(10) 7349-7354

288 fibrils for population n=1 for mean comparisons BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

Wet Tendon Dry Tendon Kemp et al J StructBio 2012 180 (3) 428-438

Collagen and bone exist in a hydrated environment in vivo bull Hydration state can impact morphology and mechanics

BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

Nonsignificant mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet (n=255) 682 plusmn 12 nm

WT Dry (n=257) 686 plusmn 08 nm

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

Brtl DryBrtl Wet

KS Test plt0001

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

WT Case 04 nm mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet 682 plusmn 12 nm

WT Dry 686 plusmn 08 nm

Brtl+ Wet 675 plusmn 14 nm

Brtl+ Dry 689 plusmn 10 nm

Brtl+ Case 14 nm increase with drying (plt0001) bull Entire population shifted upward (plt0001)

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT WetBrtl Wet

KS Test plt0001

Brtl+ distribution shifted downward vs WT when wet

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryBrtl Dry

KS Test plt0001

Brtl+ distribution shifted upward vs WT when dry

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider AFM calibration

bull Absolute distance measurements require accurate piezo calibration

bull AFM systems have large scan ranges (150 microm to lt 1 microm)

bull Scanner non-linearities can introduce substantial error over that range

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

ights Reserve

d

Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

BBML - All R

ights Reserve

d

Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

100 nm 10 80 602 1198 100 nm 7 60 827 1439 100 nm 5 40 814 1427 100 nm 35 20 917 1724 100 nm 2 10 937 1774

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

ights Reserve

d

AFM calibration ndash Take Away Message

Calibration using a scan size and feature size comparable to features of interest is highly recommended

Calibration addresses absolute accuracy not precision (differential sensitivity between measurements)

bull Does not limit ability to differentiate between groups using the same AFM with the same calibration parameters

BBML - All R

ights Reserve

d

AFM Applications in Bone Research Indentation Mechanics

BBML - All R

ights Reserve

d

AFM-Based Indentation

In addition to high resolution imaging AFM can be used to extract nanoscale mechanical data

ApproachWithdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Can

tilev

er D

efle

ctio

n (V

olts

)

1 Ramp into a rigid sample ndash measure the slope

Deflection sensitivity x 119899119899119899119899119881119881

2 Calculate cantilever spring constant

3 Hookersquos law takes deflection (volts) and converts to units of force F = kx

Spring constant k 119873119873119899119899

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Forc

e (n

N)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration Fo

rce

(nN

)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

Tip-Sample Separation (nm)

BBML - All R

ights Reserve

d

AFM-Based Indentation Parameters

Approach Withdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

Peak Force

Area Between Curves Energy Dissipation

Contact Mechanics Fit for Elastic Modulus (E)

Adhesion Force

Indentation Depth (δ)

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

z

r

δ

R

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119877119877 ∙ 12057512057532

Es Sample modulus νs Poissonrsquos ratio R tip radius of curvature δ load point displacement

Relevant Hertz Assumptions bull Strains are small and within the elastic limit bull Indenter is infinitely stiff (only sample deforms) bull Indentation depth ltlt radius of curvature of indenter bull Surfaces are continuous non-conforming (must be flat) bull Load is normal to surface

BBML - All R

ights Reserve

d

Proper Indentation Model is Essential

δ

r

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119903119903 ∙ 12057512057532

δ

α

119865119865 =2120587120587∙

1198641198641199041199041 minus 1205841205841199041199042

∙ tan120572120572 ∙ 1205751205752

Es Sample modulus νs Poissonrsquos ratio R tip radius α opening angle

Hertz Model Spherical Indenter

depth ltlt radius of curvature

Sneddon Model Conical Indenter

depth ge radius of curvature

BBML - All R

ights Reserve

d

Indentation in OI Tendon Study

x

z

yBrtl+ and WT mice 6 month old males

4 tails per group

2-3 fascicles per tail

25-30 fibrils per tail

4-5 locations per fibril

Indent force 20 nN wet (Sneddon) 50 nN dry (Hertz) BBML - A

ll Rights R

eserved

OI Tendon Differential Effects of Dehydration

Dry 1268 plusmn 718 MPa

Wet 305 plusmn 243 MPa

Dry 1737 plusmn 973 MPa

Wet 301 plusmn 202 MPa

Kemp et al J StructBio 2012 180 (3) 428-438

bull Significant stiffening of fibrils with dehydration

bull More pronounced modulus modulus as a function of disease

BBML - All R

ights Reserve

d

OI Tendon Differential Effects of Dehydration

Kemp et al J StructBio 2012 180 (3) 428-438

Diseased phenotype not present when wet phenotype observed when dried

0

10

20

30

40

50

60

0 20 40 60 80 100 120 140 160 180 200

Gro

up S

ampl

es (

)

Indent Modulus (MPa)

WT WetBrtl Wet

0

2

4

6

8

10

12

14

16

18

20

025

050

075

010

0012

5015

0017

5020

0022

5025

0027

5030

0032

5035

0037

5040

0042

5045

0047

5050

00

Gro

up S

ampl

es (

)Indent Modulus (MPa)

WT DryBrtl Dry

555 WT Indents 305 plusmn 243 MPa

614 Brtl+ Indents 301 plusmn 202 MPa

KS Test p=0129 t Test p=0464 464 WT Indents

1268 plusmn 718 MPa 479 Brtl+ Indents 1737 plusmn 973 MPa

KS Test plt0001 T Test plt0001

BBML - All R

ights Reserve

d

Indentation in Diabetic Tendon (Poster 0495)

x

z

y

Control and Diabetic Rat Tail Tendon

4-5 rats per group

2-3 fascicles per rat

~70 fibrilstail

4-5 locationsfibril

20 nN (Sneddon)

BBML - All R

ights Reserve

d

Multiscale Diabetes Study (Poster 0495)

0

5

10

15

20

25

300 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 gt25

Perc

enta

ge o

f Gro

up S

ampl

es

Elastic Modulus (MPa)

CD (1530 indents)ZDSD (913 indents)

CD (n=5) 381 plusmn 149 MPa

ZDSD (n=3) 706 plusmn 263 MPa

0

100

200

300

400

500

600

700

800

900

0 2 4 6 8 10

Mic

rosc

ale

Mod

ulus

(MPa

)

Nanoscale Modulus (MPa)

plt0001

r2=0531 p=0040

bull Increased indentation modulus in diabetic tendon

bull Strong relationship between nanoscale indent modulus and microscale tensile modulus

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

Proper probe selection for intended application bull Cantilever must be stiff relative to sample bull Sharp probes may damage sample easily dull

R or α must be determined for each probe bull Properties provided by manufacturer are unreliable bull Vary from probe to probe can dull during indent process

Spring constant must be determined for each probe

bull Value provided by manufacturer is inaccurate bull Thermal tuning OK for ldquosoftrdquo probes poor performance

for stiffer probes bull Sader method (geometry) added mass reference

cantilevers

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

Veeco Application Technical note Ben Ohler Practical Advice on the Determination of Cantilever Spring Constants

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

bull Sample processing

bull Sample hydration

bull Proper force range

bull Substrate effects for thin samples

bull Proper modulus model and range of data goodness of fit

Bearing in mind these assumptions and limitations AFM can provide true ldquonanoscalerdquo mechanical properties both in terms of forces and deformations

BBML - All R

ights Reserve

d

A Note on Peak Force QNM for Bruker AFMs bull Mechanical properties mapped pixel-by-pixel at same

resolution as the height image (PeakForce QNM)

Height Modulus BBML - All R

ights Reserve

d

A Note on Peak Force QNM

Correct probe stiffness must be used based on sample Pittenger ndash Veeco Product Bulletin

BBML - All R

ights Reserve

d

A Note on Peak Force QNM Extremely sensitive to calibration (details rarely given) Absolute radius and spring constant known

bull especially important from probe to probe bull radius uarr with indent depth ndash must determine R at depth

you expect to image bull Depth may vary as a function of sample (or at different

regions within the sample)

Relative reference material of ldquoknownrdquo modulus used to determine a ratio of 119896119896 119877119877 bull ldquoknownrdquo modulus may come from tension compression

and not indentation bull R is related to depth indentation depth on sample must

be same as on reference BBML - All R

ights Reserve

d

A Note on Peak Force QNM bull Modulus fit on the fly ndash no ability to monitor fit (or range) bull Local sample topography and sample tilt play a role in

measured parameters bull Assuming all modulus values in an image are correct how to

quantify

512 x 512 pixels = 262144 measurements

n=1 n=262144

n from selected ROIs

BBML - All R

ights Reserve

d

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ights Reserve

d

bull Imaging in air on mica ndash substrate effect

bull Same imaging force or indent depth for all

bull For each area were all modulus values pooled

Lamprou et al PLoS ONE 2013 BBML - All R

ights Reserve

d

AFM Applications in Bone Research Other Cool Mechanical Techniques

BBML - All R

ights Reserve

d

ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo Bonds within or between collagen fibrils in bone that rupture to dissipate energy as a toughening mechanism

Thompson et al Nature 2001

Fantner et al Nature Mat 2005

Hansma et al JMNI 2005

Fantner et al Nano Letters 2007 BBML - All R

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d

Other Mechanical Techniques

Jimenez-Palomar J Mech Beh Bio Mat 2012

van der Rijt Macro Bio 2004

Yang J Biomed Mater Res 2007

Yang Biophysical Journal 2008 BBML - All R

ights Reserve

d

Other Cool Techniques bull Piezoresponse Force Microscopy (PFM)

bull Electrochemical AFM (EC-AFM)

bull Force Modulation Microscopy (FMM)

bull Lateral Force Microscopy (LFM)

bull Magnetic Force Microscopy (MFM)

bull Conductive AFM (C-AFM)

bull Force Pulling

bull Cantilever Bending

bull Tensile testing of individual fibrils

bull 3 point bending of fibrils

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ights Reserve

d

Summary Things to rememberhellip

bull Imaging mode imaging medium and probe selection

bull Morphology Measurements bull Sample preparation and hydration state bull Measuring fibril diameter bull Site selection and proper sampling bull Appropriate system calibration

bull Mechanical Measurements

bull Probe selection (stiffness and tip radius) bull Probe calibration (radius angle spring constant) bull Indentation force range and mechanical model bull Hydration in biological samples bull PeakForce QNM

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Acknowledgements BBML

Max Hammond Armando Diaz Gonzalez Zachary Bart Silvia Canelon Creasy Clauser

IU School of Medicine Dr David Burr Dr Matt Allen Dr Stuart Warden Dr Lilian Plotkin

Funding Sources NIHNIDCR 1F32DE018840-01 A1 IUPUI BME Departmental Startup Funds IUPUI Research Support Funds Grant IUPUI Biomechanics and Biomaterials Research Center

NIH NICHD Dr Joan Marini

BBML - All R

ights Reserve

d

Bone Biology and Mechanics Lab (BBML) wwwiupuiedu~bbml Facebook BoneBiologyMechanicsLab

BBML - All R

ights Reserve

d

• Slide Number 1
• General Seminar Outline
• Bonersquos Hierarchical Structure
• Critical Gap in Understanding Collagen
• Methods For Analyzing Collagen Structure
• Methods For Imaging Collagen Structure
• Cryo-TEM
• Atomic Force Microscopy (AFM)
• AFM Image of Pentacene (C22H14)
• AFM Image of Hexabenzocoronene
• Basics of AFM Imaging The Probe
• Basics of AFM Imaging System Components
• van der Waals Potential Energy Curve
• Contact Mode ndash Monitor Deflection
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Peak Force Tapping Mode ndash Monitor Force
• AFM Applications in Bone Research Imaging
• AFM Imaging of Collagen
• Imaging of Osteoclast Activity
• Mineralized collagen deposition in vitro
• Imaging of Canilicular Structure
• AFM Imaging in Bone
• AFM Imaging in Bone
• Removing Mineral in Bone to Expose Collagen
• Removing Mineral in Bone to Expose Collagen
• Demineralization with EDTA
• Demineralization with EDTA
• Demineralization with EDTA
• Issues to Consider Effects of Acid
• Slide Number 32
• Slide Number 33
• Analysis of Collagen Structure in Bone
• Analysis of Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril D-Spacing
• Slide Number 39
• Collagen in Bone Fibril D-Spacing
• Collagen in Bone Fibril D-Spacing
• 2D Fast Fourier Transform Analysis
• 2D Fast Fourier Transform Analysis
• D-Spacing Distribution
• D-Spacing Distribution
• Cumulative Distribution Function
• CDF vs Histogram
• Changes in D-Spacing with Disease
• Changes in D-Spacing with Disease
• Slide Number 50
• Issues to Consider Site Selection and Sampling
• Issues to Consider Site Selection and Sampling
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• AFM calibration ndash Take Away Message
• AFM Applications in Bone ResearchIndentation Mechanics
• AFM-Based Indentation
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Parameters
• Hertz Contact Equation
• Hertz Contact Equation
• Hertz Contact Equation
• Proper Indentation Model is Essential
• Indentation in OI Tendon Study
• OI Tendon Differential Effects of Dehydration
• OI Tendon Differential Effects of Dehydration
• Indentation in Diabetic Tendon (Poster 0495)
• Multiscale Diabetes Study (Poster 0495)
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• A Note on Peak Force QNM for Bruker AFMs
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• Slide Number 83
• Slide Number 84
• AFM Applications in Bone ResearchOther Cool Mechanical Techniques
• ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo
• Other Mechanical Techniques
• Other Cool Techniques
• Summary Things to rememberhellip
• Acknowledgements
• Bone Biology and Mechanics Lab (BBML)

van der Waals Potential Energy Curve

FOR

CE

DISTANCE

Repulsive Regime

Attractive Regime

Weak atomic attraction

Net attractive force weakens as interatomic separation decreases

Net force = 0 distance between atoms is 2-3 Å (~ length of a chemical bond)

Attraction increases until electron clouds begin to electrostatically repel each other BBML - A

ll Rights R

eserved

Contact Mode ndash Monitor Deflection

Control system maintains user-defined deflection by vertically moving scanner using piezoelectric actuator

Tip-Sample Separation

Forc

e

Frictional and adhesive forces can damage sample and distort image

Probe in constant contact cantilever deflects like a spring according to Hookersquos law (F = kx)

BBML - All R

ights Reserve

d

Tapping Mode ndash Monitor Amplitude

Tip-Sample SeparationForc

e

Intermittent Contact

BBML - All R

ights Reserve

d

Tapping Mode ndash Monitor Amplitude

ApproachWithdrawal

Tip-Sample SeparationForc

e

Cantilever oscillates at user-defined amplitude near cantilever resonance frequency

-15

-1

-05

0

05

1

15

0 5 10 15

Osc

illat

ion

Am

plitu

de

Time

-15

-1

-05

0

05

1

15

0 5 10 15

Osc

illat

ion

Am

plitu

de

Time

Setpoint

Bump

-15

-1

-05

0

05

1

15

0 5 10 15

Osc

illat

ion

Am

plitu

de

Time

SetpointBumpValley

BBML - All R

ights Reserve

d

Tapping Mode ndash Monitor Amplitude bull Probe in contact for a small fraction of its tapping period

lateral forces are reduced

bull Probe must be tuned at resonance frequency ndash can be challenging for some probes when in fluid

Tuning in Air Single resonance peak Tuning in Fluid Multiple and spurious peaks wwwasylumresearchcom

BBML - All R

ights Reserve

d

Peak Force Tapping Mode ndash Monitor Force

Control system maintains user-defined maximum force

Cantilever oscillates at low frequency (1-2 kHz)

bull No need for tuning

Slow tapping force curve is captured with each tap

ApproachWithdrawal

Tip-Sample SeparationForc

ePeakForce

Direct control of maximum normal force bull Protects tip and sample wear bull Limits indentation depth to a few nm - increases resolution

by limiting contact area bull Mechanical properties mapped pixel-by-pixel at same

resolution as the height image (PeakForce QNM) BBML - All R

ights Reserve

d

AFM Applications in Bone Research Imaging

BBML - All R

ights Reserve

d

AFM Imaging of Collagen

bull AFMrsquos adoption in bone research has been slowhellip

bull First AFM-based study in collagen came in 1992 (a previous study in 1989 used Scanning Tunneling Microscopy)

bull Observed fibrillar and monomeric collagen

Chernoff J of Vac Sci Tech 1992

BBML - All R

ights Reserve

d

Imaging of Osteoclast Activity

Bozec Ultramicroscopy 2005

Hassenkam Anat Record 2006

Initial observations of cellular activity were qualitative (analyses of collagen morphology were not presented)

Sasaki J Elec Micro 1993

Sasaki CTI 1995

BBML - All R

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d

Mineralized collagen deposition in vitro

Collagen deposition from MLO-A5 cells

Barragan-Adjemian CTI 2006 BBML - All R

ights Reserve

d

Imaging of Canilicular Structure

Reilly Annals of BME 2001

Lin J of Microscopy 2010

Average canilicular diameter ~400-500 nm larger than values typically reported from TEM studies

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ights Reserve

d

AFM Imaging in Bone In general we are more interested in collagen in bone

3 microm Diamond Suspension

Unpolished

BBML - All R

ights Reserve

d

AFM Imaging in Bone

The surface is completely mineralizedhellip BBML - All R

ights Reserve

d

Removing Mineral in Bone to Expose Collagen

Dealing with mineral is a challenge to analyzing collagen ndash some mineral must be removed

bull 10 Citric acid (to remove mineral) 65 Sodium Hypochlorite (to remove non-collagenous proteins) Habelitz J Struct Bio 2002

bull 7 or 17 Phosphoric Acid Bozek IEEE 2005 El Feninat J Biomed Mat Res 1998

bull 5 Formic Acid Ge Mat Sci Eng 2007

bull 30 mM EDTA Kindt Nanotechnology 2007

BBML - All R

ights Reserve

d

Removing Mineral in Bone to Expose Collagen

Dealing with mineral is a challenge to analyzing collagen ndash some mineral must be removed

bull 30 mM EDTA Kindt Nanotechnology 2007 BBML - A

ll Rights R

eserved

Demineralization with EDTA EDTA - Ethylenediaminetetraacetic Acid

Chelating agent - sequesters divalent cations (notably Ca2+)

bull Treat with 05M EDTA pH 8 (15-20 minutes) bull Vigorously wash with ultrapure water bull Sonicate to release surface-bound mineral ions bull Repeat 3-4 times

BBML - All R

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d

Demineralization with EDTA

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ights Reserve

d

Demineralization with EDTA

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ights Reserve

d

Issues to Consider Effects of Acid

Immature collagen crosslinks are very sensitive to acid bull Using acids to demineralize bone may inadvertently

destroy crosslinks distort structure

Fixation of tissue may be necessary prior to demineralization bull Fixation (glutaraldehyde or paraformadehyde) may

preserve structural integrity

bull Fixation effects on collagen structure are not fully known

BBML - All R

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d

100 microm x 100 microm

50 microm x 50 microm

25 microm x 25 microm

10 microm x 10 microm

35 microm x 35 microm

BBML - All R

ights Reserve

d

35 microm x 35 microm

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ights Reserve

d

Analysis of Collagen Structure in Bone Spatial organization of newly formed bone following a surgically-induced injury (rat tibial hole)

Baranauskas J Vac Sci Tech 2001 Baranauskas App Surface Sci 2005

ldquohellipstatistical diameter of the fibers is 159 nm in good agreement with the literature values for type I collagen fibersrdquo

BBML - All R

ights Reserve

d

Analysis of Collagen in Bone Fibril Diameter Fibril Diameter easiest to measure property first attempts were a bit crude better with increased resolution

Sasaki J Mat Sci Tech 2002 Thalhammer J Arch Sci 2001

Bozec Ultramicroscopy 2005

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Collagen in Bone Fibril Diameter

Caution must be taken when reporting and interpreting diameters measured from fibrils in bone bull Fibril packing - width may not be fully exposed

Wallace et al Langmuir 2010

BBML - All R

ights Reserve

d

Collagen in Bone Fibril Diameter

Caution must be taken when reporting and interpreting diameters measured from fibrils in bone bull Fibril packing - width may not be fully exposed

Rigozzi ndash J Struct Bio - 2011

BBML - All R

ights Reserve

d

Collagen in Bone Fibril D-Spacing

300 nm 15 nm

N C C N C C N C C N C C

H O H2C CH2 O H

CH2

O OH HHC CH2

CH2

HO

H H

HHN C C

O

H2C CH2

CH2

HN C C

OH

HC CH2

HO

N C C N C C N C C

H O H2C CH2 O

CH2

OH HC CH2

HO

H H

H

CH2 CH2

glycine X - proline Y - hydroxyproline

Hodge and Petruska 1963

Collagen Fibril Axial D-Periodicity BBML - All R

ights Reserve

d

05

101520

0 02 04 06 08 1

Hei

ght (

nm)

Length (microns)

httpremfdartmoutheduimagesindexhtml

Randall Nature and Structure of Collagen 1953

TEM SEM

AFM

D-Spacing = 67 nm

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Collagen in Bone Fibril D-Spacing Many studies have shown observable D-spacing content that near theoretical value

Sasaki 2002 67 plusmn 2 nm Bozek 2005 665 plusmn 14 nm Ge 2007 666 plusmn 38 nm

Habelitz J Structural Bio 2002

A 2002 study in dentin got me interestedhellip

n = 322 wet n = 363 dry

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Collagen in Bone Fibril D-Spacing

bull Internal structure bull Wallace et al Bone 2010 46 1349-1354 bull Wallace et al J Stuct Bio 2011 173 146-152 bull Kemp et al J Stuct Bio 2012 180 428-438 bull Warden et al Endocrinology 2013 154(9)

3178-3187 bull Gallant et al Bone 2014 61 191-200 bull Bart et al Conn Tiss Res 2014 In Press

bull Enzymatic and non-

enzymatic cross-linking bull Hammond et al Bone 2014 60 26-32 bull Diaz Gonzalez et al J Biomech 2014 47(3)

681-686 bull Voytik-Harbin et al In Preparation 2014

D-periodic spacing important structural feature that captures aspects related to

Mouse Femur Mid-diaphysis BBML - All R

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2D Fast Fourier Transform Analysis

Harmonics

Fibril D-Spacing

Erickson B Wallace JM et al Biotechnology Journal 2013

Raw AFM Image 2D FFT Power Spectrum

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2D Fast Fourier Transform Analysis

Harmonics

Fibril D-Spacing

Raw AFM Image 2D FFT Power Spectrum

0

20000

40000

60000

80000

100000

120000

0 001 002 003 004 005 006

Am

plitu

de (

microV2 )

Position (1nm)

D-periodic spacing 0015 (1nm) ~ 67 nm

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D-Spacing Distribution

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Overall Mean 670 plusmn 00 nm

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D-Spacing Distribution

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Overall Mean 670 plusmn 00 nm

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

4 Samples Total of n = 255 fibrils Overall Mean 682 plusmn 12 nm

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Cumulative Distribution Function

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

This population 255 fibrils - Each fibril 1255 = 0392 - Data range 628-716 nm

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CDF vs Histogram 62 63 64 65 66 67 68 69 70 71 72 73 74

0

10

20

30

40

50

60

70

80

90

100

0

5

10

15

20

25

30

35

40

45

50

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

HistoCDF

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Changes in D-Spacing with Disease

0

5

10

15

20

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

0

5

10

15

20

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

Wallace et al Bone 2010 46 1349-1354

KS Test plt0001

Control 680 plusmn 26 nm

Estrogen Dep 659 plusmn 31 nm

t-test on mean p=0048

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Changes in D-Spacing with Disease

Wallace et al Bone 2010 46 1349-1354

0

10

20

30

40

50

60

70

80

90

100

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

KS Test plt0001

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Question Model and Tissue Morphology Publication

Tissue Specificity in Mice Male mouse 8 weeks Femur dentin tail tendon

No differences in bone vs dentin both ∆ vs tendon

Wallace et al Langmuir 2011 26(10) 7349-7354

Gender Effects Male and female mice 16 weeks Femur No differences NA

Estrogen Depletion OVX in sheep 5 years + 2 Radius Mean and population shift Wallace et al Bone

2010 46 1349-1354

Estrogen Depletion OVX in female 5 years + 2 Skin (dermis) Significant population shift Erickson et al Biotech

J 2013 8(1) 1174-126

Osteoporosis and Mechanical Loading

OVX in ulnar loading in rats Ulna

No loading effect population shift with OVX

Warden et al Endocrinology 2013 154(9) 3178-3187

Osteogenesis Imperfecta Brtl+ model male mice 2 months Femur

No mean ∆ widening of distribution

Wallace et al J Stuct Bio 2011 173 146-152

Osteogenesis Imperfecta Oimoim model male mice 12 weeks Femur Mean and population shift Bart et al Conn Tiss

Res 2014 In Press

Osteogenesis Imperfecta and Tissue Hydration

Brtl+ model male mice 6 months Tail Tendon Significant population shift Kemp et al J Stuct Bio

2012 180 428-438

Type 2 Diabetes Male ZDSD rats 30 weeks Tibia Significant population shift Hammond et al Bone

2014 60 26-32

Type 2 Diabetes Female ZDSD rats 32 weeks Tail Tendon Significant population shift

Diaz Gonzalez et al J Biomech 2014 47(3)

681-686

Raloxifene (in vivo and in vitro) Female dog treated with Raloxifene Femur Significant population shift Gallant et al Bone

2014 61 191-200 BBML - All R

ights Reserve

d

Issues to Consider Site Selection and Sampling

KS Test plt0001

0023 of bonersquos length

15 m

m =

150

00 micro

m

Wallace et al Langmuir 2011 26(10) 7349-7354

100 microm x 100 microm

35 microm x 35 microm

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ights Reserve

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Issues to Consider Site Selection and Sampling

60

62

64

66

68

70

72

74

1 (35)

2 (33)

3 (40)

4 (39)

5 (35)

6 (29)

7 (26)

8 (23)

9 (28)

WT (288)

D-P

erio

dic

Gap

Ove

rlap

Spac

ing

(nm

)

Axial Location (Proximal End = 1 Distal End = 9)

( of Fibrils)

91

23

45

87

6

Wallace et al Langmuir 2011 26(10) 7349-7354

288 fibrils for population n=1 for mean comparisons BBML - All R

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Issues to Consider Effects of Hydration

Wet Tendon Dry Tendon Kemp et al J StructBio 2012 180 (3) 428-438

Collagen and bone exist in a hydrated environment in vivo bull Hydration state can impact morphology and mechanics

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Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

Nonsignificant mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet (n=255) 682 plusmn 12 nm

WT Dry (n=257) 686 plusmn 08 nm

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

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Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

Brtl DryBrtl Wet

KS Test plt0001

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

WT Case 04 nm mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet 682 plusmn 12 nm

WT Dry 686 plusmn 08 nm

Brtl+ Wet 675 plusmn 14 nm

Brtl+ Dry 689 plusmn 10 nm

Brtl+ Case 14 nm increase with drying (plt0001) bull Entire population shifted upward (plt0001)

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT WetBrtl Wet

KS Test plt0001

Brtl+ distribution shifted downward vs WT when wet

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryBrtl Dry

KS Test plt0001

Brtl+ distribution shifted upward vs WT when dry

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider AFM calibration

bull Absolute distance measurements require accurate piezo calibration

bull AFM systems have large scan ranges (150 microm to lt 1 microm)

bull Scanner non-linearities can introduce substantial error over that range

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

ights Reserve

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Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

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Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

100 nm 10 80 602 1198 100 nm 7 60 827 1439 100 nm 5 40 814 1427 100 nm 35 20 917 1724 100 nm 2 10 937 1774

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

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AFM calibration ndash Take Away Message

Calibration using a scan size and feature size comparable to features of interest is highly recommended

Calibration addresses absolute accuracy not precision (differential sensitivity between measurements)

bull Does not limit ability to differentiate between groups using the same AFM with the same calibration parameters

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d

AFM Applications in Bone Research Indentation Mechanics

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AFM-Based Indentation

In addition to high resolution imaging AFM can be used to extract nanoscale mechanical data

ApproachWithdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

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ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Can

tilev

er D

efle

ctio

n (V

olts

)

1 Ramp into a rigid sample ndash measure the slope

Deflection sensitivity x 119899119899119899119899119881119881

2 Calculate cantilever spring constant

3 Hookersquos law takes deflection (volts) and converts to units of force F = kx

Spring constant k 119873119873119899119899

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ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Forc

e (n

N)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

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ights Reserve

d

AFM-Based Indentation Cantilever Calibration Fo

rce

(nN

)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

Tip-Sample Separation (nm)

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ights Reserve

d

AFM-Based Indentation Parameters

Approach Withdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

Peak Force

Area Between Curves Energy Dissipation

Contact Mechanics Fit for Elastic Modulus (E)

Adhesion Force

Indentation Depth (δ)

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ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

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ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

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ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

z

r

δ

R

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119877119877 ∙ 12057512057532

Es Sample modulus νs Poissonrsquos ratio R tip radius of curvature δ load point displacement

Relevant Hertz Assumptions bull Strains are small and within the elastic limit bull Indenter is infinitely stiff (only sample deforms) bull Indentation depth ltlt radius of curvature of indenter bull Surfaces are continuous non-conforming (must be flat) bull Load is normal to surface

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d

Proper Indentation Model is Essential

δ

r

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119903119903 ∙ 12057512057532

δ

α

119865119865 =2120587120587∙

1198641198641199041199041 minus 1205841205841199041199042

∙ tan120572120572 ∙ 1205751205752

Es Sample modulus νs Poissonrsquos ratio R tip radius α opening angle

Hertz Model Spherical Indenter

depth ltlt radius of curvature

Sneddon Model Conical Indenter

depth ge radius of curvature

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ights Reserve

d

Indentation in OI Tendon Study

x

z

yBrtl+ and WT mice 6 month old males

4 tails per group

2-3 fascicles per tail

25-30 fibrils per tail

4-5 locations per fibril

Indent force 20 nN wet (Sneddon) 50 nN dry (Hertz) BBML - A

ll Rights R

eserved

OI Tendon Differential Effects of Dehydration

Dry 1268 plusmn 718 MPa

Wet 305 plusmn 243 MPa

Dry 1737 plusmn 973 MPa

Wet 301 plusmn 202 MPa

Kemp et al J StructBio 2012 180 (3) 428-438

bull Significant stiffening of fibrils with dehydration

bull More pronounced modulus modulus as a function of disease

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OI Tendon Differential Effects of Dehydration

Kemp et al J StructBio 2012 180 (3) 428-438

Diseased phenotype not present when wet phenotype observed when dried

0

10

20

30

40

50

60

0 20 40 60 80 100 120 140 160 180 200

Gro

up S

ampl

es (

)

Indent Modulus (MPa)

WT WetBrtl Wet

0

2

4

6

8

10

12

14

16

18

20

025

050

075

010

0012

5015

0017

5020

0022

5025

0027

5030

0032

5035

0037

5040

0042

5045

0047

5050

00

Gro

up S

ampl

es (

)Indent Modulus (MPa)

WT DryBrtl Dry

555 WT Indents 305 plusmn 243 MPa

614 Brtl+ Indents 301 plusmn 202 MPa

KS Test p=0129 t Test p=0464 464 WT Indents

1268 plusmn 718 MPa 479 Brtl+ Indents 1737 plusmn 973 MPa

KS Test plt0001 T Test plt0001

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Indentation in Diabetic Tendon (Poster 0495)

x

z

y

Control and Diabetic Rat Tail Tendon

4-5 rats per group

2-3 fascicles per rat

~70 fibrilstail

4-5 locationsfibril

20 nN (Sneddon)

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Multiscale Diabetes Study (Poster 0495)

0

5

10

15

20

25

300 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 gt25

Perc

enta

ge o

f Gro

up S

ampl

es

Elastic Modulus (MPa)

CD (1530 indents)ZDSD (913 indents)

CD (n=5) 381 plusmn 149 MPa

ZDSD (n=3) 706 plusmn 263 MPa

0

100

200

300

400

500

600

700

800

900

0 2 4 6 8 10

Mic

rosc

ale

Mod

ulus

(MPa

)

Nanoscale Modulus (MPa)

plt0001

r2=0531 p=0040

bull Increased indentation modulus in diabetic tendon

bull Strong relationship between nanoscale indent modulus and microscale tensile modulus

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Indentation Limitations and Weaknesses

Proper probe selection for intended application bull Cantilever must be stiff relative to sample bull Sharp probes may damage sample easily dull

R or α must be determined for each probe bull Properties provided by manufacturer are unreliable bull Vary from probe to probe can dull during indent process

Spring constant must be determined for each probe

bull Value provided by manufacturer is inaccurate bull Thermal tuning OK for ldquosoftrdquo probes poor performance

for stiffer probes bull Sader method (geometry) added mass reference

cantilevers

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d

Indentation Limitations and Weaknesses

Veeco Application Technical note Ben Ohler Practical Advice on the Determination of Cantilever Spring Constants

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Indentation Limitations and Weaknesses

bull Sample processing

bull Sample hydration

bull Proper force range

bull Substrate effects for thin samples

bull Proper modulus model and range of data goodness of fit

Bearing in mind these assumptions and limitations AFM can provide true ldquonanoscalerdquo mechanical properties both in terms of forces and deformations

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ights Reserve

d

A Note on Peak Force QNM for Bruker AFMs bull Mechanical properties mapped pixel-by-pixel at same

resolution as the height image (PeakForce QNM)

Height Modulus BBML - All R

ights Reserve

d

A Note on Peak Force QNM

Correct probe stiffness must be used based on sample Pittenger ndash Veeco Product Bulletin

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ights Reserve

d

A Note on Peak Force QNM Extremely sensitive to calibration (details rarely given) Absolute radius and spring constant known

bull especially important from probe to probe bull radius uarr with indent depth ndash must determine R at depth

you expect to image bull Depth may vary as a function of sample (or at different

regions within the sample)

Relative reference material of ldquoknownrdquo modulus used to determine a ratio of 119896119896 119877119877 bull ldquoknownrdquo modulus may come from tension compression

and not indentation bull R is related to depth indentation depth on sample must

be same as on reference BBML - All R

ights Reserve

d

A Note on Peak Force QNM bull Modulus fit on the fly ndash no ability to monitor fit (or range) bull Local sample topography and sample tilt play a role in

measured parameters bull Assuming all modulus values in an image are correct how to

quantify

512 x 512 pixels = 262144 measurements

n=1 n=262144

n from selected ROIs

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bull Imaging in air on mica ndash substrate effect

bull Same imaging force or indent depth for all

bull For each area were all modulus values pooled

Lamprou et al PLoS ONE 2013 BBML - All R

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d

AFM Applications in Bone Research Other Cool Mechanical Techniques

BBML - All R

ights Reserve

d

ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo Bonds within or between collagen fibrils in bone that rupture to dissipate energy as a toughening mechanism

Thompson et al Nature 2001

Fantner et al Nature Mat 2005

Hansma et al JMNI 2005

Fantner et al Nano Letters 2007 BBML - All R

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d

Other Mechanical Techniques

Jimenez-Palomar J Mech Beh Bio Mat 2012

van der Rijt Macro Bio 2004

Yang J Biomed Mater Res 2007

Yang Biophysical Journal 2008 BBML - All R

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d

Other Cool Techniques bull Piezoresponse Force Microscopy (PFM)

bull Electrochemical AFM (EC-AFM)

bull Force Modulation Microscopy (FMM)

bull Lateral Force Microscopy (LFM)

bull Magnetic Force Microscopy (MFM)

bull Conductive AFM (C-AFM)

bull Force Pulling

bull Cantilever Bending

bull Tensile testing of individual fibrils

bull 3 point bending of fibrils

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Summary Things to rememberhellip

bull Imaging mode imaging medium and probe selection

bull Morphology Measurements bull Sample preparation and hydration state bull Measuring fibril diameter bull Site selection and proper sampling bull Appropriate system calibration

bull Mechanical Measurements

bull Probe selection (stiffness and tip radius) bull Probe calibration (radius angle spring constant) bull Indentation force range and mechanical model bull Hydration in biological samples bull PeakForce QNM

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Acknowledgements BBML

Max Hammond Armando Diaz Gonzalez Zachary Bart Silvia Canelon Creasy Clauser

IU School of Medicine Dr David Burr Dr Matt Allen Dr Stuart Warden Dr Lilian Plotkin

Funding Sources NIHNIDCR 1F32DE018840-01 A1 IUPUI BME Departmental Startup Funds IUPUI Research Support Funds Grant IUPUI Biomechanics and Biomaterials Research Center

NIH NICHD Dr Joan Marini

BBML - All R

ights Reserve

d

Bone Biology and Mechanics Lab (BBML) wwwiupuiedu~bbml Facebook BoneBiologyMechanicsLab

BBML - All R

ights Reserve

d

• Slide Number 1
• General Seminar Outline
• Bonersquos Hierarchical Structure
• Critical Gap in Understanding Collagen
• Methods For Analyzing Collagen Structure
• Methods For Imaging Collagen Structure
• Cryo-TEM
• Atomic Force Microscopy (AFM)
• AFM Image of Pentacene (C22H14)
• AFM Image of Hexabenzocoronene
• Basics of AFM Imaging The Probe
• Basics of AFM Imaging System Components
• van der Waals Potential Energy Curve
• Contact Mode ndash Monitor Deflection
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Peak Force Tapping Mode ndash Monitor Force
• AFM Applications in Bone Research Imaging
• AFM Imaging of Collagen
• Imaging of Osteoclast Activity
• Mineralized collagen deposition in vitro
• Imaging of Canilicular Structure
• AFM Imaging in Bone
• AFM Imaging in Bone
• Removing Mineral in Bone to Expose Collagen
• Removing Mineral in Bone to Expose Collagen
• Demineralization with EDTA
• Demineralization with EDTA
• Demineralization with EDTA
• Issues to Consider Effects of Acid
• Slide Number 32
• Slide Number 33
• Analysis of Collagen Structure in Bone
• Analysis of Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril D-Spacing
• Slide Number 39
• Collagen in Bone Fibril D-Spacing
• Collagen in Bone Fibril D-Spacing
• 2D Fast Fourier Transform Analysis
• 2D Fast Fourier Transform Analysis
• D-Spacing Distribution
• D-Spacing Distribution
• Cumulative Distribution Function
• CDF vs Histogram
• Changes in D-Spacing with Disease
• Changes in D-Spacing with Disease
• Slide Number 50
• Issues to Consider Site Selection and Sampling
• Issues to Consider Site Selection and Sampling
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• AFM calibration ndash Take Away Message
• AFM Applications in Bone ResearchIndentation Mechanics
• AFM-Based Indentation
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Parameters
• Hertz Contact Equation
• Hertz Contact Equation
• Hertz Contact Equation
• Proper Indentation Model is Essential
• Indentation in OI Tendon Study
• OI Tendon Differential Effects of Dehydration
• OI Tendon Differential Effects of Dehydration
• Indentation in Diabetic Tendon (Poster 0495)
• Multiscale Diabetes Study (Poster 0495)
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• A Note on Peak Force QNM for Bruker AFMs
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• Slide Number 83
• Slide Number 84
• AFM Applications in Bone ResearchOther Cool Mechanical Techniques
• ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo
• Other Mechanical Techniques
• Other Cool Techniques
• Summary Things to rememberhellip
• Acknowledgements
• Bone Biology and Mechanics Lab (BBML)

Contact Mode ndash Monitor Deflection

Control system maintains user-defined deflection by vertically moving scanner using piezoelectric actuator

Tip-Sample Separation

Forc

e

Frictional and adhesive forces can damage sample and distort image

Probe in constant contact cantilever deflects like a spring according to Hookersquos law (F = kx)

BBML - All R

ights Reserve

d

Tapping Mode ndash Monitor Amplitude

Tip-Sample SeparationForc

e

Intermittent Contact

BBML - All R

ights Reserve

d

Tapping Mode ndash Monitor Amplitude

ApproachWithdrawal

Tip-Sample SeparationForc

e

Cantilever oscillates at user-defined amplitude near cantilever resonance frequency

-15

-1

-05

0

05

1

15

0 5 10 15

Osc

illat

ion

Am

plitu

de

Time

-15

-1

-05

0

05

1

15

0 5 10 15

Osc

illat

ion

Am

plitu

de

Time

Setpoint

Bump

-15

-1

-05

0

05

1

15

0 5 10 15

Osc

illat

ion

Am

plitu

de

Time

SetpointBumpValley

BBML - All R

ights Reserve

d

Tapping Mode ndash Monitor Amplitude bull Probe in contact for a small fraction of its tapping period

lateral forces are reduced

bull Probe must be tuned at resonance frequency ndash can be challenging for some probes when in fluid

Tuning in Air Single resonance peak Tuning in Fluid Multiple and spurious peaks wwwasylumresearchcom

BBML - All R

ights Reserve

d

Peak Force Tapping Mode ndash Monitor Force

Control system maintains user-defined maximum force

Cantilever oscillates at low frequency (1-2 kHz)

bull No need for tuning

Slow tapping force curve is captured with each tap

ApproachWithdrawal

Tip-Sample SeparationForc

ePeakForce

Direct control of maximum normal force bull Protects tip and sample wear bull Limits indentation depth to a few nm - increases resolution

by limiting contact area bull Mechanical properties mapped pixel-by-pixel at same

resolution as the height image (PeakForce QNM) BBML - All R

ights Reserve

d

AFM Applications in Bone Research Imaging

BBML - All R

ights Reserve

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AFM Imaging of Collagen

bull AFMrsquos adoption in bone research has been slowhellip

bull First AFM-based study in collagen came in 1992 (a previous study in 1989 used Scanning Tunneling Microscopy)

bull Observed fibrillar and monomeric collagen

Chernoff J of Vac Sci Tech 1992

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ights Reserve

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Imaging of Osteoclast Activity

Bozec Ultramicroscopy 2005

Hassenkam Anat Record 2006

Initial observations of cellular activity were qualitative (analyses of collagen morphology were not presented)

Sasaki J Elec Micro 1993

Sasaki CTI 1995

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Mineralized collagen deposition in vitro

Collagen deposition from MLO-A5 cells

Barragan-Adjemian CTI 2006 BBML - All R

ights Reserve

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Imaging of Canilicular Structure

Reilly Annals of BME 2001

Lin J of Microscopy 2010

Average canilicular diameter ~400-500 nm larger than values typically reported from TEM studies

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ights Reserve

d

AFM Imaging in Bone In general we are more interested in collagen in bone

3 microm Diamond Suspension

Unpolished

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ights Reserve

d

AFM Imaging in Bone

The surface is completely mineralizedhellip BBML - All R

ights Reserve

d

Removing Mineral in Bone to Expose Collagen

Dealing with mineral is a challenge to analyzing collagen ndash some mineral must be removed

bull 10 Citric acid (to remove mineral) 65 Sodium Hypochlorite (to remove non-collagenous proteins) Habelitz J Struct Bio 2002

bull 7 or 17 Phosphoric Acid Bozek IEEE 2005 El Feninat J Biomed Mat Res 1998

bull 5 Formic Acid Ge Mat Sci Eng 2007

bull 30 mM EDTA Kindt Nanotechnology 2007

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ights Reserve

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Removing Mineral in Bone to Expose Collagen

Dealing with mineral is a challenge to analyzing collagen ndash some mineral must be removed

bull 30 mM EDTA Kindt Nanotechnology 2007 BBML - A

ll Rights R

eserved

Demineralization with EDTA EDTA - Ethylenediaminetetraacetic Acid

Chelating agent - sequesters divalent cations (notably Ca2+)

bull Treat with 05M EDTA pH 8 (15-20 minutes) bull Vigorously wash with ultrapure water bull Sonicate to release surface-bound mineral ions bull Repeat 3-4 times

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Demineralization with EDTA

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ights Reserve

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Demineralization with EDTA

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ights Reserve

d

Issues to Consider Effects of Acid

Immature collagen crosslinks are very sensitive to acid bull Using acids to demineralize bone may inadvertently

destroy crosslinks distort structure

Fixation of tissue may be necessary prior to demineralization bull Fixation (glutaraldehyde or paraformadehyde) may

preserve structural integrity

bull Fixation effects on collagen structure are not fully known

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ights Reserve

d

100 microm x 100 microm

50 microm x 50 microm

25 microm x 25 microm

10 microm x 10 microm

35 microm x 35 microm

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ights Reserve

d

35 microm x 35 microm

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ights Reserve

d

Analysis of Collagen Structure in Bone Spatial organization of newly formed bone following a surgically-induced injury (rat tibial hole)

Baranauskas J Vac Sci Tech 2001 Baranauskas App Surface Sci 2005

ldquohellipstatistical diameter of the fibers is 159 nm in good agreement with the literature values for type I collagen fibersrdquo

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ights Reserve

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Analysis of Collagen in Bone Fibril Diameter Fibril Diameter easiest to measure property first attempts were a bit crude better with increased resolution

Sasaki J Mat Sci Tech 2002 Thalhammer J Arch Sci 2001

Bozec Ultramicroscopy 2005

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Collagen in Bone Fibril Diameter

Caution must be taken when reporting and interpreting diameters measured from fibrils in bone bull Fibril packing - width may not be fully exposed

Wallace et al Langmuir 2010

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Collagen in Bone Fibril Diameter

Caution must be taken when reporting and interpreting diameters measured from fibrils in bone bull Fibril packing - width may not be fully exposed

Rigozzi ndash J Struct Bio - 2011

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Collagen in Bone Fibril D-Spacing

300 nm 15 nm

N C C N C C N C C N C C

H O H2C CH2 O H

CH2

O OH HHC CH2

CH2

HO

H H

HHN C C

O

H2C CH2

CH2

HN C C

OH

HC CH2

HO

N C C N C C N C C

H O H2C CH2 O

CH2

OH HC CH2

HO

H H

H

CH2 CH2

glycine X - proline Y - hydroxyproline

Hodge and Petruska 1963

Collagen Fibril Axial D-Periodicity BBML - All R

ights Reserve

d

05

101520

0 02 04 06 08 1

Hei

ght (

nm)

Length (microns)

httpremfdartmoutheduimagesindexhtml

Randall Nature and Structure of Collagen 1953

TEM SEM

AFM

D-Spacing = 67 nm

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ights Reserve

d

Collagen in Bone Fibril D-Spacing Many studies have shown observable D-spacing content that near theoretical value

Sasaki 2002 67 plusmn 2 nm Bozek 2005 665 plusmn 14 nm Ge 2007 666 plusmn 38 nm

Habelitz J Structural Bio 2002

A 2002 study in dentin got me interestedhellip

n = 322 wet n = 363 dry

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ights Reserve

d

Collagen in Bone Fibril D-Spacing

bull Internal structure bull Wallace et al Bone 2010 46 1349-1354 bull Wallace et al J Stuct Bio 2011 173 146-152 bull Kemp et al J Stuct Bio 2012 180 428-438 bull Warden et al Endocrinology 2013 154(9)

3178-3187 bull Gallant et al Bone 2014 61 191-200 bull Bart et al Conn Tiss Res 2014 In Press

bull Enzymatic and non-

enzymatic cross-linking bull Hammond et al Bone 2014 60 26-32 bull Diaz Gonzalez et al J Biomech 2014 47(3)

681-686 bull Voytik-Harbin et al In Preparation 2014

D-periodic spacing important structural feature that captures aspects related to

Mouse Femur Mid-diaphysis BBML - All R

ights Reserve

d

2D Fast Fourier Transform Analysis

Harmonics

Fibril D-Spacing

Erickson B Wallace JM et al Biotechnology Journal 2013

Raw AFM Image 2D FFT Power Spectrum

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ights Reserve

d

2D Fast Fourier Transform Analysis

Harmonics

Fibril D-Spacing

Raw AFM Image 2D FFT Power Spectrum

0

20000

40000

60000

80000

100000

120000

0 001 002 003 004 005 006

Am

plitu

de (

microV2 )

Position (1nm)

D-periodic spacing 0015 (1nm) ~ 67 nm

BBML - All R

ights Reserve

d

D-Spacing Distribution

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Overall Mean 670 plusmn 00 nm

BBML - All R

ights Reserve

d

D-Spacing Distribution

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Overall Mean 670 plusmn 00 nm

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

4 Samples Total of n = 255 fibrils Overall Mean 682 plusmn 12 nm

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ights Reserve

d

Cumulative Distribution Function

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

This population 255 fibrils - Each fibril 1255 = 0392 - Data range 628-716 nm

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ights Reserve

d

CDF vs Histogram 62 63 64 65 66 67 68 69 70 71 72 73 74

0

10

20

30

40

50

60

70

80

90

100

0

5

10

15

20

25

30

35

40

45

50

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

HistoCDF

BBML - All R

ights Reserve

d

Changes in D-Spacing with Disease

0

5

10

15

20

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

0

5

10

15

20

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

Wallace et al Bone 2010 46 1349-1354

KS Test plt0001

Control 680 plusmn 26 nm

Estrogen Dep 659 plusmn 31 nm

t-test on mean p=0048

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ights Reserve

d

Changes in D-Spacing with Disease

Wallace et al Bone 2010 46 1349-1354

0

10

20

30

40

50

60

70

80

90

100

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

KS Test plt0001

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ights Reserve

d

Question Model and Tissue Morphology Publication

Tissue Specificity in Mice Male mouse 8 weeks Femur dentin tail tendon

No differences in bone vs dentin both ∆ vs tendon

Wallace et al Langmuir 2011 26(10) 7349-7354

Gender Effects Male and female mice 16 weeks Femur No differences NA

Estrogen Depletion OVX in sheep 5 years + 2 Radius Mean and population shift Wallace et al Bone

2010 46 1349-1354

Estrogen Depletion OVX in female 5 years + 2 Skin (dermis) Significant population shift Erickson et al Biotech

J 2013 8(1) 1174-126

Osteoporosis and Mechanical Loading

OVX in ulnar loading in rats Ulna

No loading effect population shift with OVX

Warden et al Endocrinology 2013 154(9) 3178-3187

Osteogenesis Imperfecta Brtl+ model male mice 2 months Femur

No mean ∆ widening of distribution

Wallace et al J Stuct Bio 2011 173 146-152

Osteogenesis Imperfecta Oimoim model male mice 12 weeks Femur Mean and population shift Bart et al Conn Tiss

Res 2014 In Press

Osteogenesis Imperfecta and Tissue Hydration

Brtl+ model male mice 6 months Tail Tendon Significant population shift Kemp et al J Stuct Bio

2012 180 428-438

Type 2 Diabetes Male ZDSD rats 30 weeks Tibia Significant population shift Hammond et al Bone

2014 60 26-32

Type 2 Diabetes Female ZDSD rats 32 weeks Tail Tendon Significant population shift

Diaz Gonzalez et al J Biomech 2014 47(3)

681-686

Raloxifene (in vivo and in vitro) Female dog treated with Raloxifene Femur Significant population shift Gallant et al Bone

2014 61 191-200 BBML - All R

ights Reserve

d

Issues to Consider Site Selection and Sampling

KS Test plt0001

0023 of bonersquos length

15 m

m =

150

00 micro

m

Wallace et al Langmuir 2011 26(10) 7349-7354

100 microm x 100 microm

35 microm x 35 microm

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ights Reserve

d

Issues to Consider Site Selection and Sampling

60

62

64

66

68

70

72

74

1 (35)

2 (33)

3 (40)

4 (39)

5 (35)

6 (29)

7 (26)

8 (23)

9 (28)

WT (288)

D-P

erio

dic

Gap

Ove

rlap

Spac

ing

(nm

)

Axial Location (Proximal End = 1 Distal End = 9)

( of Fibrils)

91

23

45

87

6

Wallace et al Langmuir 2011 26(10) 7349-7354

288 fibrils for population n=1 for mean comparisons BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

Wet Tendon Dry Tendon Kemp et al J StructBio 2012 180 (3) 428-438

Collagen and bone exist in a hydrated environment in vivo bull Hydration state can impact morphology and mechanics

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ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

Nonsignificant mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet (n=255) 682 plusmn 12 nm

WT Dry (n=257) 686 plusmn 08 nm

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

Brtl DryBrtl Wet

KS Test plt0001

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

WT Case 04 nm mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet 682 plusmn 12 nm

WT Dry 686 plusmn 08 nm

Brtl+ Wet 675 plusmn 14 nm

Brtl+ Dry 689 plusmn 10 nm

Brtl+ Case 14 nm increase with drying (plt0001) bull Entire population shifted upward (plt0001)

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT WetBrtl Wet

KS Test plt0001

Brtl+ distribution shifted downward vs WT when wet

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryBrtl Dry

KS Test plt0001

Brtl+ distribution shifted upward vs WT when dry

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider AFM calibration

bull Absolute distance measurements require accurate piezo calibration

bull AFM systems have large scan ranges (150 microm to lt 1 microm)

bull Scanner non-linearities can introduce substantial error over that range

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

ights Reserve

d

Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

BBML - All R

ights Reserve

d

Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

100 nm 10 80 602 1198 100 nm 7 60 827 1439 100 nm 5 40 814 1427 100 nm 35 20 917 1724 100 nm 2 10 937 1774

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

ights Reserve

d

AFM calibration ndash Take Away Message

Calibration using a scan size and feature size comparable to features of interest is highly recommended

Calibration addresses absolute accuracy not precision (differential sensitivity between measurements)

bull Does not limit ability to differentiate between groups using the same AFM with the same calibration parameters

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ights Reserve

d

AFM Applications in Bone Research Indentation Mechanics

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ights Reserve

d

AFM-Based Indentation

In addition to high resolution imaging AFM can be used to extract nanoscale mechanical data

ApproachWithdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Can

tilev

er D

efle

ctio

n (V

olts

)

1 Ramp into a rigid sample ndash measure the slope

Deflection sensitivity x 119899119899119899119899119881119881

2 Calculate cantilever spring constant

3 Hookersquos law takes deflection (volts) and converts to units of force F = kx

Spring constant k 119873119873119899119899

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Forc

e (n

N)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration Fo

rce

(nN

)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

Tip-Sample Separation (nm)

BBML - All R

ights Reserve

d

AFM-Based Indentation Parameters

Approach Withdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

Peak Force

Area Between Curves Energy Dissipation

Contact Mechanics Fit for Elastic Modulus (E)

Adhesion Force

Indentation Depth (δ)

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

z

r

δ

R

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119877119877 ∙ 12057512057532

Es Sample modulus νs Poissonrsquos ratio R tip radius of curvature δ load point displacement

Relevant Hertz Assumptions bull Strains are small and within the elastic limit bull Indenter is infinitely stiff (only sample deforms) bull Indentation depth ltlt radius of curvature of indenter bull Surfaces are continuous non-conforming (must be flat) bull Load is normal to surface

BBML - All R

ights Reserve

d

Proper Indentation Model is Essential

δ

r

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119903119903 ∙ 12057512057532

δ

α

119865119865 =2120587120587∙

1198641198641199041199041 minus 1205841205841199041199042

∙ tan120572120572 ∙ 1205751205752

Es Sample modulus νs Poissonrsquos ratio R tip radius α opening angle

Hertz Model Spherical Indenter

depth ltlt radius of curvature

Sneddon Model Conical Indenter

depth ge radius of curvature

BBML - All R

ights Reserve

d

Indentation in OI Tendon Study

x

z

yBrtl+ and WT mice 6 month old males

4 tails per group

2-3 fascicles per tail

25-30 fibrils per tail

4-5 locations per fibril

Indent force 20 nN wet (Sneddon) 50 nN dry (Hertz) BBML - A

ll Rights R

eserved

OI Tendon Differential Effects of Dehydration

Dry 1268 plusmn 718 MPa

Wet 305 plusmn 243 MPa

Dry 1737 plusmn 973 MPa

Wet 301 plusmn 202 MPa

Kemp et al J StructBio 2012 180 (3) 428-438

bull Significant stiffening of fibrils with dehydration

bull More pronounced modulus modulus as a function of disease

BBML - All R

ights Reserve

d

OI Tendon Differential Effects of Dehydration

Kemp et al J StructBio 2012 180 (3) 428-438

Diseased phenotype not present when wet phenotype observed when dried

0

10

20

30

40

50

60

0 20 40 60 80 100 120 140 160 180 200

Gro

up S

ampl

es (

)

Indent Modulus (MPa)

WT WetBrtl Wet

0

2

4

6

8

10

12

14

16

18

20

025

050

075

010

0012

5015

0017

5020

0022

5025

0027

5030

0032

5035

0037

5040

0042

5045

0047

5050

00

Gro

up S

ampl

es (

)Indent Modulus (MPa)

WT DryBrtl Dry

555 WT Indents 305 plusmn 243 MPa

614 Brtl+ Indents 301 plusmn 202 MPa

KS Test p=0129 t Test p=0464 464 WT Indents

1268 plusmn 718 MPa 479 Brtl+ Indents 1737 plusmn 973 MPa

KS Test plt0001 T Test plt0001

BBML - All R

ights Reserve

d

Indentation in Diabetic Tendon (Poster 0495)

x

z

y

Control and Diabetic Rat Tail Tendon

4-5 rats per group

2-3 fascicles per rat

~70 fibrilstail

4-5 locationsfibril

20 nN (Sneddon)

BBML - All R

ights Reserve

d

Multiscale Diabetes Study (Poster 0495)

0

5

10

15

20

25

300 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 gt25

Perc

enta

ge o

f Gro

up S

ampl

es

Elastic Modulus (MPa)

CD (1530 indents)ZDSD (913 indents)

CD (n=5) 381 plusmn 149 MPa

ZDSD (n=3) 706 plusmn 263 MPa

0

100

200

300

400

500

600

700

800

900

0 2 4 6 8 10

Mic

rosc

ale

Mod

ulus

(MPa

)

Nanoscale Modulus (MPa)

plt0001

r2=0531 p=0040

bull Increased indentation modulus in diabetic tendon

bull Strong relationship between nanoscale indent modulus and microscale tensile modulus

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

Proper probe selection for intended application bull Cantilever must be stiff relative to sample bull Sharp probes may damage sample easily dull

R or α must be determined for each probe bull Properties provided by manufacturer are unreliable bull Vary from probe to probe can dull during indent process

Spring constant must be determined for each probe

bull Value provided by manufacturer is inaccurate bull Thermal tuning OK for ldquosoftrdquo probes poor performance

for stiffer probes bull Sader method (geometry) added mass reference

cantilevers

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

Veeco Application Technical note Ben Ohler Practical Advice on the Determination of Cantilever Spring Constants

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

bull Sample processing

bull Sample hydration

bull Proper force range

bull Substrate effects for thin samples

bull Proper modulus model and range of data goodness of fit

Bearing in mind these assumptions and limitations AFM can provide true ldquonanoscalerdquo mechanical properties both in terms of forces and deformations

BBML - All R

ights Reserve

d

A Note on Peak Force QNM for Bruker AFMs bull Mechanical properties mapped pixel-by-pixel at same

resolution as the height image (PeakForce QNM)

Height Modulus BBML - All R

ights Reserve

d

A Note on Peak Force QNM

Correct probe stiffness must be used based on sample Pittenger ndash Veeco Product Bulletin

BBML - All R

ights Reserve

d

A Note on Peak Force QNM Extremely sensitive to calibration (details rarely given) Absolute radius and spring constant known

bull especially important from probe to probe bull radius uarr with indent depth ndash must determine R at depth

you expect to image bull Depth may vary as a function of sample (or at different

regions within the sample)

Relative reference material of ldquoknownrdquo modulus used to determine a ratio of 119896119896 119877119877 bull ldquoknownrdquo modulus may come from tension compression

and not indentation bull R is related to depth indentation depth on sample must

be same as on reference BBML - All R

ights Reserve

d

A Note on Peak Force QNM bull Modulus fit on the fly ndash no ability to monitor fit (or range) bull Local sample topography and sample tilt play a role in

measured parameters bull Assuming all modulus values in an image are correct how to

quantify

512 x 512 pixels = 262144 measurements

n=1 n=262144

n from selected ROIs

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bull Imaging in air on mica ndash substrate effect

bull Same imaging force or indent depth for all

bull For each area were all modulus values pooled

Lamprou et al PLoS ONE 2013 BBML - All R

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d

AFM Applications in Bone Research Other Cool Mechanical Techniques

BBML - All R

ights Reserve

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ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo Bonds within or between collagen fibrils in bone that rupture to dissipate energy as a toughening mechanism

Thompson et al Nature 2001

Fantner et al Nature Mat 2005

Hansma et al JMNI 2005

Fantner et al Nano Letters 2007 BBML - All R

ights Reserve

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Other Mechanical Techniques

Jimenez-Palomar J Mech Beh Bio Mat 2012

van der Rijt Macro Bio 2004

Yang J Biomed Mater Res 2007

Yang Biophysical Journal 2008 BBML - All R

ights Reserve

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Other Cool Techniques bull Piezoresponse Force Microscopy (PFM)

bull Electrochemical AFM (EC-AFM)

bull Force Modulation Microscopy (FMM)

bull Lateral Force Microscopy (LFM)

bull Magnetic Force Microscopy (MFM)

bull Conductive AFM (C-AFM)

bull Force Pulling

bull Cantilever Bending

bull Tensile testing of individual fibrils

bull 3 point bending of fibrils

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Summary Things to rememberhellip

bull Imaging mode imaging medium and probe selection

bull Morphology Measurements bull Sample preparation and hydration state bull Measuring fibril diameter bull Site selection and proper sampling bull Appropriate system calibration

bull Mechanical Measurements

bull Probe selection (stiffness and tip radius) bull Probe calibration (radius angle spring constant) bull Indentation force range and mechanical model bull Hydration in biological samples bull PeakForce QNM

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Acknowledgements BBML

Max Hammond Armando Diaz Gonzalez Zachary Bart Silvia Canelon Creasy Clauser

IU School of Medicine Dr David Burr Dr Matt Allen Dr Stuart Warden Dr Lilian Plotkin

Funding Sources NIHNIDCR 1F32DE018840-01 A1 IUPUI BME Departmental Startup Funds IUPUI Research Support Funds Grant IUPUI Biomechanics and Biomaterials Research Center

NIH NICHD Dr Joan Marini

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Bone Biology and Mechanics Lab (BBML) wwwiupuiedu~bbml Facebook BoneBiologyMechanicsLab

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ights Reserve

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• Slide Number 1
• General Seminar Outline
• Bonersquos Hierarchical Structure
• Critical Gap in Understanding Collagen
• Methods For Analyzing Collagen Structure
• Methods For Imaging Collagen Structure
• Cryo-TEM
• Atomic Force Microscopy (AFM)
• AFM Image of Pentacene (C22H14)
• AFM Image of Hexabenzocoronene
• Basics of AFM Imaging The Probe
• Basics of AFM Imaging System Components
• van der Waals Potential Energy Curve
• Contact Mode ndash Monitor Deflection
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Peak Force Tapping Mode ndash Monitor Force
• AFM Applications in Bone Research Imaging
• AFM Imaging of Collagen
• Imaging of Osteoclast Activity
• Mineralized collagen deposition in vitro
• Imaging of Canilicular Structure
• AFM Imaging in Bone
• AFM Imaging in Bone
• Removing Mineral in Bone to Expose Collagen
• Removing Mineral in Bone to Expose Collagen
• Demineralization with EDTA
• Demineralization with EDTA
• Demineralization with EDTA
• Issues to Consider Effects of Acid
• Slide Number 32
• Slide Number 33
• Analysis of Collagen Structure in Bone
• Analysis of Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril D-Spacing
• Slide Number 39
• Collagen in Bone Fibril D-Spacing
• Collagen in Bone Fibril D-Spacing
• 2D Fast Fourier Transform Analysis
• 2D Fast Fourier Transform Analysis
• D-Spacing Distribution
• D-Spacing Distribution
• Cumulative Distribution Function
• CDF vs Histogram
• Changes in D-Spacing with Disease
• Changes in D-Spacing with Disease
• Slide Number 50
• Issues to Consider Site Selection and Sampling
• Issues to Consider Site Selection and Sampling
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• AFM calibration ndash Take Away Message
• AFM Applications in Bone ResearchIndentation Mechanics
• AFM-Based Indentation
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Parameters
• Hertz Contact Equation
• Hertz Contact Equation
• Hertz Contact Equation
• Proper Indentation Model is Essential
• Indentation in OI Tendon Study
• OI Tendon Differential Effects of Dehydration
• OI Tendon Differential Effects of Dehydration
• Indentation in Diabetic Tendon (Poster 0495)
• Multiscale Diabetes Study (Poster 0495)
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• A Note on Peak Force QNM for Bruker AFMs
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• Slide Number 83
• Slide Number 84
• AFM Applications in Bone ResearchOther Cool Mechanical Techniques
• ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo
• Other Mechanical Techniques
• Other Cool Techniques
• Summary Things to rememberhellip
• Acknowledgements
• Bone Biology and Mechanics Lab (BBML)

Tapping Mode ndash Monitor Amplitude

Tip-Sample SeparationForc

e

Intermittent Contact

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Tapping Mode ndash Monitor Amplitude

ApproachWithdrawal

Tip-Sample SeparationForc

e

Cantilever oscillates at user-defined amplitude near cantilever resonance frequency

-15

-1

-05

0

05

1

15

0 5 10 15

Osc

illat

ion

Am

plitu

de

Time

-15

-1

-05

0

05

1

15

0 5 10 15

Osc

illat

ion

Am

plitu

de

Time

Setpoint

Bump

-15

-1

-05

0

05

1

15

0 5 10 15

Osc

illat

ion

Am

plitu

de

Time

SetpointBumpValley

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Tapping Mode ndash Monitor Amplitude bull Probe in contact for a small fraction of its tapping period

lateral forces are reduced

bull Probe must be tuned at resonance frequency ndash can be challenging for some probes when in fluid

Tuning in Air Single resonance peak Tuning in Fluid Multiple and spurious peaks wwwasylumresearchcom

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Peak Force Tapping Mode ndash Monitor Force

Control system maintains user-defined maximum force

Cantilever oscillates at low frequency (1-2 kHz)

bull No need for tuning

Slow tapping force curve is captured with each tap

ApproachWithdrawal

Tip-Sample SeparationForc

ePeakForce

Direct control of maximum normal force bull Protects tip and sample wear bull Limits indentation depth to a few nm - increases resolution

by limiting contact area bull Mechanical properties mapped pixel-by-pixel at same

resolution as the height image (PeakForce QNM) BBML - All R

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d

AFM Applications in Bone Research Imaging

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AFM Imaging of Collagen

bull AFMrsquos adoption in bone research has been slowhellip

bull First AFM-based study in collagen came in 1992 (a previous study in 1989 used Scanning Tunneling Microscopy)

bull Observed fibrillar and monomeric collagen

Chernoff J of Vac Sci Tech 1992

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Imaging of Osteoclast Activity

Bozec Ultramicroscopy 2005

Hassenkam Anat Record 2006

Initial observations of cellular activity were qualitative (analyses of collagen morphology were not presented)

Sasaki J Elec Micro 1993

Sasaki CTI 1995

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Mineralized collagen deposition in vitro

Collagen deposition from MLO-A5 cells

Barragan-Adjemian CTI 2006 BBML - All R

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Imaging of Canilicular Structure

Reilly Annals of BME 2001

Lin J of Microscopy 2010

Average canilicular diameter ~400-500 nm larger than values typically reported from TEM studies

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AFM Imaging in Bone In general we are more interested in collagen in bone

3 microm Diamond Suspension

Unpolished

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AFM Imaging in Bone

The surface is completely mineralizedhellip BBML - All R

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Removing Mineral in Bone to Expose Collagen

Dealing with mineral is a challenge to analyzing collagen ndash some mineral must be removed

bull 10 Citric acid (to remove mineral) 65 Sodium Hypochlorite (to remove non-collagenous proteins) Habelitz J Struct Bio 2002

bull 7 or 17 Phosphoric Acid Bozek IEEE 2005 El Feninat J Biomed Mat Res 1998

bull 5 Formic Acid Ge Mat Sci Eng 2007

bull 30 mM EDTA Kindt Nanotechnology 2007

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Removing Mineral in Bone to Expose Collagen

Dealing with mineral is a challenge to analyzing collagen ndash some mineral must be removed

bull 30 mM EDTA Kindt Nanotechnology 2007 BBML - A

ll Rights R

eserved

Demineralization with EDTA EDTA - Ethylenediaminetetraacetic Acid

Chelating agent - sequesters divalent cations (notably Ca2+)

bull Treat with 05M EDTA pH 8 (15-20 minutes) bull Vigorously wash with ultrapure water bull Sonicate to release surface-bound mineral ions bull Repeat 3-4 times

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Demineralization with EDTA

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Demineralization with EDTA

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Issues to Consider Effects of Acid

Immature collagen crosslinks are very sensitive to acid bull Using acids to demineralize bone may inadvertently

destroy crosslinks distort structure

Fixation of tissue may be necessary prior to demineralization bull Fixation (glutaraldehyde or paraformadehyde) may

preserve structural integrity

bull Fixation effects on collagen structure are not fully known

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100 microm x 100 microm

50 microm x 50 microm

25 microm x 25 microm

10 microm x 10 microm

35 microm x 35 microm

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35 microm x 35 microm

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Analysis of Collagen Structure in Bone Spatial organization of newly formed bone following a surgically-induced injury (rat tibial hole)

Baranauskas J Vac Sci Tech 2001 Baranauskas App Surface Sci 2005

ldquohellipstatistical diameter of the fibers is 159 nm in good agreement with the literature values for type I collagen fibersrdquo

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Analysis of Collagen in Bone Fibril Diameter Fibril Diameter easiest to measure property first attempts were a bit crude better with increased resolution

Sasaki J Mat Sci Tech 2002 Thalhammer J Arch Sci 2001

Bozec Ultramicroscopy 2005

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Collagen in Bone Fibril Diameter

Caution must be taken when reporting and interpreting diameters measured from fibrils in bone bull Fibril packing - width may not be fully exposed

Wallace et al Langmuir 2010

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Collagen in Bone Fibril Diameter

Caution must be taken when reporting and interpreting diameters measured from fibrils in bone bull Fibril packing - width may not be fully exposed

Rigozzi ndash J Struct Bio - 2011

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Collagen in Bone Fibril D-Spacing

300 nm 15 nm

N C C N C C N C C N C C

H O H2C CH2 O H

CH2

O OH HHC CH2

CH2

HO

H H

HHN C C

O

H2C CH2

CH2

HN C C

OH

HC CH2

HO

N C C N C C N C C

H O H2C CH2 O

CH2

OH HC CH2

HO

H H

H

CH2 CH2

glycine X - proline Y - hydroxyproline

Hodge and Petruska 1963

Collagen Fibril Axial D-Periodicity BBML - All R

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d

05

101520

0 02 04 06 08 1

Hei

ght (

nm)

Length (microns)

httpremfdartmoutheduimagesindexhtml

Randall Nature and Structure of Collagen 1953

TEM SEM

AFM

D-Spacing = 67 nm

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Collagen in Bone Fibril D-Spacing Many studies have shown observable D-spacing content that near theoretical value

Sasaki 2002 67 plusmn 2 nm Bozek 2005 665 plusmn 14 nm Ge 2007 666 plusmn 38 nm

Habelitz J Structural Bio 2002

A 2002 study in dentin got me interestedhellip

n = 322 wet n = 363 dry

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Collagen in Bone Fibril D-Spacing

bull Internal structure bull Wallace et al Bone 2010 46 1349-1354 bull Wallace et al J Stuct Bio 2011 173 146-152 bull Kemp et al J Stuct Bio 2012 180 428-438 bull Warden et al Endocrinology 2013 154(9)

3178-3187 bull Gallant et al Bone 2014 61 191-200 bull Bart et al Conn Tiss Res 2014 In Press

bull Enzymatic and non-

enzymatic cross-linking bull Hammond et al Bone 2014 60 26-32 bull Diaz Gonzalez et al J Biomech 2014 47(3)

681-686 bull Voytik-Harbin et al In Preparation 2014

D-periodic spacing important structural feature that captures aspects related to

Mouse Femur Mid-diaphysis BBML - All R

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2D Fast Fourier Transform Analysis

Harmonics

Fibril D-Spacing

Erickson B Wallace JM et al Biotechnology Journal 2013

Raw AFM Image 2D FFT Power Spectrum

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2D Fast Fourier Transform Analysis

Harmonics

Fibril D-Spacing

Raw AFM Image 2D FFT Power Spectrum

0

20000

40000

60000

80000

100000

120000

0 001 002 003 004 005 006

Am

plitu

de (

microV2 )

Position (1nm)

D-periodic spacing 0015 (1nm) ~ 67 nm

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D-Spacing Distribution

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Overall Mean 670 plusmn 00 nm

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D-Spacing Distribution

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Overall Mean 670 plusmn 00 nm

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

4 Samples Total of n = 255 fibrils Overall Mean 682 plusmn 12 nm

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Cumulative Distribution Function

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

This population 255 fibrils - Each fibril 1255 = 0392 - Data range 628-716 nm

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CDF vs Histogram 62 63 64 65 66 67 68 69 70 71 72 73 74

0

10

20

30

40

50

60

70

80

90

100

0

5

10

15

20

25

30

35

40

45

50

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

HistoCDF

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Changes in D-Spacing with Disease

0

5

10

15

20

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

0

5

10

15

20

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

Wallace et al Bone 2010 46 1349-1354

KS Test plt0001

Control 680 plusmn 26 nm

Estrogen Dep 659 plusmn 31 nm

t-test on mean p=0048

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Changes in D-Spacing with Disease

Wallace et al Bone 2010 46 1349-1354

0

10

20

30

40

50

60

70

80

90

100

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

KS Test plt0001

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Question Model and Tissue Morphology Publication

Tissue Specificity in Mice Male mouse 8 weeks Femur dentin tail tendon

No differences in bone vs dentin both ∆ vs tendon

Wallace et al Langmuir 2011 26(10) 7349-7354

Gender Effects Male and female mice 16 weeks Femur No differences NA

Estrogen Depletion OVX in sheep 5 years + 2 Radius Mean and population shift Wallace et al Bone

2010 46 1349-1354

Estrogen Depletion OVX in female 5 years + 2 Skin (dermis) Significant population shift Erickson et al Biotech

J 2013 8(1) 1174-126

Osteoporosis and Mechanical Loading

OVX in ulnar loading in rats Ulna

No loading effect population shift with OVX

Warden et al Endocrinology 2013 154(9) 3178-3187

Osteogenesis Imperfecta Brtl+ model male mice 2 months Femur

No mean ∆ widening of distribution

Wallace et al J Stuct Bio 2011 173 146-152

Osteogenesis Imperfecta Oimoim model male mice 12 weeks Femur Mean and population shift Bart et al Conn Tiss

Res 2014 In Press

Osteogenesis Imperfecta and Tissue Hydration

Brtl+ model male mice 6 months Tail Tendon Significant population shift Kemp et al J Stuct Bio

2012 180 428-438

Type 2 Diabetes Male ZDSD rats 30 weeks Tibia Significant population shift Hammond et al Bone

2014 60 26-32

Type 2 Diabetes Female ZDSD rats 32 weeks Tail Tendon Significant population shift

Diaz Gonzalez et al J Biomech 2014 47(3)

681-686

Raloxifene (in vivo and in vitro) Female dog treated with Raloxifene Femur Significant population shift Gallant et al Bone

2014 61 191-200 BBML - All R

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d

Issues to Consider Site Selection and Sampling

KS Test plt0001

0023 of bonersquos length

15 m

m =

150

00 micro

m

Wallace et al Langmuir 2011 26(10) 7349-7354

100 microm x 100 microm

35 microm x 35 microm

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ights Reserve

d

Issues to Consider Site Selection and Sampling

60

62

64

66

68

70

72

74

1 (35)

2 (33)

3 (40)

4 (39)

5 (35)

6 (29)

7 (26)

8 (23)

9 (28)

WT (288)

D-P

erio

dic

Gap

Ove

rlap

Spac

ing

(nm

)

Axial Location (Proximal End = 1 Distal End = 9)

( of Fibrils)

91

23

45

87

6

Wallace et al Langmuir 2011 26(10) 7349-7354

288 fibrils for population n=1 for mean comparisons BBML - All R

ights Reserve

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Issues to Consider Effects of Hydration

Wet Tendon Dry Tendon Kemp et al J StructBio 2012 180 (3) 428-438

Collagen and bone exist in a hydrated environment in vivo bull Hydration state can impact morphology and mechanics

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ights Reserve

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Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

Nonsignificant mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet (n=255) 682 plusmn 12 nm

WT Dry (n=257) 686 plusmn 08 nm

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

Brtl DryBrtl Wet

KS Test plt0001

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

WT Case 04 nm mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet 682 plusmn 12 nm

WT Dry 686 plusmn 08 nm

Brtl+ Wet 675 plusmn 14 nm

Brtl+ Dry 689 plusmn 10 nm

Brtl+ Case 14 nm increase with drying (plt0001) bull Entire population shifted upward (plt0001)

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

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d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT WetBrtl Wet

KS Test plt0001

Brtl+ distribution shifted downward vs WT when wet

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryBrtl Dry

KS Test plt0001

Brtl+ distribution shifted upward vs WT when dry

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider AFM calibration

bull Absolute distance measurements require accurate piezo calibration

bull AFM systems have large scan ranges (150 microm to lt 1 microm)

bull Scanner non-linearities can introduce substantial error over that range

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

ights Reserve

d

Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

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Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

100 nm 10 80 602 1198 100 nm 7 60 827 1439 100 nm 5 40 814 1427 100 nm 35 20 917 1724 100 nm 2 10 937 1774

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

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d

AFM calibration ndash Take Away Message

Calibration using a scan size and feature size comparable to features of interest is highly recommended

Calibration addresses absolute accuracy not precision (differential sensitivity between measurements)

bull Does not limit ability to differentiate between groups using the same AFM with the same calibration parameters

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AFM Applications in Bone Research Indentation Mechanics

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ights Reserve

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AFM-Based Indentation

In addition to high resolution imaging AFM can be used to extract nanoscale mechanical data

ApproachWithdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Can

tilev

er D

efle

ctio

n (V

olts

)

1 Ramp into a rigid sample ndash measure the slope

Deflection sensitivity x 119899119899119899119899119881119881

2 Calculate cantilever spring constant

3 Hookersquos law takes deflection (volts) and converts to units of force F = kx

Spring constant k 119873119873119899119899

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Forc

e (n

N)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration Fo

rce

(nN

)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

Tip-Sample Separation (nm)

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ights Reserve

d

AFM-Based Indentation Parameters

Approach Withdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

Peak Force

Area Between Curves Energy Dissipation

Contact Mechanics Fit for Elastic Modulus (E)

Adhesion Force

Indentation Depth (δ)

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

z

r

δ

R

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119877119877 ∙ 12057512057532

Es Sample modulus νs Poissonrsquos ratio R tip radius of curvature δ load point displacement

Relevant Hertz Assumptions bull Strains are small and within the elastic limit bull Indenter is infinitely stiff (only sample deforms) bull Indentation depth ltlt radius of curvature of indenter bull Surfaces are continuous non-conforming (must be flat) bull Load is normal to surface

BBML - All R

ights Reserve

d

Proper Indentation Model is Essential

δ

r

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119903119903 ∙ 12057512057532

δ

α

119865119865 =2120587120587∙

1198641198641199041199041 minus 1205841205841199041199042

∙ tan120572120572 ∙ 1205751205752

Es Sample modulus νs Poissonrsquos ratio R tip radius α opening angle

Hertz Model Spherical Indenter

depth ltlt radius of curvature

Sneddon Model Conical Indenter

depth ge radius of curvature

BBML - All R

ights Reserve

d

Indentation in OI Tendon Study

x

z

yBrtl+ and WT mice 6 month old males

4 tails per group

2-3 fascicles per tail

25-30 fibrils per tail

4-5 locations per fibril

Indent force 20 nN wet (Sneddon) 50 nN dry (Hertz) BBML - A

ll Rights R

eserved

OI Tendon Differential Effects of Dehydration

Dry 1268 plusmn 718 MPa

Wet 305 plusmn 243 MPa

Dry 1737 plusmn 973 MPa

Wet 301 plusmn 202 MPa

Kemp et al J StructBio 2012 180 (3) 428-438

bull Significant stiffening of fibrils with dehydration

bull More pronounced modulus modulus as a function of disease

BBML - All R

ights Reserve

d

OI Tendon Differential Effects of Dehydration

Kemp et al J StructBio 2012 180 (3) 428-438

Diseased phenotype not present when wet phenotype observed when dried

0

10

20

30

40

50

60

0 20 40 60 80 100 120 140 160 180 200

Gro

up S

ampl

es (

)

Indent Modulus (MPa)

WT WetBrtl Wet

0

2

4

6

8

10

12

14

16

18

20

025

050

075

010

0012

5015

0017

5020

0022

5025

0027

5030

0032

5035

0037

5040

0042

5045

0047

5050

00

Gro

up S

ampl

es (

)Indent Modulus (MPa)

WT DryBrtl Dry

555 WT Indents 305 plusmn 243 MPa

614 Brtl+ Indents 301 plusmn 202 MPa

KS Test p=0129 t Test p=0464 464 WT Indents

1268 plusmn 718 MPa 479 Brtl+ Indents 1737 plusmn 973 MPa

KS Test plt0001 T Test plt0001

BBML - All R

ights Reserve

d

Indentation in Diabetic Tendon (Poster 0495)

x

z

y

Control and Diabetic Rat Tail Tendon

4-5 rats per group

2-3 fascicles per rat

~70 fibrilstail

4-5 locationsfibril

20 nN (Sneddon)

BBML - All R

ights Reserve

d

Multiscale Diabetes Study (Poster 0495)

0

5

10

15

20

25

300 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 gt25

Perc

enta

ge o

f Gro

up S

ampl

es

Elastic Modulus (MPa)

CD (1530 indents)ZDSD (913 indents)

CD (n=5) 381 plusmn 149 MPa

ZDSD (n=3) 706 plusmn 263 MPa

0

100

200

300

400

500

600

700

800

900

0 2 4 6 8 10

Mic

rosc

ale

Mod

ulus

(MPa

)

Nanoscale Modulus (MPa)

plt0001

r2=0531 p=0040

bull Increased indentation modulus in diabetic tendon

bull Strong relationship between nanoscale indent modulus and microscale tensile modulus

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

Proper probe selection for intended application bull Cantilever must be stiff relative to sample bull Sharp probes may damage sample easily dull

R or α must be determined for each probe bull Properties provided by manufacturer are unreliable bull Vary from probe to probe can dull during indent process

Spring constant must be determined for each probe

bull Value provided by manufacturer is inaccurate bull Thermal tuning OK for ldquosoftrdquo probes poor performance

for stiffer probes bull Sader method (geometry) added mass reference

cantilevers

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

Veeco Application Technical note Ben Ohler Practical Advice on the Determination of Cantilever Spring Constants

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

bull Sample processing

bull Sample hydration

bull Proper force range

bull Substrate effects for thin samples

bull Proper modulus model and range of data goodness of fit

Bearing in mind these assumptions and limitations AFM can provide true ldquonanoscalerdquo mechanical properties both in terms of forces and deformations

BBML - All R

ights Reserve

d

A Note on Peak Force QNM for Bruker AFMs bull Mechanical properties mapped pixel-by-pixel at same

resolution as the height image (PeakForce QNM)

Height Modulus BBML - All R

ights Reserve

d

A Note on Peak Force QNM

Correct probe stiffness must be used based on sample Pittenger ndash Veeco Product Bulletin

BBML - All R

ights Reserve

d

A Note on Peak Force QNM Extremely sensitive to calibration (details rarely given) Absolute radius and spring constant known

bull especially important from probe to probe bull radius uarr with indent depth ndash must determine R at depth

you expect to image bull Depth may vary as a function of sample (or at different

regions within the sample)

Relative reference material of ldquoknownrdquo modulus used to determine a ratio of 119896119896 119877119877 bull ldquoknownrdquo modulus may come from tension compression

and not indentation bull R is related to depth indentation depth on sample must

be same as on reference BBML - All R

ights Reserve

d

A Note on Peak Force QNM bull Modulus fit on the fly ndash no ability to monitor fit (or range) bull Local sample topography and sample tilt play a role in

measured parameters bull Assuming all modulus values in an image are correct how to

quantify

512 x 512 pixels = 262144 measurements

n=1 n=262144

n from selected ROIs

BBML - All R

ights Reserve

d

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ights Reserve

d

bull Imaging in air on mica ndash substrate effect

bull Same imaging force or indent depth for all

bull For each area were all modulus values pooled

Lamprou et al PLoS ONE 2013 BBML - All R

ights Reserve

d

AFM Applications in Bone Research Other Cool Mechanical Techniques

BBML - All R

ights Reserve

d

ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo Bonds within or between collagen fibrils in bone that rupture to dissipate energy as a toughening mechanism

Thompson et al Nature 2001

Fantner et al Nature Mat 2005

Hansma et al JMNI 2005

Fantner et al Nano Letters 2007 BBML - All R

ights Reserve

d

Other Mechanical Techniques

Jimenez-Palomar J Mech Beh Bio Mat 2012

van der Rijt Macro Bio 2004

Yang J Biomed Mater Res 2007

Yang Biophysical Journal 2008 BBML - All R

ights Reserve

d

Other Cool Techniques bull Piezoresponse Force Microscopy (PFM)

bull Electrochemical AFM (EC-AFM)

bull Force Modulation Microscopy (FMM)

bull Lateral Force Microscopy (LFM)

bull Magnetic Force Microscopy (MFM)

bull Conductive AFM (C-AFM)

bull Force Pulling

bull Cantilever Bending

bull Tensile testing of individual fibrils

bull 3 point bending of fibrils

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ights Reserve

d

Summary Things to rememberhellip

bull Imaging mode imaging medium and probe selection

bull Morphology Measurements bull Sample preparation and hydration state bull Measuring fibril diameter bull Site selection and proper sampling bull Appropriate system calibration

bull Mechanical Measurements

bull Probe selection (stiffness and tip radius) bull Probe calibration (radius angle spring constant) bull Indentation force range and mechanical model bull Hydration in biological samples bull PeakForce QNM

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ights Reserve

d

Acknowledgements BBML

Max Hammond Armando Diaz Gonzalez Zachary Bart Silvia Canelon Creasy Clauser

IU School of Medicine Dr David Burr Dr Matt Allen Dr Stuart Warden Dr Lilian Plotkin

Funding Sources NIHNIDCR 1F32DE018840-01 A1 IUPUI BME Departmental Startup Funds IUPUI Research Support Funds Grant IUPUI Biomechanics and Biomaterials Research Center

NIH NICHD Dr Joan Marini

BBML - All R

ights Reserve

d

Bone Biology and Mechanics Lab (BBML) wwwiupuiedu~bbml Facebook BoneBiologyMechanicsLab

BBML - All R

ights Reserve

d

• Slide Number 1
• General Seminar Outline
• Bonersquos Hierarchical Structure
• Critical Gap in Understanding Collagen
• Methods For Analyzing Collagen Structure
• Methods For Imaging Collagen Structure
• Cryo-TEM
• Atomic Force Microscopy (AFM)
• AFM Image of Pentacene (C22H14)
• AFM Image of Hexabenzocoronene
• Basics of AFM Imaging The Probe
• Basics of AFM Imaging System Components
• van der Waals Potential Energy Curve
• Contact Mode ndash Monitor Deflection
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Peak Force Tapping Mode ndash Monitor Force
• AFM Applications in Bone Research Imaging
• AFM Imaging of Collagen
• Imaging of Osteoclast Activity
• Mineralized collagen deposition in vitro
• Imaging of Canilicular Structure
• AFM Imaging in Bone
• AFM Imaging in Bone
• Removing Mineral in Bone to Expose Collagen
• Removing Mineral in Bone to Expose Collagen
• Demineralization with EDTA
• Demineralization with EDTA
• Demineralization with EDTA
• Issues to Consider Effects of Acid
• Slide Number 32
• Slide Number 33
• Analysis of Collagen Structure in Bone
• Analysis of Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril D-Spacing
• Slide Number 39
• Collagen in Bone Fibril D-Spacing
• Collagen in Bone Fibril D-Spacing
• 2D Fast Fourier Transform Analysis
• 2D Fast Fourier Transform Analysis
• D-Spacing Distribution
• D-Spacing Distribution
• Cumulative Distribution Function
• CDF vs Histogram
• Changes in D-Spacing with Disease
• Changes in D-Spacing with Disease
• Slide Number 50
• Issues to Consider Site Selection and Sampling
• Issues to Consider Site Selection and Sampling
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• AFM calibration ndash Take Away Message
• AFM Applications in Bone ResearchIndentation Mechanics
• AFM-Based Indentation
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Parameters
• Hertz Contact Equation
• Hertz Contact Equation
• Hertz Contact Equation
• Proper Indentation Model is Essential
• Indentation in OI Tendon Study
• OI Tendon Differential Effects of Dehydration
• OI Tendon Differential Effects of Dehydration
• Indentation in Diabetic Tendon (Poster 0495)
• Multiscale Diabetes Study (Poster 0495)
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• A Note on Peak Force QNM for Bruker AFMs
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• Slide Number 83
• Slide Number 84
• AFM Applications in Bone ResearchOther Cool Mechanical Techniques
• ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo
• Other Mechanical Techniques
• Other Cool Techniques
• Summary Things to rememberhellip
• Acknowledgements
• Bone Biology and Mechanics Lab (BBML)

Tapping Mode ndash Monitor Amplitude

ApproachWithdrawal

Tip-Sample SeparationForc

e

Cantilever oscillates at user-defined amplitude near cantilever resonance frequency

-15

-1

-05

0

05

1

15

0 5 10 15

Osc

illat

ion

Am

plitu

de

Time

-15

-1

-05

0

05

1

15

0 5 10 15

Osc

illat

ion

Am

plitu

de

Time

Setpoint

Bump

-15

-1

-05

0

05

1

15

0 5 10 15

Osc

illat

ion

Am

plitu

de

Time

SetpointBumpValley

BBML - All R

ights Reserve

d

Tapping Mode ndash Monitor Amplitude bull Probe in contact for a small fraction of its tapping period

lateral forces are reduced

bull Probe must be tuned at resonance frequency ndash can be challenging for some probes when in fluid

Tuning in Air Single resonance peak Tuning in Fluid Multiple and spurious peaks wwwasylumresearchcom

BBML - All R

ights Reserve

d

Peak Force Tapping Mode ndash Monitor Force

Control system maintains user-defined maximum force

Cantilever oscillates at low frequency (1-2 kHz)

bull No need for tuning

Slow tapping force curve is captured with each tap

ApproachWithdrawal

Tip-Sample SeparationForc

ePeakForce

Direct control of maximum normal force bull Protects tip and sample wear bull Limits indentation depth to a few nm - increases resolution

by limiting contact area bull Mechanical properties mapped pixel-by-pixel at same

resolution as the height image (PeakForce QNM) BBML - All R

ights Reserve

d

AFM Applications in Bone Research Imaging

BBML - All R

ights Reserve

d

AFM Imaging of Collagen

bull AFMrsquos adoption in bone research has been slowhellip

bull First AFM-based study in collagen came in 1992 (a previous study in 1989 used Scanning Tunneling Microscopy)

bull Observed fibrillar and monomeric collagen

Chernoff J of Vac Sci Tech 1992

BBML - All R

ights Reserve

d

Imaging of Osteoclast Activity

Bozec Ultramicroscopy 2005

Hassenkam Anat Record 2006

Initial observations of cellular activity were qualitative (analyses of collagen morphology were not presented)

Sasaki J Elec Micro 1993

Sasaki CTI 1995

BBML - All R

ights Reserve

d

Mineralized collagen deposition in vitro

Collagen deposition from MLO-A5 cells

Barragan-Adjemian CTI 2006 BBML - All R

ights Reserve

d

Imaging of Canilicular Structure

Reilly Annals of BME 2001

Lin J of Microscopy 2010

Average canilicular diameter ~400-500 nm larger than values typically reported from TEM studies

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ights Reserve

d

AFM Imaging in Bone In general we are more interested in collagen in bone

3 microm Diamond Suspension

Unpolished

BBML - All R

ights Reserve

d

AFM Imaging in Bone

The surface is completely mineralizedhellip BBML - All R

ights Reserve

d

Removing Mineral in Bone to Expose Collagen

Dealing with mineral is a challenge to analyzing collagen ndash some mineral must be removed

bull 10 Citric acid (to remove mineral) 65 Sodium Hypochlorite (to remove non-collagenous proteins) Habelitz J Struct Bio 2002

bull 7 or 17 Phosphoric Acid Bozek IEEE 2005 El Feninat J Biomed Mat Res 1998

bull 5 Formic Acid Ge Mat Sci Eng 2007

bull 30 mM EDTA Kindt Nanotechnology 2007

BBML - All R

ights Reserve

d

Removing Mineral in Bone to Expose Collagen

Dealing with mineral is a challenge to analyzing collagen ndash some mineral must be removed

bull 30 mM EDTA Kindt Nanotechnology 2007 BBML - A

ll Rights R

eserved

Demineralization with EDTA EDTA - Ethylenediaminetetraacetic Acid

Chelating agent - sequesters divalent cations (notably Ca2+)

bull Treat with 05M EDTA pH 8 (15-20 minutes) bull Vigorously wash with ultrapure water bull Sonicate to release surface-bound mineral ions bull Repeat 3-4 times

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ights Reserve

d

Demineralization with EDTA

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ights Reserve

d

Demineralization with EDTA

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ights Reserve

d

Issues to Consider Effects of Acid

Immature collagen crosslinks are very sensitive to acid bull Using acids to demineralize bone may inadvertently

destroy crosslinks distort structure

Fixation of tissue may be necessary prior to demineralization bull Fixation (glutaraldehyde or paraformadehyde) may

preserve structural integrity

bull Fixation effects on collagen structure are not fully known

BBML - All R

ights Reserve

d

100 microm x 100 microm

50 microm x 50 microm

25 microm x 25 microm

10 microm x 10 microm

35 microm x 35 microm

BBML - All R

ights Reserve

d

35 microm x 35 microm

BBML - All R

ights Reserve

d

Analysis of Collagen Structure in Bone Spatial organization of newly formed bone following a surgically-induced injury (rat tibial hole)

Baranauskas J Vac Sci Tech 2001 Baranauskas App Surface Sci 2005

ldquohellipstatistical diameter of the fibers is 159 nm in good agreement with the literature values for type I collagen fibersrdquo

BBML - All R

ights Reserve

d

Analysis of Collagen in Bone Fibril Diameter Fibril Diameter easiest to measure property first attempts were a bit crude better with increased resolution

Sasaki J Mat Sci Tech 2002 Thalhammer J Arch Sci 2001

Bozec Ultramicroscopy 2005

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ights Reserve

d

Collagen in Bone Fibril Diameter

Caution must be taken when reporting and interpreting diameters measured from fibrils in bone bull Fibril packing - width may not be fully exposed

Wallace et al Langmuir 2010

BBML - All R

ights Reserve

d

Collagen in Bone Fibril Diameter

Caution must be taken when reporting and interpreting diameters measured from fibrils in bone bull Fibril packing - width may not be fully exposed

Rigozzi ndash J Struct Bio - 2011

BBML - All R

ights Reserve

d

Collagen in Bone Fibril D-Spacing

300 nm 15 nm

N C C N C C N C C N C C

H O H2C CH2 O H

CH2

O OH HHC CH2

CH2

HO

H H

HHN C C

O

H2C CH2

CH2

HN C C

OH

HC CH2

HO

N C C N C C N C C

H O H2C CH2 O

CH2

OH HC CH2

HO

H H

H

CH2 CH2

glycine X - proline Y - hydroxyproline

Hodge and Petruska 1963

Collagen Fibril Axial D-Periodicity BBML - All R

ights Reserve

d

05

101520

0 02 04 06 08 1

Hei

ght (

nm)

Length (microns)

httpremfdartmoutheduimagesindexhtml

Randall Nature and Structure of Collagen 1953

TEM SEM

AFM

D-Spacing = 67 nm

BBML - All R

ights Reserve

d

Collagen in Bone Fibril D-Spacing Many studies have shown observable D-spacing content that near theoretical value

Sasaki 2002 67 plusmn 2 nm Bozek 2005 665 plusmn 14 nm Ge 2007 666 plusmn 38 nm

Habelitz J Structural Bio 2002

A 2002 study in dentin got me interestedhellip

n = 322 wet n = 363 dry

BBML - All R

ights Reserve

d

Collagen in Bone Fibril D-Spacing

bull Internal structure bull Wallace et al Bone 2010 46 1349-1354 bull Wallace et al J Stuct Bio 2011 173 146-152 bull Kemp et al J Stuct Bio 2012 180 428-438 bull Warden et al Endocrinology 2013 154(9)

3178-3187 bull Gallant et al Bone 2014 61 191-200 bull Bart et al Conn Tiss Res 2014 In Press

bull Enzymatic and non-

enzymatic cross-linking bull Hammond et al Bone 2014 60 26-32 bull Diaz Gonzalez et al J Biomech 2014 47(3)

681-686 bull Voytik-Harbin et al In Preparation 2014

D-periodic spacing important structural feature that captures aspects related to

Mouse Femur Mid-diaphysis BBML - All R

ights Reserve

d

2D Fast Fourier Transform Analysis

Harmonics

Fibril D-Spacing

Erickson B Wallace JM et al Biotechnology Journal 2013

Raw AFM Image 2D FFT Power Spectrum

BBML - All R

ights Reserve

d

2D Fast Fourier Transform Analysis

Harmonics

Fibril D-Spacing

Raw AFM Image 2D FFT Power Spectrum

0

20000

40000

60000

80000

100000

120000

0 001 002 003 004 005 006

Am

plitu

de (

microV2 )

Position (1nm)

D-periodic spacing 0015 (1nm) ~ 67 nm

BBML - All R

ights Reserve

d

D-Spacing Distribution

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Overall Mean 670 plusmn 00 nm

BBML - All R

ights Reserve

d

D-Spacing Distribution

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Overall Mean 670 plusmn 00 nm

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

4 Samples Total of n = 255 fibrils Overall Mean 682 plusmn 12 nm

BBML - All R

ights Reserve

d

Cumulative Distribution Function

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

This population 255 fibrils - Each fibril 1255 = 0392 - Data range 628-716 nm

BBML - All R

ights Reserve

d

CDF vs Histogram 62 63 64 65 66 67 68 69 70 71 72 73 74

0

10

20

30

40

50

60

70

80

90

100

0

5

10

15

20

25

30

35

40

45

50

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

HistoCDF

BBML - All R

ights Reserve

d

Changes in D-Spacing with Disease

0

5

10

15

20

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

0

5

10

15

20

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

Wallace et al Bone 2010 46 1349-1354

KS Test plt0001

Control 680 plusmn 26 nm

Estrogen Dep 659 plusmn 31 nm

t-test on mean p=0048

BBML - All R

ights Reserve

d

Changes in D-Spacing with Disease

Wallace et al Bone 2010 46 1349-1354

0

10

20

30

40

50

60

70

80

90

100

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

KS Test plt0001

BBML - All R

ights Reserve

d

Question Model and Tissue Morphology Publication

Tissue Specificity in Mice Male mouse 8 weeks Femur dentin tail tendon

No differences in bone vs dentin both ∆ vs tendon

Wallace et al Langmuir 2011 26(10) 7349-7354

Gender Effects Male and female mice 16 weeks Femur No differences NA

Estrogen Depletion OVX in sheep 5 years + 2 Radius Mean and population shift Wallace et al Bone

2010 46 1349-1354

Estrogen Depletion OVX in female 5 years + 2 Skin (dermis) Significant population shift Erickson et al Biotech

J 2013 8(1) 1174-126

Osteoporosis and Mechanical Loading

OVX in ulnar loading in rats Ulna

No loading effect population shift with OVX

Warden et al Endocrinology 2013 154(9) 3178-3187

Osteogenesis Imperfecta Brtl+ model male mice 2 months Femur

No mean ∆ widening of distribution

Wallace et al J Stuct Bio 2011 173 146-152

Osteogenesis Imperfecta Oimoim model male mice 12 weeks Femur Mean and population shift Bart et al Conn Tiss

Res 2014 In Press

Osteogenesis Imperfecta and Tissue Hydration

Brtl+ model male mice 6 months Tail Tendon Significant population shift Kemp et al J Stuct Bio

2012 180 428-438

Type 2 Diabetes Male ZDSD rats 30 weeks Tibia Significant population shift Hammond et al Bone

2014 60 26-32

Type 2 Diabetes Female ZDSD rats 32 weeks Tail Tendon Significant population shift

Diaz Gonzalez et al J Biomech 2014 47(3)

681-686

Raloxifene (in vivo and in vitro) Female dog treated with Raloxifene Femur Significant population shift Gallant et al Bone

2014 61 191-200 BBML - All R

ights Reserve

d

Issues to Consider Site Selection and Sampling

KS Test plt0001

0023 of bonersquos length

15 m

m =

150

00 micro

m

Wallace et al Langmuir 2011 26(10) 7349-7354

100 microm x 100 microm

35 microm x 35 microm

BBML - All R

ights Reserve

d

Issues to Consider Site Selection and Sampling

60

62

64

66

68

70

72

74

1 (35)

2 (33)

3 (40)

4 (39)

5 (35)

6 (29)

7 (26)

8 (23)

9 (28)

WT (288)

D-P

erio

dic

Gap

Ove

rlap

Spac

ing

(nm

)

Axial Location (Proximal End = 1 Distal End = 9)

( of Fibrils)

91

23

45

87

6

Wallace et al Langmuir 2011 26(10) 7349-7354

288 fibrils for population n=1 for mean comparisons BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

Wet Tendon Dry Tendon Kemp et al J StructBio 2012 180 (3) 428-438

Collagen and bone exist in a hydrated environment in vivo bull Hydration state can impact morphology and mechanics

BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

Nonsignificant mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet (n=255) 682 plusmn 12 nm

WT Dry (n=257) 686 plusmn 08 nm

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

Brtl DryBrtl Wet

KS Test plt0001

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

WT Case 04 nm mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet 682 plusmn 12 nm

WT Dry 686 plusmn 08 nm

Brtl+ Wet 675 plusmn 14 nm

Brtl+ Dry 689 plusmn 10 nm

Brtl+ Case 14 nm increase with drying (plt0001) bull Entire population shifted upward (plt0001)

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT WetBrtl Wet

KS Test plt0001

Brtl+ distribution shifted downward vs WT when wet

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryBrtl Dry

KS Test plt0001

Brtl+ distribution shifted upward vs WT when dry

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider AFM calibration

bull Absolute distance measurements require accurate piezo calibration

bull AFM systems have large scan ranges (150 microm to lt 1 microm)

bull Scanner non-linearities can introduce substantial error over that range

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

ights Reserve

d

Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

BBML - All R

ights Reserve

d

Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

100 nm 10 80 602 1198 100 nm 7 60 827 1439 100 nm 5 40 814 1427 100 nm 35 20 917 1724 100 nm 2 10 937 1774

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

ights Reserve

d

AFM calibration ndash Take Away Message

Calibration using a scan size and feature size comparable to features of interest is highly recommended

Calibration addresses absolute accuracy not precision (differential sensitivity between measurements)

bull Does not limit ability to differentiate between groups using the same AFM with the same calibration parameters

BBML - All R

ights Reserve

d

AFM Applications in Bone Research Indentation Mechanics

BBML - All R

ights Reserve

d

AFM-Based Indentation

In addition to high resolution imaging AFM can be used to extract nanoscale mechanical data

ApproachWithdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Can

tilev

er D

efle

ctio

n (V

olts

)

1 Ramp into a rigid sample ndash measure the slope

Deflection sensitivity x 119899119899119899119899119881119881

2 Calculate cantilever spring constant

3 Hookersquos law takes deflection (volts) and converts to units of force F = kx

Spring constant k 119873119873119899119899

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Forc

e (n

N)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration Fo

rce

(nN

)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

Tip-Sample Separation (nm)

BBML - All R

ights Reserve

d

AFM-Based Indentation Parameters

Approach Withdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

Peak Force

Area Between Curves Energy Dissipation

Contact Mechanics Fit for Elastic Modulus (E)

Adhesion Force

Indentation Depth (δ)

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

z

r

δ

R

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119877119877 ∙ 12057512057532

Es Sample modulus νs Poissonrsquos ratio R tip radius of curvature δ load point displacement

Relevant Hertz Assumptions bull Strains are small and within the elastic limit bull Indenter is infinitely stiff (only sample deforms) bull Indentation depth ltlt radius of curvature of indenter bull Surfaces are continuous non-conforming (must be flat) bull Load is normal to surface

BBML - All R

ights Reserve

d

Proper Indentation Model is Essential

δ

r

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119903119903 ∙ 12057512057532

δ

α

119865119865 =2120587120587∙

1198641198641199041199041 minus 1205841205841199041199042

∙ tan120572120572 ∙ 1205751205752

Es Sample modulus νs Poissonrsquos ratio R tip radius α opening angle

Hertz Model Spherical Indenter

depth ltlt radius of curvature

Sneddon Model Conical Indenter

depth ge radius of curvature

BBML - All R

ights Reserve

d

Indentation in OI Tendon Study

x

z

yBrtl+ and WT mice 6 month old males

4 tails per group

2-3 fascicles per tail

25-30 fibrils per tail

4-5 locations per fibril

Indent force 20 nN wet (Sneddon) 50 nN dry (Hertz) BBML - A

ll Rights R

eserved

OI Tendon Differential Effects of Dehydration

Dry 1268 plusmn 718 MPa

Wet 305 plusmn 243 MPa

Dry 1737 plusmn 973 MPa

Wet 301 plusmn 202 MPa

Kemp et al J StructBio 2012 180 (3) 428-438

bull Significant stiffening of fibrils with dehydration

bull More pronounced modulus modulus as a function of disease

BBML - All R

ights Reserve

d

OI Tendon Differential Effects of Dehydration

Kemp et al J StructBio 2012 180 (3) 428-438

Diseased phenotype not present when wet phenotype observed when dried

0

10

20

30

40

50

60

0 20 40 60 80 100 120 140 160 180 200

Gro

up S

ampl

es (

)

Indent Modulus (MPa)

WT WetBrtl Wet

0

2

4

6

8

10

12

14

16

18

20

025

050

075

010

0012

5015

0017

5020

0022

5025

0027

5030

0032

5035

0037

5040

0042

5045

0047

5050

00

Gro

up S

ampl

es (

)Indent Modulus (MPa)

WT DryBrtl Dry

555 WT Indents 305 plusmn 243 MPa

614 Brtl+ Indents 301 plusmn 202 MPa

KS Test p=0129 t Test p=0464 464 WT Indents

1268 plusmn 718 MPa 479 Brtl+ Indents 1737 plusmn 973 MPa

KS Test plt0001 T Test plt0001

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ights Reserve

d

Indentation in Diabetic Tendon (Poster 0495)

x

z

y

Control and Diabetic Rat Tail Tendon

4-5 rats per group

2-3 fascicles per rat

~70 fibrilstail

4-5 locationsfibril

20 nN (Sneddon)

BBML - All R

ights Reserve

d

Multiscale Diabetes Study (Poster 0495)

0

5

10

15

20

25

300 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 gt25

Perc

enta

ge o

f Gro

up S

ampl

es

Elastic Modulus (MPa)

CD (1530 indents)ZDSD (913 indents)

CD (n=5) 381 plusmn 149 MPa

ZDSD (n=3) 706 plusmn 263 MPa

0

100

200

300

400

500

600

700

800

900

0 2 4 6 8 10

Mic

rosc

ale

Mod

ulus

(MPa

)

Nanoscale Modulus (MPa)

plt0001

r2=0531 p=0040

bull Increased indentation modulus in diabetic tendon

bull Strong relationship between nanoscale indent modulus and microscale tensile modulus

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

Proper probe selection for intended application bull Cantilever must be stiff relative to sample bull Sharp probes may damage sample easily dull

R or α must be determined for each probe bull Properties provided by manufacturer are unreliable bull Vary from probe to probe can dull during indent process

Spring constant must be determined for each probe

bull Value provided by manufacturer is inaccurate bull Thermal tuning OK for ldquosoftrdquo probes poor performance

for stiffer probes bull Sader method (geometry) added mass reference

cantilevers

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

Veeco Application Technical note Ben Ohler Practical Advice on the Determination of Cantilever Spring Constants

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

bull Sample processing

bull Sample hydration

bull Proper force range

bull Substrate effects for thin samples

bull Proper modulus model and range of data goodness of fit

Bearing in mind these assumptions and limitations AFM can provide true ldquonanoscalerdquo mechanical properties both in terms of forces and deformations

BBML - All R

ights Reserve

d

A Note on Peak Force QNM for Bruker AFMs bull Mechanical properties mapped pixel-by-pixel at same

resolution as the height image (PeakForce QNM)

Height Modulus BBML - All R

ights Reserve

d

A Note on Peak Force QNM

Correct probe stiffness must be used based on sample Pittenger ndash Veeco Product Bulletin

BBML - All R

ights Reserve

d

A Note on Peak Force QNM Extremely sensitive to calibration (details rarely given) Absolute radius and spring constant known

bull especially important from probe to probe bull radius uarr with indent depth ndash must determine R at depth

you expect to image bull Depth may vary as a function of sample (or at different

regions within the sample)

Relative reference material of ldquoknownrdquo modulus used to determine a ratio of 119896119896 119877119877 bull ldquoknownrdquo modulus may come from tension compression

and not indentation bull R is related to depth indentation depth on sample must

be same as on reference BBML - All R

ights Reserve

d

A Note on Peak Force QNM bull Modulus fit on the fly ndash no ability to monitor fit (or range) bull Local sample topography and sample tilt play a role in

measured parameters bull Assuming all modulus values in an image are correct how to

quantify

512 x 512 pixels = 262144 measurements

n=1 n=262144

n from selected ROIs

BBML - All R

ights Reserve

d

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ights Reserve

d

bull Imaging in air on mica ndash substrate effect

bull Same imaging force or indent depth for all

bull For each area were all modulus values pooled

Lamprou et al PLoS ONE 2013 BBML - All R

ights Reserve

d

AFM Applications in Bone Research Other Cool Mechanical Techniques

BBML - All R

ights Reserve

d

ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo Bonds within or between collagen fibrils in bone that rupture to dissipate energy as a toughening mechanism

Thompson et al Nature 2001

Fantner et al Nature Mat 2005

Hansma et al JMNI 2005

Fantner et al Nano Letters 2007 BBML - All R

ights Reserve

d

Other Mechanical Techniques

Jimenez-Palomar J Mech Beh Bio Mat 2012

van der Rijt Macro Bio 2004

Yang J Biomed Mater Res 2007

Yang Biophysical Journal 2008 BBML - All R

ights Reserve

d

Other Cool Techniques bull Piezoresponse Force Microscopy (PFM)

bull Electrochemical AFM (EC-AFM)

bull Force Modulation Microscopy (FMM)

bull Lateral Force Microscopy (LFM)

bull Magnetic Force Microscopy (MFM)

bull Conductive AFM (C-AFM)

bull Force Pulling

bull Cantilever Bending

bull Tensile testing of individual fibrils

bull 3 point bending of fibrils

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ights Reserve

d

Summary Things to rememberhellip

bull Imaging mode imaging medium and probe selection

bull Morphology Measurements bull Sample preparation and hydration state bull Measuring fibril diameter bull Site selection and proper sampling bull Appropriate system calibration

bull Mechanical Measurements

bull Probe selection (stiffness and tip radius) bull Probe calibration (radius angle spring constant) bull Indentation force range and mechanical model bull Hydration in biological samples bull PeakForce QNM

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d

Acknowledgements BBML

Max Hammond Armando Diaz Gonzalez Zachary Bart Silvia Canelon Creasy Clauser

IU School of Medicine Dr David Burr Dr Matt Allen Dr Stuart Warden Dr Lilian Plotkin

Funding Sources NIHNIDCR 1F32DE018840-01 A1 IUPUI BME Departmental Startup Funds IUPUI Research Support Funds Grant IUPUI Biomechanics and Biomaterials Research Center

NIH NICHD Dr Joan Marini

BBML - All R

ights Reserve

d

Bone Biology and Mechanics Lab (BBML) wwwiupuiedu~bbml Facebook BoneBiologyMechanicsLab

BBML - All R

ights Reserve

d

• Slide Number 1
• General Seminar Outline
• Bonersquos Hierarchical Structure
• Critical Gap in Understanding Collagen
• Methods For Analyzing Collagen Structure
• Methods For Imaging Collagen Structure
• Cryo-TEM
• Atomic Force Microscopy (AFM)
• AFM Image of Pentacene (C22H14)
• AFM Image of Hexabenzocoronene
• Basics of AFM Imaging The Probe
• Basics of AFM Imaging System Components
• van der Waals Potential Energy Curve
• Contact Mode ndash Monitor Deflection
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Peak Force Tapping Mode ndash Monitor Force
• AFM Applications in Bone Research Imaging
• AFM Imaging of Collagen
• Imaging of Osteoclast Activity
• Mineralized collagen deposition in vitro
• Imaging of Canilicular Structure
• AFM Imaging in Bone
• AFM Imaging in Bone
• Removing Mineral in Bone to Expose Collagen
• Removing Mineral in Bone to Expose Collagen
• Demineralization with EDTA
• Demineralization with EDTA
• Demineralization with EDTA
• Issues to Consider Effects of Acid
• Slide Number 32
• Slide Number 33
• Analysis of Collagen Structure in Bone
• Analysis of Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril D-Spacing
• Slide Number 39
• Collagen in Bone Fibril D-Spacing
• Collagen in Bone Fibril D-Spacing
• 2D Fast Fourier Transform Analysis
• 2D Fast Fourier Transform Analysis
• D-Spacing Distribution
• D-Spacing Distribution
• Cumulative Distribution Function
• CDF vs Histogram
• Changes in D-Spacing with Disease
• Changes in D-Spacing with Disease
• Slide Number 50
• Issues to Consider Site Selection and Sampling
• Issues to Consider Site Selection and Sampling
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• AFM calibration ndash Take Away Message
• AFM Applications in Bone ResearchIndentation Mechanics
• AFM-Based Indentation
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Parameters
• Hertz Contact Equation
• Hertz Contact Equation
• Hertz Contact Equation
• Proper Indentation Model is Essential
• Indentation in OI Tendon Study
• OI Tendon Differential Effects of Dehydration
• OI Tendon Differential Effects of Dehydration
• Indentation in Diabetic Tendon (Poster 0495)
• Multiscale Diabetes Study (Poster 0495)
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• A Note on Peak Force QNM for Bruker AFMs
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• Slide Number 83
• Slide Number 84
• AFM Applications in Bone ResearchOther Cool Mechanical Techniques
• ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo
• Other Mechanical Techniques
• Other Cool Techniques
• Summary Things to rememberhellip
• Acknowledgements
• Bone Biology and Mechanics Lab (BBML)

Tapping Mode ndash Monitor Amplitude bull Probe in contact for a small fraction of its tapping period

lateral forces are reduced

bull Probe must be tuned at resonance frequency ndash can be challenging for some probes when in fluid

Tuning in Air Single resonance peak Tuning in Fluid Multiple and spurious peaks wwwasylumresearchcom

BBML - All R

ights Reserve

d

Peak Force Tapping Mode ndash Monitor Force

Control system maintains user-defined maximum force

Cantilever oscillates at low frequency (1-2 kHz)

bull No need for tuning

Slow tapping force curve is captured with each tap

ApproachWithdrawal

Tip-Sample SeparationForc

ePeakForce

Direct control of maximum normal force bull Protects tip and sample wear bull Limits indentation depth to a few nm - increases resolution

by limiting contact area bull Mechanical properties mapped pixel-by-pixel at same

resolution as the height image (PeakForce QNM) BBML - All R

ights Reserve

d

AFM Applications in Bone Research Imaging

BBML - All R

ights Reserve

d

AFM Imaging of Collagen

bull AFMrsquos adoption in bone research has been slowhellip

bull First AFM-based study in collagen came in 1992 (a previous study in 1989 used Scanning Tunneling Microscopy)

bull Observed fibrillar and monomeric collagen

Chernoff J of Vac Sci Tech 1992

BBML - All R

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d

Imaging of Osteoclast Activity

Bozec Ultramicroscopy 2005

Hassenkam Anat Record 2006

Initial observations of cellular activity were qualitative (analyses of collagen morphology were not presented)

Sasaki J Elec Micro 1993

Sasaki CTI 1995

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d

Mineralized collagen deposition in vitro

Collagen deposition from MLO-A5 cells

Barragan-Adjemian CTI 2006 BBML - All R

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d

Imaging of Canilicular Structure

Reilly Annals of BME 2001

Lin J of Microscopy 2010

Average canilicular diameter ~400-500 nm larger than values typically reported from TEM studies

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ights Reserve

d

AFM Imaging in Bone In general we are more interested in collagen in bone

3 microm Diamond Suspension

Unpolished

BBML - All R

ights Reserve

d

AFM Imaging in Bone

The surface is completely mineralizedhellip BBML - All R

ights Reserve

d

Removing Mineral in Bone to Expose Collagen

Dealing with mineral is a challenge to analyzing collagen ndash some mineral must be removed

bull 10 Citric acid (to remove mineral) 65 Sodium Hypochlorite (to remove non-collagenous proteins) Habelitz J Struct Bio 2002

bull 7 or 17 Phosphoric Acid Bozek IEEE 2005 El Feninat J Biomed Mat Res 1998

bull 5 Formic Acid Ge Mat Sci Eng 2007

bull 30 mM EDTA Kindt Nanotechnology 2007

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ights Reserve

d

Removing Mineral in Bone to Expose Collagen

Dealing with mineral is a challenge to analyzing collagen ndash some mineral must be removed

bull 30 mM EDTA Kindt Nanotechnology 2007 BBML - A

ll Rights R

eserved

Demineralization with EDTA EDTA - Ethylenediaminetetraacetic Acid

Chelating agent - sequesters divalent cations (notably Ca2+)

bull Treat with 05M EDTA pH 8 (15-20 minutes) bull Vigorously wash with ultrapure water bull Sonicate to release surface-bound mineral ions bull Repeat 3-4 times

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ights Reserve

d

Demineralization with EDTA

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ights Reserve

d

Demineralization with EDTA

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ights Reserve

d

Issues to Consider Effects of Acid

Immature collagen crosslinks are very sensitive to acid bull Using acids to demineralize bone may inadvertently

destroy crosslinks distort structure

Fixation of tissue may be necessary prior to demineralization bull Fixation (glutaraldehyde or paraformadehyde) may

preserve structural integrity

bull Fixation effects on collagen structure are not fully known

BBML - All R

ights Reserve

d

100 microm x 100 microm

50 microm x 50 microm

25 microm x 25 microm

10 microm x 10 microm

35 microm x 35 microm

BBML - All R

ights Reserve

d

35 microm x 35 microm

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ights Reserve

d

Analysis of Collagen Structure in Bone Spatial organization of newly formed bone following a surgically-induced injury (rat tibial hole)

Baranauskas J Vac Sci Tech 2001 Baranauskas App Surface Sci 2005

ldquohellipstatistical diameter of the fibers is 159 nm in good agreement with the literature values for type I collagen fibersrdquo

BBML - All R

ights Reserve

d

Analysis of Collagen in Bone Fibril Diameter Fibril Diameter easiest to measure property first attempts were a bit crude better with increased resolution

Sasaki J Mat Sci Tech 2002 Thalhammer J Arch Sci 2001

Bozec Ultramicroscopy 2005

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ights Reserve

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Collagen in Bone Fibril Diameter

Caution must be taken when reporting and interpreting diameters measured from fibrils in bone bull Fibril packing - width may not be fully exposed

Wallace et al Langmuir 2010

BBML - All R

ights Reserve

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Collagen in Bone Fibril Diameter

Caution must be taken when reporting and interpreting diameters measured from fibrils in bone bull Fibril packing - width may not be fully exposed

Rigozzi ndash J Struct Bio - 2011

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ights Reserve

d

Collagen in Bone Fibril D-Spacing

300 nm 15 nm

N C C N C C N C C N C C

H O H2C CH2 O H

CH2

O OH HHC CH2

CH2

HO

H H

HHN C C

O

H2C CH2

CH2

HN C C

OH

HC CH2

HO

N C C N C C N C C

H O H2C CH2 O

CH2

OH HC CH2

HO

H H

H

CH2 CH2

glycine X - proline Y - hydroxyproline

Hodge and Petruska 1963

Collagen Fibril Axial D-Periodicity BBML - All R

ights Reserve

d

05

101520

0 02 04 06 08 1

Hei

ght (

nm)

Length (microns)

httpremfdartmoutheduimagesindexhtml

Randall Nature and Structure of Collagen 1953

TEM SEM

AFM

D-Spacing = 67 nm

BBML - All R

ights Reserve

d

Collagen in Bone Fibril D-Spacing Many studies have shown observable D-spacing content that near theoretical value

Sasaki 2002 67 plusmn 2 nm Bozek 2005 665 plusmn 14 nm Ge 2007 666 plusmn 38 nm

Habelitz J Structural Bio 2002

A 2002 study in dentin got me interestedhellip

n = 322 wet n = 363 dry

BBML - All R

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Collagen in Bone Fibril D-Spacing

bull Internal structure bull Wallace et al Bone 2010 46 1349-1354 bull Wallace et al J Stuct Bio 2011 173 146-152 bull Kemp et al J Stuct Bio 2012 180 428-438 bull Warden et al Endocrinology 2013 154(9)

3178-3187 bull Gallant et al Bone 2014 61 191-200 bull Bart et al Conn Tiss Res 2014 In Press

bull Enzymatic and non-

enzymatic cross-linking bull Hammond et al Bone 2014 60 26-32 bull Diaz Gonzalez et al J Biomech 2014 47(3)

681-686 bull Voytik-Harbin et al In Preparation 2014

D-periodic spacing important structural feature that captures aspects related to

Mouse Femur Mid-diaphysis BBML - All R

ights Reserve

d

2D Fast Fourier Transform Analysis

Harmonics

Fibril D-Spacing

Erickson B Wallace JM et al Biotechnology Journal 2013

Raw AFM Image 2D FFT Power Spectrum

BBML - All R

ights Reserve

d

2D Fast Fourier Transform Analysis

Harmonics

Fibril D-Spacing

Raw AFM Image 2D FFT Power Spectrum

0

20000

40000

60000

80000

100000

120000

0 001 002 003 004 005 006

Am

plitu

de (

microV2 )

Position (1nm)

D-periodic spacing 0015 (1nm) ~ 67 nm

BBML - All R

ights Reserve

d

D-Spacing Distribution

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Overall Mean 670 plusmn 00 nm

BBML - All R

ights Reserve

d

D-Spacing Distribution

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Overall Mean 670 plusmn 00 nm

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

4 Samples Total of n = 255 fibrils Overall Mean 682 plusmn 12 nm

BBML - All R

ights Reserve

d

Cumulative Distribution Function

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

This population 255 fibrils - Each fibril 1255 = 0392 - Data range 628-716 nm

BBML - All R

ights Reserve

d

CDF vs Histogram 62 63 64 65 66 67 68 69 70 71 72 73 74

0

10

20

30

40

50

60

70

80

90

100

0

5

10

15

20

25

30

35

40

45

50

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

HistoCDF

BBML - All R

ights Reserve

d

Changes in D-Spacing with Disease

0

5

10

15

20

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

0

5

10

15

20

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

Wallace et al Bone 2010 46 1349-1354

KS Test plt0001

Control 680 plusmn 26 nm

Estrogen Dep 659 plusmn 31 nm

t-test on mean p=0048

BBML - All R

ights Reserve

d

Changes in D-Spacing with Disease

Wallace et al Bone 2010 46 1349-1354

0

10

20

30

40

50

60

70

80

90

100

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

KS Test plt0001

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ights Reserve

d

Question Model and Tissue Morphology Publication

Tissue Specificity in Mice Male mouse 8 weeks Femur dentin tail tendon

No differences in bone vs dentin both ∆ vs tendon

Wallace et al Langmuir 2011 26(10) 7349-7354

Gender Effects Male and female mice 16 weeks Femur No differences NA

Estrogen Depletion OVX in sheep 5 years + 2 Radius Mean and population shift Wallace et al Bone

2010 46 1349-1354

Estrogen Depletion OVX in female 5 years + 2 Skin (dermis) Significant population shift Erickson et al Biotech

J 2013 8(1) 1174-126

Osteoporosis and Mechanical Loading

OVX in ulnar loading in rats Ulna

No loading effect population shift with OVX

Warden et al Endocrinology 2013 154(9) 3178-3187

Osteogenesis Imperfecta Brtl+ model male mice 2 months Femur

No mean ∆ widening of distribution

Wallace et al J Stuct Bio 2011 173 146-152

Osteogenesis Imperfecta Oimoim model male mice 12 weeks Femur Mean and population shift Bart et al Conn Tiss

Res 2014 In Press

Osteogenesis Imperfecta and Tissue Hydration

Brtl+ model male mice 6 months Tail Tendon Significant population shift Kemp et al J Stuct Bio

2012 180 428-438

Type 2 Diabetes Male ZDSD rats 30 weeks Tibia Significant population shift Hammond et al Bone

2014 60 26-32

Type 2 Diabetes Female ZDSD rats 32 weeks Tail Tendon Significant population shift

Diaz Gonzalez et al J Biomech 2014 47(3)

681-686

Raloxifene (in vivo and in vitro) Female dog treated with Raloxifene Femur Significant population shift Gallant et al Bone

2014 61 191-200 BBML - All R

ights Reserve

d

Issues to Consider Site Selection and Sampling

KS Test plt0001

0023 of bonersquos length

15 m

m =

150

00 micro

m

Wallace et al Langmuir 2011 26(10) 7349-7354

100 microm x 100 microm

35 microm x 35 microm

BBML - All R

ights Reserve

d

Issues to Consider Site Selection and Sampling

60

62

64

66

68

70

72

74

1 (35)

2 (33)

3 (40)

4 (39)

5 (35)

6 (29)

7 (26)

8 (23)

9 (28)

WT (288)

D-P

erio

dic

Gap

Ove

rlap

Spac

ing

(nm

)

Axial Location (Proximal End = 1 Distal End = 9)

( of Fibrils)

91

23

45

87

6

Wallace et al Langmuir 2011 26(10) 7349-7354

288 fibrils for population n=1 for mean comparisons BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

Wet Tendon Dry Tendon Kemp et al J StructBio 2012 180 (3) 428-438

Collagen and bone exist in a hydrated environment in vivo bull Hydration state can impact morphology and mechanics

BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

Nonsignificant mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet (n=255) 682 plusmn 12 nm

WT Dry (n=257) 686 plusmn 08 nm

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

Brtl DryBrtl Wet

KS Test plt0001

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

WT Case 04 nm mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet 682 plusmn 12 nm

WT Dry 686 plusmn 08 nm

Brtl+ Wet 675 plusmn 14 nm

Brtl+ Dry 689 plusmn 10 nm

Brtl+ Case 14 nm increase with drying (plt0001) bull Entire population shifted upward (plt0001)

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT WetBrtl Wet

KS Test plt0001

Brtl+ distribution shifted downward vs WT when wet

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryBrtl Dry

KS Test plt0001

Brtl+ distribution shifted upward vs WT when dry

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider AFM calibration

bull Absolute distance measurements require accurate piezo calibration

bull AFM systems have large scan ranges (150 microm to lt 1 microm)

bull Scanner non-linearities can introduce substantial error over that range

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

ights Reserve

d

Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

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ights Reserve

d

Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

100 nm 10 80 602 1198 100 nm 7 60 827 1439 100 nm 5 40 814 1427 100 nm 35 20 917 1724 100 nm 2 10 937 1774

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

ights Reserve

d

AFM calibration ndash Take Away Message

Calibration using a scan size and feature size comparable to features of interest is highly recommended

Calibration addresses absolute accuracy not precision (differential sensitivity between measurements)

bull Does not limit ability to differentiate between groups using the same AFM with the same calibration parameters

BBML - All R

ights Reserve

d

AFM Applications in Bone Research Indentation Mechanics

BBML - All R

ights Reserve

d

AFM-Based Indentation

In addition to high resolution imaging AFM can be used to extract nanoscale mechanical data

ApproachWithdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Can

tilev

er D

efle

ctio

n (V

olts

)

1 Ramp into a rigid sample ndash measure the slope

Deflection sensitivity x 119899119899119899119899119881119881

2 Calculate cantilever spring constant

3 Hookersquos law takes deflection (volts) and converts to units of force F = kx

Spring constant k 119873119873119899119899

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Forc

e (n

N)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration Fo

rce

(nN

)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

Tip-Sample Separation (nm)

BBML - All R

ights Reserve

d

AFM-Based Indentation Parameters

Approach Withdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

Peak Force

Area Between Curves Energy Dissipation

Contact Mechanics Fit for Elastic Modulus (E)

Adhesion Force

Indentation Depth (δ)

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

z

r

δ

R

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119877119877 ∙ 12057512057532

Es Sample modulus νs Poissonrsquos ratio R tip radius of curvature δ load point displacement

Relevant Hertz Assumptions bull Strains are small and within the elastic limit bull Indenter is infinitely stiff (only sample deforms) bull Indentation depth ltlt radius of curvature of indenter bull Surfaces are continuous non-conforming (must be flat) bull Load is normal to surface

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ights Reserve

d

Proper Indentation Model is Essential

δ

r

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119903119903 ∙ 12057512057532

δ

α

119865119865 =2120587120587∙

1198641198641199041199041 minus 1205841205841199041199042

∙ tan120572120572 ∙ 1205751205752

Es Sample modulus νs Poissonrsquos ratio R tip radius α opening angle

Hertz Model Spherical Indenter

depth ltlt radius of curvature

Sneddon Model Conical Indenter

depth ge radius of curvature

BBML - All R

ights Reserve

d

Indentation in OI Tendon Study

x

z

yBrtl+ and WT mice 6 month old males

4 tails per group

2-3 fascicles per tail

25-30 fibrils per tail

4-5 locations per fibril

Indent force 20 nN wet (Sneddon) 50 nN dry (Hertz) BBML - A

ll Rights R

eserved

OI Tendon Differential Effects of Dehydration

Dry 1268 plusmn 718 MPa

Wet 305 plusmn 243 MPa

Dry 1737 plusmn 973 MPa

Wet 301 plusmn 202 MPa

Kemp et al J StructBio 2012 180 (3) 428-438

bull Significant stiffening of fibrils with dehydration

bull More pronounced modulus modulus as a function of disease

BBML - All R

ights Reserve

d

OI Tendon Differential Effects of Dehydration

Kemp et al J StructBio 2012 180 (3) 428-438

Diseased phenotype not present when wet phenotype observed when dried

0

10

20

30

40

50

60

0 20 40 60 80 100 120 140 160 180 200

Gro

up S

ampl

es (

)

Indent Modulus (MPa)

WT WetBrtl Wet

0

2

4

6

8

10

12

14

16

18

20

025

050

075

010

0012

5015

0017

5020

0022

5025

0027

5030

0032

5035

0037

5040

0042

5045

0047

5050

00

Gro

up S

ampl

es (

)Indent Modulus (MPa)

WT DryBrtl Dry

555 WT Indents 305 plusmn 243 MPa

614 Brtl+ Indents 301 plusmn 202 MPa

KS Test p=0129 t Test p=0464 464 WT Indents

1268 plusmn 718 MPa 479 Brtl+ Indents 1737 plusmn 973 MPa

KS Test plt0001 T Test plt0001

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ights Reserve

d

Indentation in Diabetic Tendon (Poster 0495)

x

z

y

Control and Diabetic Rat Tail Tendon

4-5 rats per group

2-3 fascicles per rat

~70 fibrilstail

4-5 locationsfibril

20 nN (Sneddon)

BBML - All R

ights Reserve

d

Multiscale Diabetes Study (Poster 0495)

0

5

10

15

20

25

300 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 gt25

Perc

enta

ge o

f Gro

up S

ampl

es

Elastic Modulus (MPa)

CD (1530 indents)ZDSD (913 indents)

CD (n=5) 381 plusmn 149 MPa

ZDSD (n=3) 706 plusmn 263 MPa

0

100

200

300

400

500

600

700

800

900

0 2 4 6 8 10

Mic

rosc

ale

Mod

ulus

(MPa

)

Nanoscale Modulus (MPa)

plt0001

r2=0531 p=0040

bull Increased indentation modulus in diabetic tendon

bull Strong relationship between nanoscale indent modulus and microscale tensile modulus

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

Proper probe selection for intended application bull Cantilever must be stiff relative to sample bull Sharp probes may damage sample easily dull

R or α must be determined for each probe bull Properties provided by manufacturer are unreliable bull Vary from probe to probe can dull during indent process

Spring constant must be determined for each probe

bull Value provided by manufacturer is inaccurate bull Thermal tuning OK for ldquosoftrdquo probes poor performance

for stiffer probes bull Sader method (geometry) added mass reference

cantilevers

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

Veeco Application Technical note Ben Ohler Practical Advice on the Determination of Cantilever Spring Constants

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

bull Sample processing

bull Sample hydration

bull Proper force range

bull Substrate effects for thin samples

bull Proper modulus model and range of data goodness of fit

Bearing in mind these assumptions and limitations AFM can provide true ldquonanoscalerdquo mechanical properties both in terms of forces and deformations

BBML - All R

ights Reserve

d

A Note on Peak Force QNM for Bruker AFMs bull Mechanical properties mapped pixel-by-pixel at same

resolution as the height image (PeakForce QNM)

Height Modulus BBML - All R

ights Reserve

d

A Note on Peak Force QNM

Correct probe stiffness must be used based on sample Pittenger ndash Veeco Product Bulletin

BBML - All R

ights Reserve

d

A Note on Peak Force QNM Extremely sensitive to calibration (details rarely given) Absolute radius and spring constant known

bull especially important from probe to probe bull radius uarr with indent depth ndash must determine R at depth

you expect to image bull Depth may vary as a function of sample (or at different

regions within the sample)

Relative reference material of ldquoknownrdquo modulus used to determine a ratio of 119896119896 119877119877 bull ldquoknownrdquo modulus may come from tension compression

and not indentation bull R is related to depth indentation depth on sample must

be same as on reference BBML - All R

ights Reserve

d

A Note on Peak Force QNM bull Modulus fit on the fly ndash no ability to monitor fit (or range) bull Local sample topography and sample tilt play a role in

measured parameters bull Assuming all modulus values in an image are correct how to

quantify

512 x 512 pixels = 262144 measurements

n=1 n=262144

n from selected ROIs

BBML - All R

ights Reserve

d

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ights Reserve

d

bull Imaging in air on mica ndash substrate effect

bull Same imaging force or indent depth for all

bull For each area were all modulus values pooled

Lamprou et al PLoS ONE 2013 BBML - All R

ights Reserve

d

AFM Applications in Bone Research Other Cool Mechanical Techniques

BBML - All R

ights Reserve

d

ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo Bonds within or between collagen fibrils in bone that rupture to dissipate energy as a toughening mechanism

Thompson et al Nature 2001

Fantner et al Nature Mat 2005

Hansma et al JMNI 2005

Fantner et al Nano Letters 2007 BBML - All R

ights Reserve

d

Other Mechanical Techniques

Jimenez-Palomar J Mech Beh Bio Mat 2012

van der Rijt Macro Bio 2004

Yang J Biomed Mater Res 2007

Yang Biophysical Journal 2008 BBML - All R

ights Reserve

d

Other Cool Techniques bull Piezoresponse Force Microscopy (PFM)

bull Electrochemical AFM (EC-AFM)

bull Force Modulation Microscopy (FMM)

bull Lateral Force Microscopy (LFM)

bull Magnetic Force Microscopy (MFM)

bull Conductive AFM (C-AFM)

bull Force Pulling

bull Cantilever Bending

bull Tensile testing of individual fibrils

bull 3 point bending of fibrils

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ights Reserve

d

Summary Things to rememberhellip

bull Imaging mode imaging medium and probe selection

bull Morphology Measurements bull Sample preparation and hydration state bull Measuring fibril diameter bull Site selection and proper sampling bull Appropriate system calibration

bull Mechanical Measurements

bull Probe selection (stiffness and tip radius) bull Probe calibration (radius angle spring constant) bull Indentation force range and mechanical model bull Hydration in biological samples bull PeakForce QNM

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d

Acknowledgements BBML

Max Hammond Armando Diaz Gonzalez Zachary Bart Silvia Canelon Creasy Clauser

IU School of Medicine Dr David Burr Dr Matt Allen Dr Stuart Warden Dr Lilian Plotkin

Funding Sources NIHNIDCR 1F32DE018840-01 A1 IUPUI BME Departmental Startup Funds IUPUI Research Support Funds Grant IUPUI Biomechanics and Biomaterials Research Center

NIH NICHD Dr Joan Marini

BBML - All R

ights Reserve

d

Bone Biology and Mechanics Lab (BBML) wwwiupuiedu~bbml Facebook BoneBiologyMechanicsLab

BBML - All R

ights Reserve

d

• Slide Number 1
• General Seminar Outline
• Bonersquos Hierarchical Structure
• Critical Gap in Understanding Collagen
• Methods For Analyzing Collagen Structure
• Methods For Imaging Collagen Structure
• Cryo-TEM
• Atomic Force Microscopy (AFM)
• AFM Image of Pentacene (C22H14)
• AFM Image of Hexabenzocoronene
• Basics of AFM Imaging The Probe
• Basics of AFM Imaging System Components
• van der Waals Potential Energy Curve
• Contact Mode ndash Monitor Deflection
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Peak Force Tapping Mode ndash Monitor Force
• AFM Applications in Bone Research Imaging
• AFM Imaging of Collagen
• Imaging of Osteoclast Activity
• Mineralized collagen deposition in vitro
• Imaging of Canilicular Structure
• AFM Imaging in Bone
• AFM Imaging in Bone
• Removing Mineral in Bone to Expose Collagen
• Removing Mineral in Bone to Expose Collagen
• Demineralization with EDTA
• Demineralization with EDTA
• Demineralization with EDTA
• Issues to Consider Effects of Acid
• Slide Number 32
• Slide Number 33
• Analysis of Collagen Structure in Bone
• Analysis of Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril D-Spacing
• Slide Number 39
• Collagen in Bone Fibril D-Spacing
• Collagen in Bone Fibril D-Spacing
• 2D Fast Fourier Transform Analysis
• 2D Fast Fourier Transform Analysis
• D-Spacing Distribution
• D-Spacing Distribution
• Cumulative Distribution Function
• CDF vs Histogram
• Changes in D-Spacing with Disease
• Changes in D-Spacing with Disease
• Slide Number 50
• Issues to Consider Site Selection and Sampling
• Issues to Consider Site Selection and Sampling
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• AFM calibration ndash Take Away Message
• AFM Applications in Bone ResearchIndentation Mechanics
• AFM-Based Indentation
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Parameters
• Hertz Contact Equation
• Hertz Contact Equation
• Hertz Contact Equation
• Proper Indentation Model is Essential
• Indentation in OI Tendon Study
• OI Tendon Differential Effects of Dehydration
• OI Tendon Differential Effects of Dehydration
• Indentation in Diabetic Tendon (Poster 0495)
• Multiscale Diabetes Study (Poster 0495)
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• A Note on Peak Force QNM for Bruker AFMs
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• Slide Number 83
• Slide Number 84
• AFM Applications in Bone ResearchOther Cool Mechanical Techniques
• ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo
• Other Mechanical Techniques
• Other Cool Techniques
• Summary Things to rememberhellip
• Acknowledgements
• Bone Biology and Mechanics Lab (BBML)

Peak Force Tapping Mode ndash Monitor Force

Control system maintains user-defined maximum force

Cantilever oscillates at low frequency (1-2 kHz)

bull No need for tuning

Slow tapping force curve is captured with each tap

ApproachWithdrawal

Tip-Sample SeparationForc

ePeakForce

Direct control of maximum normal force bull Protects tip and sample wear bull Limits indentation depth to a few nm - increases resolution

by limiting contact area bull Mechanical properties mapped pixel-by-pixel at same

resolution as the height image (PeakForce QNM) BBML - All R

ights Reserve

d

AFM Applications in Bone Research Imaging

BBML - All R

ights Reserve

d

AFM Imaging of Collagen

bull AFMrsquos adoption in bone research has been slowhellip

bull First AFM-based study in collagen came in 1992 (a previous study in 1989 used Scanning Tunneling Microscopy)

bull Observed fibrillar and monomeric collagen

Chernoff J of Vac Sci Tech 1992

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ights Reserve

d

Imaging of Osteoclast Activity

Bozec Ultramicroscopy 2005

Hassenkam Anat Record 2006

Initial observations of cellular activity were qualitative (analyses of collagen morphology were not presented)

Sasaki J Elec Micro 1993

Sasaki CTI 1995

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ights Reserve

d

Mineralized collagen deposition in vitro

Collagen deposition from MLO-A5 cells

Barragan-Adjemian CTI 2006 BBML - All R

ights Reserve

d

Imaging of Canilicular Structure

Reilly Annals of BME 2001

Lin J of Microscopy 2010

Average canilicular diameter ~400-500 nm larger than values typically reported from TEM studies

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ights Reserve

d

AFM Imaging in Bone In general we are more interested in collagen in bone

3 microm Diamond Suspension

Unpolished

BBML - All R

ights Reserve

d

AFM Imaging in Bone

The surface is completely mineralizedhellip BBML - All R

ights Reserve

d

Removing Mineral in Bone to Expose Collagen

Dealing with mineral is a challenge to analyzing collagen ndash some mineral must be removed

bull 10 Citric acid (to remove mineral) 65 Sodium Hypochlorite (to remove non-collagenous proteins) Habelitz J Struct Bio 2002

bull 7 or 17 Phosphoric Acid Bozek IEEE 2005 El Feninat J Biomed Mat Res 1998

bull 5 Formic Acid Ge Mat Sci Eng 2007

bull 30 mM EDTA Kindt Nanotechnology 2007

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ights Reserve

d

Removing Mineral in Bone to Expose Collagen

Dealing with mineral is a challenge to analyzing collagen ndash some mineral must be removed

bull 30 mM EDTA Kindt Nanotechnology 2007 BBML - A

ll Rights R

eserved

Demineralization with EDTA EDTA - Ethylenediaminetetraacetic Acid

Chelating agent - sequesters divalent cations (notably Ca2+)

bull Treat with 05M EDTA pH 8 (15-20 minutes) bull Vigorously wash with ultrapure water bull Sonicate to release surface-bound mineral ions bull Repeat 3-4 times

BBML - All R

ights Reserve

d

Demineralization with EDTA

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ights Reserve

d

Demineralization with EDTA

BBML - All R

ights Reserve

d

Issues to Consider Effects of Acid

Immature collagen crosslinks are very sensitive to acid bull Using acids to demineralize bone may inadvertently

destroy crosslinks distort structure

Fixation of tissue may be necessary prior to demineralization bull Fixation (glutaraldehyde or paraformadehyde) may

preserve structural integrity

bull Fixation effects on collagen structure are not fully known

BBML - All R

ights Reserve

d

100 microm x 100 microm

50 microm x 50 microm

25 microm x 25 microm

10 microm x 10 microm

35 microm x 35 microm

BBML - All R

ights Reserve

d

35 microm x 35 microm

BBML - All R

ights Reserve

d

Analysis of Collagen Structure in Bone Spatial organization of newly formed bone following a surgically-induced injury (rat tibial hole)

Baranauskas J Vac Sci Tech 2001 Baranauskas App Surface Sci 2005

ldquohellipstatistical diameter of the fibers is 159 nm in good agreement with the literature values for type I collagen fibersrdquo

BBML - All R

ights Reserve

d

Analysis of Collagen in Bone Fibril Diameter Fibril Diameter easiest to measure property first attempts were a bit crude better with increased resolution

Sasaki J Mat Sci Tech 2002 Thalhammer J Arch Sci 2001

Bozec Ultramicroscopy 2005

BBML - All R

ights Reserve

d

Collagen in Bone Fibril Diameter

Caution must be taken when reporting and interpreting diameters measured from fibrils in bone bull Fibril packing - width may not be fully exposed

Wallace et al Langmuir 2010

BBML - All R

ights Reserve

d

Collagen in Bone Fibril Diameter

Caution must be taken when reporting and interpreting diameters measured from fibrils in bone bull Fibril packing - width may not be fully exposed

Rigozzi ndash J Struct Bio - 2011

BBML - All R

ights Reserve

d

Collagen in Bone Fibril D-Spacing

300 nm 15 nm

N C C N C C N C C N C C

H O H2C CH2 O H

CH2

O OH HHC CH2

CH2

HO

H H

HHN C C

O

H2C CH2

CH2

HN C C

OH

HC CH2

HO

N C C N C C N C C

H O H2C CH2 O

CH2

OH HC CH2

HO

H H

H

CH2 CH2

glycine X - proline Y - hydroxyproline

Hodge and Petruska 1963

Collagen Fibril Axial D-Periodicity BBML - All R

ights Reserve

d

05

101520

0 02 04 06 08 1

Hei

ght (

nm)

Length (microns)

httpremfdartmoutheduimagesindexhtml

Randall Nature and Structure of Collagen 1953

TEM SEM

AFM

D-Spacing = 67 nm

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Collagen in Bone Fibril D-Spacing Many studies have shown observable D-spacing content that near theoretical value

Sasaki 2002 67 plusmn 2 nm Bozek 2005 665 plusmn 14 nm Ge 2007 666 plusmn 38 nm

Habelitz J Structural Bio 2002

A 2002 study in dentin got me interestedhellip

n = 322 wet n = 363 dry

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Collagen in Bone Fibril D-Spacing

bull Internal structure bull Wallace et al Bone 2010 46 1349-1354 bull Wallace et al J Stuct Bio 2011 173 146-152 bull Kemp et al J Stuct Bio 2012 180 428-438 bull Warden et al Endocrinology 2013 154(9)

3178-3187 bull Gallant et al Bone 2014 61 191-200 bull Bart et al Conn Tiss Res 2014 In Press

bull Enzymatic and non-

enzymatic cross-linking bull Hammond et al Bone 2014 60 26-32 bull Diaz Gonzalez et al J Biomech 2014 47(3)

681-686 bull Voytik-Harbin et al In Preparation 2014

D-periodic spacing important structural feature that captures aspects related to

Mouse Femur Mid-diaphysis BBML - All R

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2D Fast Fourier Transform Analysis

Harmonics

Fibril D-Spacing

Erickson B Wallace JM et al Biotechnology Journal 2013

Raw AFM Image 2D FFT Power Spectrum

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2D Fast Fourier Transform Analysis

Harmonics

Fibril D-Spacing

Raw AFM Image 2D FFT Power Spectrum

0

20000

40000

60000

80000

100000

120000

0 001 002 003 004 005 006

Am

plitu

de (

microV2 )

Position (1nm)

D-periodic spacing 0015 (1nm) ~ 67 nm

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D-Spacing Distribution

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Overall Mean 670 plusmn 00 nm

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D-Spacing Distribution

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Overall Mean 670 plusmn 00 nm

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

4 Samples Total of n = 255 fibrils Overall Mean 682 plusmn 12 nm

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Cumulative Distribution Function

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

This population 255 fibrils - Each fibril 1255 = 0392 - Data range 628-716 nm

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CDF vs Histogram 62 63 64 65 66 67 68 69 70 71 72 73 74

0

10

20

30

40

50

60

70

80

90

100

0

5

10

15

20

25

30

35

40

45

50

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

HistoCDF

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Changes in D-Spacing with Disease

0

5

10

15

20

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

0

5

10

15

20

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

Wallace et al Bone 2010 46 1349-1354

KS Test plt0001

Control 680 plusmn 26 nm

Estrogen Dep 659 plusmn 31 nm

t-test on mean p=0048

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Changes in D-Spacing with Disease

Wallace et al Bone 2010 46 1349-1354

0

10

20

30

40

50

60

70

80

90

100

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

KS Test plt0001

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Question Model and Tissue Morphology Publication

Tissue Specificity in Mice Male mouse 8 weeks Femur dentin tail tendon

No differences in bone vs dentin both ∆ vs tendon

Wallace et al Langmuir 2011 26(10) 7349-7354

Gender Effects Male and female mice 16 weeks Femur No differences NA

Estrogen Depletion OVX in sheep 5 years + 2 Radius Mean and population shift Wallace et al Bone

2010 46 1349-1354

Estrogen Depletion OVX in female 5 years + 2 Skin (dermis) Significant population shift Erickson et al Biotech

J 2013 8(1) 1174-126

Osteoporosis and Mechanical Loading

OVX in ulnar loading in rats Ulna

No loading effect population shift with OVX

Warden et al Endocrinology 2013 154(9) 3178-3187

Osteogenesis Imperfecta Brtl+ model male mice 2 months Femur

No mean ∆ widening of distribution

Wallace et al J Stuct Bio 2011 173 146-152

Osteogenesis Imperfecta Oimoim model male mice 12 weeks Femur Mean and population shift Bart et al Conn Tiss

Res 2014 In Press

Osteogenesis Imperfecta and Tissue Hydration

Brtl+ model male mice 6 months Tail Tendon Significant population shift Kemp et al J Stuct Bio

2012 180 428-438

Type 2 Diabetes Male ZDSD rats 30 weeks Tibia Significant population shift Hammond et al Bone

2014 60 26-32

Type 2 Diabetes Female ZDSD rats 32 weeks Tail Tendon Significant population shift

Diaz Gonzalez et al J Biomech 2014 47(3)

681-686

Raloxifene (in vivo and in vitro) Female dog treated with Raloxifene Femur Significant population shift Gallant et al Bone

2014 61 191-200 BBML - All R

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Issues to Consider Site Selection and Sampling

KS Test plt0001

0023 of bonersquos length

15 m

m =

150

00 micro

m

Wallace et al Langmuir 2011 26(10) 7349-7354

100 microm x 100 microm

35 microm x 35 microm

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Issues to Consider Site Selection and Sampling

60

62

64

66

68

70

72

74

1 (35)

2 (33)

3 (40)

4 (39)

5 (35)

6 (29)

7 (26)

8 (23)

9 (28)

WT (288)

D-P

erio

dic

Gap

Ove

rlap

Spac

ing

(nm

)

Axial Location (Proximal End = 1 Distal End = 9)

( of Fibrils)

91

23

45

87

6

Wallace et al Langmuir 2011 26(10) 7349-7354

288 fibrils for population n=1 for mean comparisons BBML - All R

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Issues to Consider Effects of Hydration

Wet Tendon Dry Tendon Kemp et al J StructBio 2012 180 (3) 428-438

Collagen and bone exist in a hydrated environment in vivo bull Hydration state can impact morphology and mechanics

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Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

Nonsignificant mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet (n=255) 682 plusmn 12 nm

WT Dry (n=257) 686 plusmn 08 nm

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

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Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

Brtl DryBrtl Wet

KS Test plt0001

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

WT Case 04 nm mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet 682 plusmn 12 nm

WT Dry 686 plusmn 08 nm

Brtl+ Wet 675 plusmn 14 nm

Brtl+ Dry 689 plusmn 10 nm

Brtl+ Case 14 nm increase with drying (plt0001) bull Entire population shifted upward (plt0001)

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

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Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT WetBrtl Wet

KS Test plt0001

Brtl+ distribution shifted downward vs WT when wet

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryBrtl Dry

KS Test plt0001

Brtl+ distribution shifted upward vs WT when dry

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

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Issues to Consider AFM calibration

bull Absolute distance measurements require accurate piezo calibration

bull AFM systems have large scan ranges (150 microm to lt 1 microm)

bull Scanner non-linearities can introduce substantial error over that range

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

ights Reserve

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Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

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Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

100 nm 10 80 602 1198 100 nm 7 60 827 1439 100 nm 5 40 814 1427 100 nm 35 20 917 1724 100 nm 2 10 937 1774

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

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AFM calibration ndash Take Away Message

Calibration using a scan size and feature size comparable to features of interest is highly recommended

Calibration addresses absolute accuracy not precision (differential sensitivity between measurements)

bull Does not limit ability to differentiate between groups using the same AFM with the same calibration parameters

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AFM Applications in Bone Research Indentation Mechanics

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AFM-Based Indentation

In addition to high resolution imaging AFM can be used to extract nanoscale mechanical data

ApproachWithdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

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AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Can

tilev

er D

efle

ctio

n (V

olts

)

1 Ramp into a rigid sample ndash measure the slope

Deflection sensitivity x 119899119899119899119899119881119881

2 Calculate cantilever spring constant

3 Hookersquos law takes deflection (volts) and converts to units of force F = kx

Spring constant k 119873119873119899119899

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ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Forc

e (n

N)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

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ights Reserve

d

AFM-Based Indentation Cantilever Calibration Fo

rce

(nN

)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

Tip-Sample Separation (nm)

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AFM-Based Indentation Parameters

Approach Withdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

Peak Force

Area Between Curves Energy Dissipation

Contact Mechanics Fit for Elastic Modulus (E)

Adhesion Force

Indentation Depth (δ)

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ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

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ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

z

r

δ

R

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119877119877 ∙ 12057512057532

Es Sample modulus νs Poissonrsquos ratio R tip radius of curvature δ load point displacement

Relevant Hertz Assumptions bull Strains are small and within the elastic limit bull Indenter is infinitely stiff (only sample deforms) bull Indentation depth ltlt radius of curvature of indenter bull Surfaces are continuous non-conforming (must be flat) bull Load is normal to surface

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Proper Indentation Model is Essential

δ

r

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119903119903 ∙ 12057512057532

δ

α

119865119865 =2120587120587∙

1198641198641199041199041 minus 1205841205841199041199042

∙ tan120572120572 ∙ 1205751205752

Es Sample modulus νs Poissonrsquos ratio R tip radius α opening angle

Hertz Model Spherical Indenter

depth ltlt radius of curvature

Sneddon Model Conical Indenter

depth ge radius of curvature

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ights Reserve

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Indentation in OI Tendon Study

x

z

yBrtl+ and WT mice 6 month old males

4 tails per group

2-3 fascicles per tail

25-30 fibrils per tail

4-5 locations per fibril

Indent force 20 nN wet (Sneddon) 50 nN dry (Hertz) BBML - A

ll Rights R

eserved

OI Tendon Differential Effects of Dehydration

Dry 1268 plusmn 718 MPa

Wet 305 plusmn 243 MPa

Dry 1737 plusmn 973 MPa

Wet 301 plusmn 202 MPa

Kemp et al J StructBio 2012 180 (3) 428-438

bull Significant stiffening of fibrils with dehydration

bull More pronounced modulus modulus as a function of disease

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OI Tendon Differential Effects of Dehydration

Kemp et al J StructBio 2012 180 (3) 428-438

Diseased phenotype not present when wet phenotype observed when dried

0

10

20

30

40

50

60

0 20 40 60 80 100 120 140 160 180 200

Gro

up S

ampl

es (

)

Indent Modulus (MPa)

WT WetBrtl Wet

0

2

4

6

8

10

12

14

16

18

20

025

050

075

010

0012

5015

0017

5020

0022

5025

0027

5030

0032

5035

0037

5040

0042

5045

0047

5050

00

Gro

up S

ampl

es (

)Indent Modulus (MPa)

WT DryBrtl Dry

555 WT Indents 305 plusmn 243 MPa

614 Brtl+ Indents 301 plusmn 202 MPa

KS Test p=0129 t Test p=0464 464 WT Indents

1268 plusmn 718 MPa 479 Brtl+ Indents 1737 plusmn 973 MPa

KS Test plt0001 T Test plt0001

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Indentation in Diabetic Tendon (Poster 0495)

x

z

y

Control and Diabetic Rat Tail Tendon

4-5 rats per group

2-3 fascicles per rat

~70 fibrilstail

4-5 locationsfibril

20 nN (Sneddon)

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Multiscale Diabetes Study (Poster 0495)

0

5

10

15

20

25

300 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 gt25

Perc

enta

ge o

f Gro

up S

ampl

es

Elastic Modulus (MPa)

CD (1530 indents)ZDSD (913 indents)

CD (n=5) 381 plusmn 149 MPa

ZDSD (n=3) 706 plusmn 263 MPa

0

100

200

300

400

500

600

700

800

900

0 2 4 6 8 10

Mic

rosc

ale

Mod

ulus

(MPa

)

Nanoscale Modulus (MPa)

plt0001

r2=0531 p=0040

bull Increased indentation modulus in diabetic tendon

bull Strong relationship between nanoscale indent modulus and microscale tensile modulus

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Indentation Limitations and Weaknesses

Proper probe selection for intended application bull Cantilever must be stiff relative to sample bull Sharp probes may damage sample easily dull

R or α must be determined for each probe bull Properties provided by manufacturer are unreliable bull Vary from probe to probe can dull during indent process

Spring constant must be determined for each probe

bull Value provided by manufacturer is inaccurate bull Thermal tuning OK for ldquosoftrdquo probes poor performance

for stiffer probes bull Sader method (geometry) added mass reference

cantilevers

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Indentation Limitations and Weaknesses

Veeco Application Technical note Ben Ohler Practical Advice on the Determination of Cantilever Spring Constants

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Indentation Limitations and Weaknesses

bull Sample processing

bull Sample hydration

bull Proper force range

bull Substrate effects for thin samples

bull Proper modulus model and range of data goodness of fit

Bearing in mind these assumptions and limitations AFM can provide true ldquonanoscalerdquo mechanical properties both in terms of forces and deformations

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ights Reserve

d

A Note on Peak Force QNM for Bruker AFMs bull Mechanical properties mapped pixel-by-pixel at same

resolution as the height image (PeakForce QNM)

Height Modulus BBML - All R

ights Reserve

d

A Note on Peak Force QNM

Correct probe stiffness must be used based on sample Pittenger ndash Veeco Product Bulletin

BBML - All R

ights Reserve

d

A Note on Peak Force QNM Extremely sensitive to calibration (details rarely given) Absolute radius and spring constant known

bull especially important from probe to probe bull radius uarr with indent depth ndash must determine R at depth

you expect to image bull Depth may vary as a function of sample (or at different

regions within the sample)

Relative reference material of ldquoknownrdquo modulus used to determine a ratio of 119896119896 119877119877 bull ldquoknownrdquo modulus may come from tension compression

and not indentation bull R is related to depth indentation depth on sample must

be same as on reference BBML - All R

ights Reserve

d

A Note on Peak Force QNM bull Modulus fit on the fly ndash no ability to monitor fit (or range) bull Local sample topography and sample tilt play a role in

measured parameters bull Assuming all modulus values in an image are correct how to

quantify

512 x 512 pixels = 262144 measurements

n=1 n=262144

n from selected ROIs

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bull Imaging in air on mica ndash substrate effect

bull Same imaging force or indent depth for all

bull For each area were all modulus values pooled

Lamprou et al PLoS ONE 2013 BBML - All R

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d

AFM Applications in Bone Research Other Cool Mechanical Techniques

BBML - All R

ights Reserve

d

ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo Bonds within or between collagen fibrils in bone that rupture to dissipate energy as a toughening mechanism

Thompson et al Nature 2001

Fantner et al Nature Mat 2005

Hansma et al JMNI 2005

Fantner et al Nano Letters 2007 BBML - All R

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d

Other Mechanical Techniques

Jimenez-Palomar J Mech Beh Bio Mat 2012

van der Rijt Macro Bio 2004

Yang J Biomed Mater Res 2007

Yang Biophysical Journal 2008 BBML - All R

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d

Other Cool Techniques bull Piezoresponse Force Microscopy (PFM)

bull Electrochemical AFM (EC-AFM)

bull Force Modulation Microscopy (FMM)

bull Lateral Force Microscopy (LFM)

bull Magnetic Force Microscopy (MFM)

bull Conductive AFM (C-AFM)

bull Force Pulling

bull Cantilever Bending

bull Tensile testing of individual fibrils

bull 3 point bending of fibrils

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Summary Things to rememberhellip

bull Imaging mode imaging medium and probe selection

bull Morphology Measurements bull Sample preparation and hydration state bull Measuring fibril diameter bull Site selection and proper sampling bull Appropriate system calibration

bull Mechanical Measurements

bull Probe selection (stiffness and tip radius) bull Probe calibration (radius angle spring constant) bull Indentation force range and mechanical model bull Hydration in biological samples bull PeakForce QNM

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Acknowledgements BBML

Max Hammond Armando Diaz Gonzalez Zachary Bart Silvia Canelon Creasy Clauser

IU School of Medicine Dr David Burr Dr Matt Allen Dr Stuart Warden Dr Lilian Plotkin

Funding Sources NIHNIDCR 1F32DE018840-01 A1 IUPUI BME Departmental Startup Funds IUPUI Research Support Funds Grant IUPUI Biomechanics and Biomaterials Research Center

NIH NICHD Dr Joan Marini

BBML - All R

ights Reserve

d

Bone Biology and Mechanics Lab (BBML) wwwiupuiedu~bbml Facebook BoneBiologyMechanicsLab

BBML - All R

ights Reserve

d

• Slide Number 1
• General Seminar Outline
• Bonersquos Hierarchical Structure
• Critical Gap in Understanding Collagen
• Methods For Analyzing Collagen Structure
• Methods For Imaging Collagen Structure
• Cryo-TEM
• Atomic Force Microscopy (AFM)
• AFM Image of Pentacene (C22H14)
• AFM Image of Hexabenzocoronene
• Basics of AFM Imaging The Probe
• Basics of AFM Imaging System Components
• van der Waals Potential Energy Curve
• Contact Mode ndash Monitor Deflection
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Peak Force Tapping Mode ndash Monitor Force
• AFM Applications in Bone Research Imaging
• AFM Imaging of Collagen
• Imaging of Osteoclast Activity
• Mineralized collagen deposition in vitro
• Imaging of Canilicular Structure
• AFM Imaging in Bone
• AFM Imaging in Bone
• Removing Mineral in Bone to Expose Collagen
• Removing Mineral in Bone to Expose Collagen
• Demineralization with EDTA
• Demineralization with EDTA
• Demineralization with EDTA
• Issues to Consider Effects of Acid
• Slide Number 32
• Slide Number 33
• Analysis of Collagen Structure in Bone
• Analysis of Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril D-Spacing
• Slide Number 39
• Collagen in Bone Fibril D-Spacing
• Collagen in Bone Fibril D-Spacing
• 2D Fast Fourier Transform Analysis
• 2D Fast Fourier Transform Analysis
• D-Spacing Distribution
• D-Spacing Distribution
• Cumulative Distribution Function
• CDF vs Histogram
• Changes in D-Spacing with Disease
• Changes in D-Spacing with Disease
• Slide Number 50
• Issues to Consider Site Selection and Sampling
• Issues to Consider Site Selection and Sampling
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• AFM calibration ndash Take Away Message
• AFM Applications in Bone ResearchIndentation Mechanics
• AFM-Based Indentation
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Parameters
• Hertz Contact Equation
• Hertz Contact Equation
• Hertz Contact Equation
• Proper Indentation Model is Essential
• Indentation in OI Tendon Study
• OI Tendon Differential Effects of Dehydration
• OI Tendon Differential Effects of Dehydration
• Indentation in Diabetic Tendon (Poster 0495)
• Multiscale Diabetes Study (Poster 0495)
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• A Note on Peak Force QNM for Bruker AFMs
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• Slide Number 83
• Slide Number 84
• AFM Applications in Bone ResearchOther Cool Mechanical Techniques
• ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo
• Other Mechanical Techniques
• Other Cool Techniques
• Summary Things to rememberhellip
• Acknowledgements
• Bone Biology and Mechanics Lab (BBML)

AFM Applications in Bone Research Imaging

BBML - All R

ights Reserve

d

AFM Imaging of Collagen

bull AFMrsquos adoption in bone research has been slowhellip

bull First AFM-based study in collagen came in 1992 (a previous study in 1989 used Scanning Tunneling Microscopy)

bull Observed fibrillar and monomeric collagen

Chernoff J of Vac Sci Tech 1992

BBML - All R

ights Reserve

d

Imaging of Osteoclast Activity

Bozec Ultramicroscopy 2005

Hassenkam Anat Record 2006

Initial observations of cellular activity were qualitative (analyses of collagen morphology were not presented)

Sasaki J Elec Micro 1993

Sasaki CTI 1995

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Mineralized collagen deposition in vitro

Collagen deposition from MLO-A5 cells

Barragan-Adjemian CTI 2006 BBML - All R

ights Reserve

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Imaging of Canilicular Structure

Reilly Annals of BME 2001

Lin J of Microscopy 2010

Average canilicular diameter ~400-500 nm larger than values typically reported from TEM studies

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ights Reserve

d

AFM Imaging in Bone In general we are more interested in collagen in bone

3 microm Diamond Suspension

Unpolished

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ights Reserve

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AFM Imaging in Bone

The surface is completely mineralizedhellip BBML - All R

ights Reserve

d

Removing Mineral in Bone to Expose Collagen

Dealing with mineral is a challenge to analyzing collagen ndash some mineral must be removed

bull 10 Citric acid (to remove mineral) 65 Sodium Hypochlorite (to remove non-collagenous proteins) Habelitz J Struct Bio 2002

bull 7 or 17 Phosphoric Acid Bozek IEEE 2005 El Feninat J Biomed Mat Res 1998

bull 5 Formic Acid Ge Mat Sci Eng 2007

bull 30 mM EDTA Kindt Nanotechnology 2007

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ights Reserve

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Removing Mineral in Bone to Expose Collagen

Dealing with mineral is a challenge to analyzing collagen ndash some mineral must be removed

bull 30 mM EDTA Kindt Nanotechnology 2007 BBML - A

ll Rights R

eserved

Demineralization with EDTA EDTA - Ethylenediaminetetraacetic Acid

Chelating agent - sequesters divalent cations (notably Ca2+)

bull Treat with 05M EDTA pH 8 (15-20 minutes) bull Vigorously wash with ultrapure water bull Sonicate to release surface-bound mineral ions bull Repeat 3-4 times

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Demineralization with EDTA

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ights Reserve

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Demineralization with EDTA

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ights Reserve

d

Issues to Consider Effects of Acid

Immature collagen crosslinks are very sensitive to acid bull Using acids to demineralize bone may inadvertently

destroy crosslinks distort structure

Fixation of tissue may be necessary prior to demineralization bull Fixation (glutaraldehyde or paraformadehyde) may

preserve structural integrity

bull Fixation effects on collagen structure are not fully known

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100 microm x 100 microm

50 microm x 50 microm

25 microm x 25 microm

10 microm x 10 microm

35 microm x 35 microm

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ights Reserve

d

35 microm x 35 microm

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ights Reserve

d

Analysis of Collagen Structure in Bone Spatial organization of newly formed bone following a surgically-induced injury (rat tibial hole)

Baranauskas J Vac Sci Tech 2001 Baranauskas App Surface Sci 2005

ldquohellipstatistical diameter of the fibers is 159 nm in good agreement with the literature values for type I collagen fibersrdquo

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ights Reserve

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Analysis of Collagen in Bone Fibril Diameter Fibril Diameter easiest to measure property first attempts were a bit crude better with increased resolution

Sasaki J Mat Sci Tech 2002 Thalhammer J Arch Sci 2001

Bozec Ultramicroscopy 2005

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Collagen in Bone Fibril Diameter

Caution must be taken when reporting and interpreting diameters measured from fibrils in bone bull Fibril packing - width may not be fully exposed

Wallace et al Langmuir 2010

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Collagen in Bone Fibril Diameter

Caution must be taken when reporting and interpreting diameters measured from fibrils in bone bull Fibril packing - width may not be fully exposed

Rigozzi ndash J Struct Bio - 2011

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Collagen in Bone Fibril D-Spacing

300 nm 15 nm

N C C N C C N C C N C C

H O H2C CH2 O H

CH2

O OH HHC CH2

CH2

HO

H H

HHN C C

O

H2C CH2

CH2

HN C C

OH

HC CH2

HO

N C C N C C N C C

H O H2C CH2 O

CH2

OH HC CH2

HO

H H

H

CH2 CH2

glycine X - proline Y - hydroxyproline

Hodge and Petruska 1963

Collagen Fibril Axial D-Periodicity BBML - All R

ights Reserve

d

05

101520

0 02 04 06 08 1

Hei

ght (

nm)

Length (microns)

httpremfdartmoutheduimagesindexhtml

Randall Nature and Structure of Collagen 1953

TEM SEM

AFM

D-Spacing = 67 nm

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ights Reserve

d

Collagen in Bone Fibril D-Spacing Many studies have shown observable D-spacing content that near theoretical value

Sasaki 2002 67 plusmn 2 nm Bozek 2005 665 plusmn 14 nm Ge 2007 666 plusmn 38 nm

Habelitz J Structural Bio 2002

A 2002 study in dentin got me interestedhellip

n = 322 wet n = 363 dry

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ights Reserve

d

Collagen in Bone Fibril D-Spacing

bull Internal structure bull Wallace et al Bone 2010 46 1349-1354 bull Wallace et al J Stuct Bio 2011 173 146-152 bull Kemp et al J Stuct Bio 2012 180 428-438 bull Warden et al Endocrinology 2013 154(9)

3178-3187 bull Gallant et al Bone 2014 61 191-200 bull Bart et al Conn Tiss Res 2014 In Press

bull Enzymatic and non-

enzymatic cross-linking bull Hammond et al Bone 2014 60 26-32 bull Diaz Gonzalez et al J Biomech 2014 47(3)

681-686 bull Voytik-Harbin et al In Preparation 2014

D-periodic spacing important structural feature that captures aspects related to

Mouse Femur Mid-diaphysis BBML - All R

ights Reserve

d

2D Fast Fourier Transform Analysis

Harmonics

Fibril D-Spacing

Erickson B Wallace JM et al Biotechnology Journal 2013

Raw AFM Image 2D FFT Power Spectrum

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ights Reserve

d

2D Fast Fourier Transform Analysis

Harmonics

Fibril D-Spacing

Raw AFM Image 2D FFT Power Spectrum

0

20000

40000

60000

80000

100000

120000

0 001 002 003 004 005 006

Am

plitu

de (

microV2 )

Position (1nm)

D-periodic spacing 0015 (1nm) ~ 67 nm

BBML - All R

ights Reserve

d

D-Spacing Distribution

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Overall Mean 670 plusmn 00 nm

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ights Reserve

d

D-Spacing Distribution

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Overall Mean 670 plusmn 00 nm

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

4 Samples Total of n = 255 fibrils Overall Mean 682 plusmn 12 nm

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ights Reserve

d

Cumulative Distribution Function

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

This population 255 fibrils - Each fibril 1255 = 0392 - Data range 628-716 nm

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ights Reserve

d

CDF vs Histogram 62 63 64 65 66 67 68 69 70 71 72 73 74

0

10

20

30

40

50

60

70

80

90

100

0

5

10

15

20

25

30

35

40

45

50

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

HistoCDF

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ights Reserve

d

Changes in D-Spacing with Disease

0

5

10

15

20

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

0

5

10

15

20

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

Wallace et al Bone 2010 46 1349-1354

KS Test plt0001

Control 680 plusmn 26 nm

Estrogen Dep 659 plusmn 31 nm

t-test on mean p=0048

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ights Reserve

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Changes in D-Spacing with Disease

Wallace et al Bone 2010 46 1349-1354

0

10

20

30

40

50

60

70

80

90

100

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

KS Test plt0001

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ights Reserve

d

Question Model and Tissue Morphology Publication

Tissue Specificity in Mice Male mouse 8 weeks Femur dentin tail tendon

No differences in bone vs dentin both ∆ vs tendon

Wallace et al Langmuir 2011 26(10) 7349-7354

Gender Effects Male and female mice 16 weeks Femur No differences NA

Estrogen Depletion OVX in sheep 5 years + 2 Radius Mean and population shift Wallace et al Bone

2010 46 1349-1354

Estrogen Depletion OVX in female 5 years + 2 Skin (dermis) Significant population shift Erickson et al Biotech

J 2013 8(1) 1174-126

Osteoporosis and Mechanical Loading

OVX in ulnar loading in rats Ulna

No loading effect population shift with OVX

Warden et al Endocrinology 2013 154(9) 3178-3187

Osteogenesis Imperfecta Brtl+ model male mice 2 months Femur

No mean ∆ widening of distribution

Wallace et al J Stuct Bio 2011 173 146-152

Osteogenesis Imperfecta Oimoim model male mice 12 weeks Femur Mean and population shift Bart et al Conn Tiss

Res 2014 In Press

Osteogenesis Imperfecta and Tissue Hydration

Brtl+ model male mice 6 months Tail Tendon Significant population shift Kemp et al J Stuct Bio

2012 180 428-438

Type 2 Diabetes Male ZDSD rats 30 weeks Tibia Significant population shift Hammond et al Bone

2014 60 26-32

Type 2 Diabetes Female ZDSD rats 32 weeks Tail Tendon Significant population shift

Diaz Gonzalez et al J Biomech 2014 47(3)

681-686

Raloxifene (in vivo and in vitro) Female dog treated with Raloxifene Femur Significant population shift Gallant et al Bone

2014 61 191-200 BBML - All R

ights Reserve

d

Issues to Consider Site Selection and Sampling

KS Test plt0001

0023 of bonersquos length

15 m

m =

150

00 micro

m

Wallace et al Langmuir 2011 26(10) 7349-7354

100 microm x 100 microm

35 microm x 35 microm

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ights Reserve

d

Issues to Consider Site Selection and Sampling

60

62

64

66

68

70

72

74

1 (35)

2 (33)

3 (40)

4 (39)

5 (35)

6 (29)

7 (26)

8 (23)

9 (28)

WT (288)

D-P

erio

dic

Gap

Ove

rlap

Spac

ing

(nm

)

Axial Location (Proximal End = 1 Distal End = 9)

( of Fibrils)

91

23

45

87

6

Wallace et al Langmuir 2011 26(10) 7349-7354

288 fibrils for population n=1 for mean comparisons BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

Wet Tendon Dry Tendon Kemp et al J StructBio 2012 180 (3) 428-438

Collagen and bone exist in a hydrated environment in vivo bull Hydration state can impact morphology and mechanics

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ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

Nonsignificant mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet (n=255) 682 plusmn 12 nm

WT Dry (n=257) 686 plusmn 08 nm

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

Brtl DryBrtl Wet

KS Test plt0001

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

WT Case 04 nm mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet 682 plusmn 12 nm

WT Dry 686 plusmn 08 nm

Brtl+ Wet 675 plusmn 14 nm

Brtl+ Dry 689 plusmn 10 nm

Brtl+ Case 14 nm increase with drying (plt0001) bull Entire population shifted upward (plt0001)

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT WetBrtl Wet

KS Test plt0001

Brtl+ distribution shifted downward vs WT when wet

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryBrtl Dry

KS Test plt0001

Brtl+ distribution shifted upward vs WT when dry

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider AFM calibration

bull Absolute distance measurements require accurate piezo calibration

bull AFM systems have large scan ranges (150 microm to lt 1 microm)

bull Scanner non-linearities can introduce substantial error over that range

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

ights Reserve

d

Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

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ights Reserve

d

Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

100 nm 10 80 602 1198 100 nm 7 60 827 1439 100 nm 5 40 814 1427 100 nm 35 20 917 1724 100 nm 2 10 937 1774

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

ights Reserve

d

AFM calibration ndash Take Away Message

Calibration using a scan size and feature size comparable to features of interest is highly recommended

Calibration addresses absolute accuracy not precision (differential sensitivity between measurements)

bull Does not limit ability to differentiate between groups using the same AFM with the same calibration parameters

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d

AFM Applications in Bone Research Indentation Mechanics

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ights Reserve

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AFM-Based Indentation

In addition to high resolution imaging AFM can be used to extract nanoscale mechanical data

ApproachWithdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

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ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Can

tilev

er D

efle

ctio

n (V

olts

)

1 Ramp into a rigid sample ndash measure the slope

Deflection sensitivity x 119899119899119899119899119881119881

2 Calculate cantilever spring constant

3 Hookersquos law takes deflection (volts) and converts to units of force F = kx

Spring constant k 119873119873119899119899

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ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Forc

e (n

N)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

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ights Reserve

d

AFM-Based Indentation Cantilever Calibration Fo

rce

(nN

)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

Tip-Sample Separation (nm)

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ights Reserve

d

AFM-Based Indentation Parameters

Approach Withdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

Peak Force

Area Between Curves Energy Dissipation

Contact Mechanics Fit for Elastic Modulus (E)

Adhesion Force

Indentation Depth (δ)

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

z

r

δ

R

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119877119877 ∙ 12057512057532

Es Sample modulus νs Poissonrsquos ratio R tip radius of curvature δ load point displacement

Relevant Hertz Assumptions bull Strains are small and within the elastic limit bull Indenter is infinitely stiff (only sample deforms) bull Indentation depth ltlt radius of curvature of indenter bull Surfaces are continuous non-conforming (must be flat) bull Load is normal to surface

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ights Reserve

d

Proper Indentation Model is Essential

δ

r

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119903119903 ∙ 12057512057532

δ

α

119865119865 =2120587120587∙

1198641198641199041199041 minus 1205841205841199041199042

∙ tan120572120572 ∙ 1205751205752

Es Sample modulus νs Poissonrsquos ratio R tip radius α opening angle

Hertz Model Spherical Indenter

depth ltlt radius of curvature

Sneddon Model Conical Indenter

depth ge radius of curvature

BBML - All R

ights Reserve

d

Indentation in OI Tendon Study

x

z

yBrtl+ and WT mice 6 month old males

4 tails per group

2-3 fascicles per tail

25-30 fibrils per tail

4-5 locations per fibril

Indent force 20 nN wet (Sneddon) 50 nN dry (Hertz) BBML - A

ll Rights R

eserved

OI Tendon Differential Effects of Dehydration

Dry 1268 plusmn 718 MPa

Wet 305 plusmn 243 MPa

Dry 1737 plusmn 973 MPa

Wet 301 plusmn 202 MPa

Kemp et al J StructBio 2012 180 (3) 428-438

bull Significant stiffening of fibrils with dehydration

bull More pronounced modulus modulus as a function of disease

BBML - All R

ights Reserve

d

OI Tendon Differential Effects of Dehydration

Kemp et al J StructBio 2012 180 (3) 428-438

Diseased phenotype not present when wet phenotype observed when dried

0

10

20

30

40

50

60

0 20 40 60 80 100 120 140 160 180 200

Gro

up S

ampl

es (

)

Indent Modulus (MPa)

WT WetBrtl Wet

0

2

4

6

8

10

12

14

16

18

20

025

050

075

010

0012

5015

0017

5020

0022

5025

0027

5030

0032

5035

0037

5040

0042

5045

0047

5050

00

Gro

up S

ampl

es (

)Indent Modulus (MPa)

WT DryBrtl Dry

555 WT Indents 305 plusmn 243 MPa

614 Brtl+ Indents 301 plusmn 202 MPa

KS Test p=0129 t Test p=0464 464 WT Indents

1268 plusmn 718 MPa 479 Brtl+ Indents 1737 plusmn 973 MPa

KS Test plt0001 T Test plt0001

BBML - All R

ights Reserve

d

Indentation in Diabetic Tendon (Poster 0495)

x

z

y

Control and Diabetic Rat Tail Tendon

4-5 rats per group

2-3 fascicles per rat

~70 fibrilstail

4-5 locationsfibril

20 nN (Sneddon)

BBML - All R

ights Reserve

d

Multiscale Diabetes Study (Poster 0495)

0

5

10

15

20

25

300 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 gt25

Perc

enta

ge o

f Gro

up S

ampl

es

Elastic Modulus (MPa)

CD (1530 indents)ZDSD (913 indents)

CD (n=5) 381 plusmn 149 MPa

ZDSD (n=3) 706 plusmn 263 MPa

0

100

200

300

400

500

600

700

800

900

0 2 4 6 8 10

Mic

rosc

ale

Mod

ulus

(MPa

)

Nanoscale Modulus (MPa)

plt0001

r2=0531 p=0040

bull Increased indentation modulus in diabetic tendon

bull Strong relationship between nanoscale indent modulus and microscale tensile modulus

BBML - All R

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d

Indentation Limitations and Weaknesses

Proper probe selection for intended application bull Cantilever must be stiff relative to sample bull Sharp probes may damage sample easily dull

R or α must be determined for each probe bull Properties provided by manufacturer are unreliable bull Vary from probe to probe can dull during indent process

Spring constant must be determined for each probe

bull Value provided by manufacturer is inaccurate bull Thermal tuning OK for ldquosoftrdquo probes poor performance

for stiffer probes bull Sader method (geometry) added mass reference

cantilevers

BBML - All R

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d

Indentation Limitations and Weaknesses

Veeco Application Technical note Ben Ohler Practical Advice on the Determination of Cantilever Spring Constants

BBML - All R

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d

Indentation Limitations and Weaknesses

bull Sample processing

bull Sample hydration

bull Proper force range

bull Substrate effects for thin samples

bull Proper modulus model and range of data goodness of fit

Bearing in mind these assumptions and limitations AFM can provide true ldquonanoscalerdquo mechanical properties both in terms of forces and deformations

BBML - All R

ights Reserve

d

A Note on Peak Force QNM for Bruker AFMs bull Mechanical properties mapped pixel-by-pixel at same

resolution as the height image (PeakForce QNM)

Height Modulus BBML - All R

ights Reserve

d

A Note on Peak Force QNM

Correct probe stiffness must be used based on sample Pittenger ndash Veeco Product Bulletin

BBML - All R

ights Reserve

d

A Note on Peak Force QNM Extremely sensitive to calibration (details rarely given) Absolute radius and spring constant known

bull especially important from probe to probe bull radius uarr with indent depth ndash must determine R at depth

you expect to image bull Depth may vary as a function of sample (or at different

regions within the sample)

Relative reference material of ldquoknownrdquo modulus used to determine a ratio of 119896119896 119877119877 bull ldquoknownrdquo modulus may come from tension compression

and not indentation bull R is related to depth indentation depth on sample must

be same as on reference BBML - All R

ights Reserve

d

A Note on Peak Force QNM bull Modulus fit on the fly ndash no ability to monitor fit (or range) bull Local sample topography and sample tilt play a role in

measured parameters bull Assuming all modulus values in an image are correct how to

quantify

512 x 512 pixels = 262144 measurements

n=1 n=262144

n from selected ROIs

BBML - All R

ights Reserve

d

BBML - All R

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d

bull Imaging in air on mica ndash substrate effect

bull Same imaging force or indent depth for all

bull For each area were all modulus values pooled

Lamprou et al PLoS ONE 2013 BBML - All R

ights Reserve

d

AFM Applications in Bone Research Other Cool Mechanical Techniques

BBML - All R

ights Reserve

d

ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo Bonds within or between collagen fibrils in bone that rupture to dissipate energy as a toughening mechanism

Thompson et al Nature 2001

Fantner et al Nature Mat 2005

Hansma et al JMNI 2005

Fantner et al Nano Letters 2007 BBML - All R

ights Reserve

d

Other Mechanical Techniques

Jimenez-Palomar J Mech Beh Bio Mat 2012

van der Rijt Macro Bio 2004

Yang J Biomed Mater Res 2007

Yang Biophysical Journal 2008 BBML - All R

ights Reserve

d

Other Cool Techniques bull Piezoresponse Force Microscopy (PFM)

bull Electrochemical AFM (EC-AFM)

bull Force Modulation Microscopy (FMM)

bull Lateral Force Microscopy (LFM)

bull Magnetic Force Microscopy (MFM)

bull Conductive AFM (C-AFM)

bull Force Pulling

bull Cantilever Bending

bull Tensile testing of individual fibrils

bull 3 point bending of fibrils

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ights Reserve

d

Summary Things to rememberhellip

bull Imaging mode imaging medium and probe selection

bull Morphology Measurements bull Sample preparation and hydration state bull Measuring fibril diameter bull Site selection and proper sampling bull Appropriate system calibration

bull Mechanical Measurements

bull Probe selection (stiffness and tip radius) bull Probe calibration (radius angle spring constant) bull Indentation force range and mechanical model bull Hydration in biological samples bull PeakForce QNM

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d

Acknowledgements BBML

Max Hammond Armando Diaz Gonzalez Zachary Bart Silvia Canelon Creasy Clauser

IU School of Medicine Dr David Burr Dr Matt Allen Dr Stuart Warden Dr Lilian Plotkin

Funding Sources NIHNIDCR 1F32DE018840-01 A1 IUPUI BME Departmental Startup Funds IUPUI Research Support Funds Grant IUPUI Biomechanics and Biomaterials Research Center

NIH NICHD Dr Joan Marini

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Bone Biology and Mechanics Lab (BBML) wwwiupuiedu~bbml Facebook BoneBiologyMechanicsLab

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• Slide Number 1
• General Seminar Outline
• Bonersquos Hierarchical Structure
• Critical Gap in Understanding Collagen
• Methods For Analyzing Collagen Structure
• Methods For Imaging Collagen Structure
• Cryo-TEM
• Atomic Force Microscopy (AFM)
• AFM Image of Pentacene (C22H14)
• AFM Image of Hexabenzocoronene
• Basics of AFM Imaging The Probe
• Basics of AFM Imaging System Components
• van der Waals Potential Energy Curve
• Contact Mode ndash Monitor Deflection
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Peak Force Tapping Mode ndash Monitor Force
• AFM Applications in Bone Research Imaging
• AFM Imaging of Collagen
• Imaging of Osteoclast Activity
• Mineralized collagen deposition in vitro
• Imaging of Canilicular Structure
• AFM Imaging in Bone
• AFM Imaging in Bone
• Removing Mineral in Bone to Expose Collagen
• Removing Mineral in Bone to Expose Collagen
• Demineralization with EDTA
• Demineralization with EDTA
• Demineralization with EDTA
• Issues to Consider Effects of Acid
• Slide Number 32
• Slide Number 33
• Analysis of Collagen Structure in Bone
• Analysis of Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril D-Spacing
• Slide Number 39
• Collagen in Bone Fibril D-Spacing
• Collagen in Bone Fibril D-Spacing
• 2D Fast Fourier Transform Analysis
• 2D Fast Fourier Transform Analysis
• D-Spacing Distribution
• D-Spacing Distribution
• Cumulative Distribution Function
• CDF vs Histogram
• Changes in D-Spacing with Disease
• Changes in D-Spacing with Disease
• Slide Number 50
• Issues to Consider Site Selection and Sampling
• Issues to Consider Site Selection and Sampling
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• AFM calibration ndash Take Away Message
• AFM Applications in Bone ResearchIndentation Mechanics
• AFM-Based Indentation
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Parameters
• Hertz Contact Equation
• Hertz Contact Equation
• Hertz Contact Equation
• Proper Indentation Model is Essential
• Indentation in OI Tendon Study
• OI Tendon Differential Effects of Dehydration
• OI Tendon Differential Effects of Dehydration
• Indentation in Diabetic Tendon (Poster 0495)
• Multiscale Diabetes Study (Poster 0495)
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• A Note on Peak Force QNM for Bruker AFMs
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• Slide Number 83
• Slide Number 84
• AFM Applications in Bone ResearchOther Cool Mechanical Techniques
• ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo
• Other Mechanical Techniques
• Other Cool Techniques
• Summary Things to rememberhellip
• Acknowledgements
• Bone Biology and Mechanics Lab (BBML)

AFM Imaging of Collagen

bull AFMrsquos adoption in bone research has been slowhellip

bull First AFM-based study in collagen came in 1992 (a previous study in 1989 used Scanning Tunneling Microscopy)

bull Observed fibrillar and monomeric collagen

Chernoff J of Vac Sci Tech 1992

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Imaging of Osteoclast Activity

Bozec Ultramicroscopy 2005

Hassenkam Anat Record 2006

Initial observations of cellular activity were qualitative (analyses of collagen morphology were not presented)

Sasaki J Elec Micro 1993

Sasaki CTI 1995

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Mineralized collagen deposition in vitro

Collagen deposition from MLO-A5 cells

Barragan-Adjemian CTI 2006 BBML - All R

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Imaging of Canilicular Structure

Reilly Annals of BME 2001

Lin J of Microscopy 2010

Average canilicular diameter ~400-500 nm larger than values typically reported from TEM studies

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AFM Imaging in Bone In general we are more interested in collagen in bone

3 microm Diamond Suspension

Unpolished

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AFM Imaging in Bone

The surface is completely mineralizedhellip BBML - All R

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Removing Mineral in Bone to Expose Collagen

Dealing with mineral is a challenge to analyzing collagen ndash some mineral must be removed

bull 10 Citric acid (to remove mineral) 65 Sodium Hypochlorite (to remove non-collagenous proteins) Habelitz J Struct Bio 2002

bull 7 or 17 Phosphoric Acid Bozek IEEE 2005 El Feninat J Biomed Mat Res 1998

bull 5 Formic Acid Ge Mat Sci Eng 2007

bull 30 mM EDTA Kindt Nanotechnology 2007

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Removing Mineral in Bone to Expose Collagen

Dealing with mineral is a challenge to analyzing collagen ndash some mineral must be removed

bull 30 mM EDTA Kindt Nanotechnology 2007 BBML - A

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Demineralization with EDTA EDTA - Ethylenediaminetetraacetic Acid

Chelating agent - sequesters divalent cations (notably Ca2+)

bull Treat with 05M EDTA pH 8 (15-20 minutes) bull Vigorously wash with ultrapure water bull Sonicate to release surface-bound mineral ions bull Repeat 3-4 times

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Demineralization with EDTA

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Demineralization with EDTA

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Issues to Consider Effects of Acid

Immature collagen crosslinks are very sensitive to acid bull Using acids to demineralize bone may inadvertently

destroy crosslinks distort structure

Fixation of tissue may be necessary prior to demineralization bull Fixation (glutaraldehyde or paraformadehyde) may

preserve structural integrity

bull Fixation effects on collagen structure are not fully known

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100 microm x 100 microm

50 microm x 50 microm

25 microm x 25 microm

10 microm x 10 microm

35 microm x 35 microm

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35 microm x 35 microm

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Analysis of Collagen Structure in Bone Spatial organization of newly formed bone following a surgically-induced injury (rat tibial hole)

Baranauskas J Vac Sci Tech 2001 Baranauskas App Surface Sci 2005

ldquohellipstatistical diameter of the fibers is 159 nm in good agreement with the literature values for type I collagen fibersrdquo

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Analysis of Collagen in Bone Fibril Diameter Fibril Diameter easiest to measure property first attempts were a bit crude better with increased resolution

Sasaki J Mat Sci Tech 2002 Thalhammer J Arch Sci 2001

Bozec Ultramicroscopy 2005

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Collagen in Bone Fibril Diameter

Caution must be taken when reporting and interpreting diameters measured from fibrils in bone bull Fibril packing - width may not be fully exposed

Wallace et al Langmuir 2010

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Collagen in Bone Fibril Diameter

Caution must be taken when reporting and interpreting diameters measured from fibrils in bone bull Fibril packing - width may not be fully exposed

Rigozzi ndash J Struct Bio - 2011

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Collagen in Bone Fibril D-Spacing

300 nm 15 nm

N C C N C C N C C N C C

H O H2C CH2 O H

CH2

O OH HHC CH2

CH2

HO

H H

HHN C C

O

H2C CH2

CH2

HN C C

OH

HC CH2

HO

N C C N C C N C C

H O H2C CH2 O

CH2

OH HC CH2

HO

H H

H

CH2 CH2

glycine X - proline Y - hydroxyproline

Hodge and Petruska 1963

Collagen Fibril Axial D-Periodicity BBML - All R

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d

05

101520

0 02 04 06 08 1

Hei

ght (

nm)

Length (microns)

httpremfdartmoutheduimagesindexhtml

Randall Nature and Structure of Collagen 1953

TEM SEM

AFM

D-Spacing = 67 nm

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Collagen in Bone Fibril D-Spacing Many studies have shown observable D-spacing content that near theoretical value

Sasaki 2002 67 plusmn 2 nm Bozek 2005 665 plusmn 14 nm Ge 2007 666 plusmn 38 nm

Habelitz J Structural Bio 2002

A 2002 study in dentin got me interestedhellip

n = 322 wet n = 363 dry

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Collagen in Bone Fibril D-Spacing

bull Internal structure bull Wallace et al Bone 2010 46 1349-1354 bull Wallace et al J Stuct Bio 2011 173 146-152 bull Kemp et al J Stuct Bio 2012 180 428-438 bull Warden et al Endocrinology 2013 154(9)

3178-3187 bull Gallant et al Bone 2014 61 191-200 bull Bart et al Conn Tiss Res 2014 In Press

bull Enzymatic and non-

enzymatic cross-linking bull Hammond et al Bone 2014 60 26-32 bull Diaz Gonzalez et al J Biomech 2014 47(3)

681-686 bull Voytik-Harbin et al In Preparation 2014

D-periodic spacing important structural feature that captures aspects related to

Mouse Femur Mid-diaphysis BBML - All R

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2D Fast Fourier Transform Analysis

Harmonics

Fibril D-Spacing

Erickson B Wallace JM et al Biotechnology Journal 2013

Raw AFM Image 2D FFT Power Spectrum

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2D Fast Fourier Transform Analysis

Harmonics

Fibril D-Spacing

Raw AFM Image 2D FFT Power Spectrum

0

20000

40000

60000

80000

100000

120000

0 001 002 003 004 005 006

Am

plitu

de (

microV2 )

Position (1nm)

D-periodic spacing 0015 (1nm) ~ 67 nm

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D-Spacing Distribution

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Overall Mean 670 plusmn 00 nm

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D-Spacing Distribution

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Overall Mean 670 plusmn 00 nm

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

4 Samples Total of n = 255 fibrils Overall Mean 682 plusmn 12 nm

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Cumulative Distribution Function

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

This population 255 fibrils - Each fibril 1255 = 0392 - Data range 628-716 nm

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CDF vs Histogram 62 63 64 65 66 67 68 69 70 71 72 73 74

0

10

20

30

40

50

60

70

80

90

100

0

5

10

15

20

25

30

35

40

45

50

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

HistoCDF

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Changes in D-Spacing with Disease

0

5

10

15

20

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

0

5

10

15

20

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

Wallace et al Bone 2010 46 1349-1354

KS Test plt0001

Control 680 plusmn 26 nm

Estrogen Dep 659 plusmn 31 nm

t-test on mean p=0048

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Changes in D-Spacing with Disease

Wallace et al Bone 2010 46 1349-1354

0

10

20

30

40

50

60

70

80

90

100

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

KS Test plt0001

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Question Model and Tissue Morphology Publication

Tissue Specificity in Mice Male mouse 8 weeks Femur dentin tail tendon

No differences in bone vs dentin both ∆ vs tendon

Wallace et al Langmuir 2011 26(10) 7349-7354

Gender Effects Male and female mice 16 weeks Femur No differences NA

Estrogen Depletion OVX in sheep 5 years + 2 Radius Mean and population shift Wallace et al Bone

2010 46 1349-1354

Estrogen Depletion OVX in female 5 years + 2 Skin (dermis) Significant population shift Erickson et al Biotech

J 2013 8(1) 1174-126

Osteoporosis and Mechanical Loading

OVX in ulnar loading in rats Ulna

No loading effect population shift with OVX

Warden et al Endocrinology 2013 154(9) 3178-3187

Osteogenesis Imperfecta Brtl+ model male mice 2 months Femur

No mean ∆ widening of distribution

Wallace et al J Stuct Bio 2011 173 146-152

Osteogenesis Imperfecta Oimoim model male mice 12 weeks Femur Mean and population shift Bart et al Conn Tiss

Res 2014 In Press

Osteogenesis Imperfecta and Tissue Hydration

Brtl+ model male mice 6 months Tail Tendon Significant population shift Kemp et al J Stuct Bio

2012 180 428-438

Type 2 Diabetes Male ZDSD rats 30 weeks Tibia Significant population shift Hammond et al Bone

2014 60 26-32

Type 2 Diabetes Female ZDSD rats 32 weeks Tail Tendon Significant population shift

Diaz Gonzalez et al J Biomech 2014 47(3)

681-686

Raloxifene (in vivo and in vitro) Female dog treated with Raloxifene Femur Significant population shift Gallant et al Bone

2014 61 191-200 BBML - All R

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Issues to Consider Site Selection and Sampling

KS Test plt0001

0023 of bonersquos length

15 m

m =

150

00 micro

m

Wallace et al Langmuir 2011 26(10) 7349-7354

100 microm x 100 microm

35 microm x 35 microm

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Issues to Consider Site Selection and Sampling

60

62

64

66

68

70

72

74

1 (35)

2 (33)

3 (40)

4 (39)

5 (35)

6 (29)

7 (26)

8 (23)

9 (28)

WT (288)

D-P

erio

dic

Gap

Ove

rlap

Spac

ing

(nm

)

Axial Location (Proximal End = 1 Distal End = 9)

( of Fibrils)

91

23

45

87

6

Wallace et al Langmuir 2011 26(10) 7349-7354

288 fibrils for population n=1 for mean comparisons BBML - All R

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Issues to Consider Effects of Hydration

Wet Tendon Dry Tendon Kemp et al J StructBio 2012 180 (3) 428-438

Collagen and bone exist in a hydrated environment in vivo bull Hydration state can impact morphology and mechanics

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Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

Nonsignificant mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet (n=255) 682 plusmn 12 nm

WT Dry (n=257) 686 plusmn 08 nm

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

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Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

Brtl DryBrtl Wet

KS Test plt0001

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

WT Case 04 nm mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet 682 plusmn 12 nm

WT Dry 686 plusmn 08 nm

Brtl+ Wet 675 plusmn 14 nm

Brtl+ Dry 689 plusmn 10 nm

Brtl+ Case 14 nm increase with drying (plt0001) bull Entire population shifted upward (plt0001)

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

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Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT WetBrtl Wet

KS Test plt0001

Brtl+ distribution shifted downward vs WT when wet

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryBrtl Dry

KS Test plt0001

Brtl+ distribution shifted upward vs WT when dry

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

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Issues to Consider AFM calibration

bull Absolute distance measurements require accurate piezo calibration

bull AFM systems have large scan ranges (150 microm to lt 1 microm)

bull Scanner non-linearities can introduce substantial error over that range

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

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Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

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Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

100 nm 10 80 602 1198 100 nm 7 60 827 1439 100 nm 5 40 814 1427 100 nm 35 20 917 1724 100 nm 2 10 937 1774

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

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AFM calibration ndash Take Away Message

Calibration using a scan size and feature size comparable to features of interest is highly recommended

Calibration addresses absolute accuracy not precision (differential sensitivity between measurements)

bull Does not limit ability to differentiate between groups using the same AFM with the same calibration parameters

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AFM Applications in Bone Research Indentation Mechanics

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AFM-Based Indentation

In addition to high resolution imaging AFM can be used to extract nanoscale mechanical data

ApproachWithdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

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AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Can

tilev

er D

efle

ctio

n (V

olts

)

1 Ramp into a rigid sample ndash measure the slope

Deflection sensitivity x 119899119899119899119899119881119881

2 Calculate cantilever spring constant

3 Hookersquos law takes deflection (volts) and converts to units of force F = kx

Spring constant k 119873119873119899119899

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AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Forc

e (n

N)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

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AFM-Based Indentation Cantilever Calibration Fo

rce

(nN

)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

Tip-Sample Separation (nm)

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AFM-Based Indentation Parameters

Approach Withdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

Peak Force

Area Between Curves Energy Dissipation

Contact Mechanics Fit for Elastic Modulus (E)

Adhesion Force

Indentation Depth (δ)

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Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

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Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

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Hertz Contact Equation

wwwwikipediacom

z

r

δ

R

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119877119877 ∙ 12057512057532

Es Sample modulus νs Poissonrsquos ratio R tip radius of curvature δ load point displacement

Relevant Hertz Assumptions bull Strains are small and within the elastic limit bull Indenter is infinitely stiff (only sample deforms) bull Indentation depth ltlt radius of curvature of indenter bull Surfaces are continuous non-conforming (must be flat) bull Load is normal to surface

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Proper Indentation Model is Essential

δ

r

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119903119903 ∙ 12057512057532

δ

α

119865119865 =2120587120587∙

1198641198641199041199041 minus 1205841205841199041199042

∙ tan120572120572 ∙ 1205751205752

Es Sample modulus νs Poissonrsquos ratio R tip radius α opening angle

Hertz Model Spherical Indenter

depth ltlt radius of curvature

Sneddon Model Conical Indenter

depth ge radius of curvature

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Indentation in OI Tendon Study

x

z

yBrtl+ and WT mice 6 month old males

4 tails per group

2-3 fascicles per tail

25-30 fibrils per tail

4-5 locations per fibril

Indent force 20 nN wet (Sneddon) 50 nN dry (Hertz) BBML - A

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OI Tendon Differential Effects of Dehydration

Dry 1268 plusmn 718 MPa

Wet 305 plusmn 243 MPa

Dry 1737 plusmn 973 MPa

Wet 301 plusmn 202 MPa

Kemp et al J StructBio 2012 180 (3) 428-438

bull Significant stiffening of fibrils with dehydration

bull More pronounced modulus modulus as a function of disease

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OI Tendon Differential Effects of Dehydration

Kemp et al J StructBio 2012 180 (3) 428-438

Diseased phenotype not present when wet phenotype observed when dried

0

10

20

30

40

50

60

0 20 40 60 80 100 120 140 160 180 200

Gro

up S

ampl

es (

)

Indent Modulus (MPa)

WT WetBrtl Wet

0

2

4

6

8

10

12

14

16

18

20

025

050

075

010

0012

5015

0017

5020

0022

5025

0027

5030

0032

5035

0037

5040

0042

5045

0047

5050

00

Gro

up S

ampl

es (

)Indent Modulus (MPa)

WT DryBrtl Dry

555 WT Indents 305 plusmn 243 MPa

614 Brtl+ Indents 301 plusmn 202 MPa

KS Test p=0129 t Test p=0464 464 WT Indents

1268 plusmn 718 MPa 479 Brtl+ Indents 1737 plusmn 973 MPa

KS Test plt0001 T Test plt0001

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Indentation in Diabetic Tendon (Poster 0495)

x

z

y

Control and Diabetic Rat Tail Tendon

4-5 rats per group

2-3 fascicles per rat

~70 fibrilstail

4-5 locationsfibril

20 nN (Sneddon)

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Multiscale Diabetes Study (Poster 0495)

0

5

10

15

20

25

300 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 gt25

Perc

enta

ge o

f Gro

up S

ampl

es

Elastic Modulus (MPa)

CD (1530 indents)ZDSD (913 indents)

CD (n=5) 381 plusmn 149 MPa

ZDSD (n=3) 706 plusmn 263 MPa

0

100

200

300

400

500

600

700

800

900

0 2 4 6 8 10

Mic

rosc

ale

Mod

ulus

(MPa

)

Nanoscale Modulus (MPa)

plt0001

r2=0531 p=0040

bull Increased indentation modulus in diabetic tendon

bull Strong relationship between nanoscale indent modulus and microscale tensile modulus

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Indentation Limitations and Weaknesses

Proper probe selection for intended application bull Cantilever must be stiff relative to sample bull Sharp probes may damage sample easily dull

R or α must be determined for each probe bull Properties provided by manufacturer are unreliable bull Vary from probe to probe can dull during indent process

Spring constant must be determined for each probe

bull Value provided by manufacturer is inaccurate bull Thermal tuning OK for ldquosoftrdquo probes poor performance

for stiffer probes bull Sader method (geometry) added mass reference

cantilevers

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Indentation Limitations and Weaknesses

Veeco Application Technical note Ben Ohler Practical Advice on the Determination of Cantilever Spring Constants

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Indentation Limitations and Weaknesses

bull Sample processing

bull Sample hydration

bull Proper force range

bull Substrate effects for thin samples

bull Proper modulus model and range of data goodness of fit

Bearing in mind these assumptions and limitations AFM can provide true ldquonanoscalerdquo mechanical properties both in terms of forces and deformations

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ights Reserve

d

A Note on Peak Force QNM for Bruker AFMs bull Mechanical properties mapped pixel-by-pixel at same

resolution as the height image (PeakForce QNM)

Height Modulus BBML - All R

ights Reserve

d

A Note on Peak Force QNM

Correct probe stiffness must be used based on sample Pittenger ndash Veeco Product Bulletin

BBML - All R

ights Reserve

d

A Note on Peak Force QNM Extremely sensitive to calibration (details rarely given) Absolute radius and spring constant known

bull especially important from probe to probe bull radius uarr with indent depth ndash must determine R at depth

you expect to image bull Depth may vary as a function of sample (or at different

regions within the sample)

Relative reference material of ldquoknownrdquo modulus used to determine a ratio of 119896119896 119877119877 bull ldquoknownrdquo modulus may come from tension compression

and not indentation bull R is related to depth indentation depth on sample must

be same as on reference BBML - All R

ights Reserve

d

A Note on Peak Force QNM bull Modulus fit on the fly ndash no ability to monitor fit (or range) bull Local sample topography and sample tilt play a role in

measured parameters bull Assuming all modulus values in an image are correct how to

quantify

512 x 512 pixels = 262144 measurements

n=1 n=262144

n from selected ROIs

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ights Reserve

d

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d

bull Imaging in air on mica ndash substrate effect

bull Same imaging force or indent depth for all

bull For each area were all modulus values pooled

Lamprou et al PLoS ONE 2013 BBML - All R

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d

AFM Applications in Bone Research Other Cool Mechanical Techniques

BBML - All R

ights Reserve

d

ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo Bonds within or between collagen fibrils in bone that rupture to dissipate energy as a toughening mechanism

Thompson et al Nature 2001

Fantner et al Nature Mat 2005

Hansma et al JMNI 2005

Fantner et al Nano Letters 2007 BBML - All R

ights Reserve

d

Other Mechanical Techniques

Jimenez-Palomar J Mech Beh Bio Mat 2012

van der Rijt Macro Bio 2004

Yang J Biomed Mater Res 2007

Yang Biophysical Journal 2008 BBML - All R

ights Reserve

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Other Cool Techniques bull Piezoresponse Force Microscopy (PFM)

bull Electrochemical AFM (EC-AFM)

bull Force Modulation Microscopy (FMM)

bull Lateral Force Microscopy (LFM)

bull Magnetic Force Microscopy (MFM)

bull Conductive AFM (C-AFM)

bull Force Pulling

bull Cantilever Bending

bull Tensile testing of individual fibrils

bull 3 point bending of fibrils

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Summary Things to rememberhellip

bull Imaging mode imaging medium and probe selection

bull Morphology Measurements bull Sample preparation and hydration state bull Measuring fibril diameter bull Site selection and proper sampling bull Appropriate system calibration

bull Mechanical Measurements

bull Probe selection (stiffness and tip radius) bull Probe calibration (radius angle spring constant) bull Indentation force range and mechanical model bull Hydration in biological samples bull PeakForce QNM

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Acknowledgements BBML

Max Hammond Armando Diaz Gonzalez Zachary Bart Silvia Canelon Creasy Clauser

IU School of Medicine Dr David Burr Dr Matt Allen Dr Stuart Warden Dr Lilian Plotkin

Funding Sources NIHNIDCR 1F32DE018840-01 A1 IUPUI BME Departmental Startup Funds IUPUI Research Support Funds Grant IUPUI Biomechanics and Biomaterials Research Center

NIH NICHD Dr Joan Marini

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Bone Biology and Mechanics Lab (BBML) wwwiupuiedu~bbml Facebook BoneBiologyMechanicsLab

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• Slide Number 1
• General Seminar Outline
• Bonersquos Hierarchical Structure
• Critical Gap in Understanding Collagen
• Methods For Analyzing Collagen Structure
• Methods For Imaging Collagen Structure
• Cryo-TEM
• Atomic Force Microscopy (AFM)
• AFM Image of Pentacene (C22H14)
• AFM Image of Hexabenzocoronene
• Basics of AFM Imaging The Probe
• Basics of AFM Imaging System Components
• van der Waals Potential Energy Curve
• Contact Mode ndash Monitor Deflection
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Peak Force Tapping Mode ndash Monitor Force
• AFM Applications in Bone Research Imaging
• AFM Imaging of Collagen
• Imaging of Osteoclast Activity
• Mineralized collagen deposition in vitro
• Imaging of Canilicular Structure
• AFM Imaging in Bone
• AFM Imaging in Bone
• Removing Mineral in Bone to Expose Collagen
• Removing Mineral in Bone to Expose Collagen
• Demineralization with EDTA
• Demineralization with EDTA
• Demineralization with EDTA
• Issues to Consider Effects of Acid
• Slide Number 32
• Slide Number 33
• Analysis of Collagen Structure in Bone
• Analysis of Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril D-Spacing
• Slide Number 39
• Collagen in Bone Fibril D-Spacing
• Collagen in Bone Fibril D-Spacing
• 2D Fast Fourier Transform Analysis
• 2D Fast Fourier Transform Analysis
• D-Spacing Distribution
• D-Spacing Distribution
• Cumulative Distribution Function
• CDF vs Histogram
• Changes in D-Spacing with Disease
• Changes in D-Spacing with Disease
• Slide Number 50
• Issues to Consider Site Selection and Sampling
• Issues to Consider Site Selection and Sampling
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• AFM calibration ndash Take Away Message
• AFM Applications in Bone ResearchIndentation Mechanics
• AFM-Based Indentation
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Parameters
• Hertz Contact Equation
• Hertz Contact Equation
• Hertz Contact Equation
• Proper Indentation Model is Essential
• Indentation in OI Tendon Study
• OI Tendon Differential Effects of Dehydration
• OI Tendon Differential Effects of Dehydration
• Indentation in Diabetic Tendon (Poster 0495)
• Multiscale Diabetes Study (Poster 0495)
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• A Note on Peak Force QNM for Bruker AFMs
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• Slide Number 83
• Slide Number 84
• AFM Applications in Bone ResearchOther Cool Mechanical Techniques
• ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo
• Other Mechanical Techniques
• Other Cool Techniques
• Summary Things to rememberhellip
• Acknowledgements
• Bone Biology and Mechanics Lab (BBML)

Imaging of Osteoclast Activity

Bozec Ultramicroscopy 2005

Hassenkam Anat Record 2006

Initial observations of cellular activity were qualitative (analyses of collagen morphology were not presented)

Sasaki J Elec Micro 1993

Sasaki CTI 1995

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Mineralized collagen deposition in vitro

Collagen deposition from MLO-A5 cells

Barragan-Adjemian CTI 2006 BBML - All R

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Imaging of Canilicular Structure

Reilly Annals of BME 2001

Lin J of Microscopy 2010

Average canilicular diameter ~400-500 nm larger than values typically reported from TEM studies

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d

AFM Imaging in Bone In general we are more interested in collagen in bone

3 microm Diamond Suspension

Unpolished

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ights Reserve

d

AFM Imaging in Bone

The surface is completely mineralizedhellip BBML - All R

ights Reserve

d

Removing Mineral in Bone to Expose Collagen

Dealing with mineral is a challenge to analyzing collagen ndash some mineral must be removed

bull 10 Citric acid (to remove mineral) 65 Sodium Hypochlorite (to remove non-collagenous proteins) Habelitz J Struct Bio 2002

bull 7 or 17 Phosphoric Acid Bozek IEEE 2005 El Feninat J Biomed Mat Res 1998

bull 5 Formic Acid Ge Mat Sci Eng 2007

bull 30 mM EDTA Kindt Nanotechnology 2007

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ights Reserve

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Removing Mineral in Bone to Expose Collagen

Dealing with mineral is a challenge to analyzing collagen ndash some mineral must be removed

bull 30 mM EDTA Kindt Nanotechnology 2007 BBML - A

ll Rights R

eserved

Demineralization with EDTA EDTA - Ethylenediaminetetraacetic Acid

Chelating agent - sequesters divalent cations (notably Ca2+)

bull Treat with 05M EDTA pH 8 (15-20 minutes) bull Vigorously wash with ultrapure water bull Sonicate to release surface-bound mineral ions bull Repeat 3-4 times

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Demineralization with EDTA

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Demineralization with EDTA

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ights Reserve

d

Issues to Consider Effects of Acid

Immature collagen crosslinks are very sensitive to acid bull Using acids to demineralize bone may inadvertently

destroy crosslinks distort structure

Fixation of tissue may be necessary prior to demineralization bull Fixation (glutaraldehyde or paraformadehyde) may

preserve structural integrity

bull Fixation effects on collagen structure are not fully known

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100 microm x 100 microm

50 microm x 50 microm

25 microm x 25 microm

10 microm x 10 microm

35 microm x 35 microm

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ights Reserve

d

35 microm x 35 microm

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ights Reserve

d

Analysis of Collagen Structure in Bone Spatial organization of newly formed bone following a surgically-induced injury (rat tibial hole)

Baranauskas J Vac Sci Tech 2001 Baranauskas App Surface Sci 2005

ldquohellipstatistical diameter of the fibers is 159 nm in good agreement with the literature values for type I collagen fibersrdquo

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ights Reserve

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Analysis of Collagen in Bone Fibril Diameter Fibril Diameter easiest to measure property first attempts were a bit crude better with increased resolution

Sasaki J Mat Sci Tech 2002 Thalhammer J Arch Sci 2001

Bozec Ultramicroscopy 2005

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Collagen in Bone Fibril Diameter

Caution must be taken when reporting and interpreting diameters measured from fibrils in bone bull Fibril packing - width may not be fully exposed

Wallace et al Langmuir 2010

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Collagen in Bone Fibril Diameter

Caution must be taken when reporting and interpreting diameters measured from fibrils in bone bull Fibril packing - width may not be fully exposed

Rigozzi ndash J Struct Bio - 2011

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Collagen in Bone Fibril D-Spacing

300 nm 15 nm

N C C N C C N C C N C C

H O H2C CH2 O H

CH2

O OH HHC CH2

CH2

HO

H H

HHN C C

O

H2C CH2

CH2

HN C C

OH

HC CH2

HO

N C C N C C N C C

H O H2C CH2 O

CH2

OH HC CH2

HO

H H

H

CH2 CH2

glycine X - proline Y - hydroxyproline

Hodge and Petruska 1963

Collagen Fibril Axial D-Periodicity BBML - All R

ights Reserve

d

05

101520

0 02 04 06 08 1

Hei

ght (

nm)

Length (microns)

httpremfdartmoutheduimagesindexhtml

Randall Nature and Structure of Collagen 1953

TEM SEM

AFM

D-Spacing = 67 nm

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Collagen in Bone Fibril D-Spacing Many studies have shown observable D-spacing content that near theoretical value

Sasaki 2002 67 plusmn 2 nm Bozek 2005 665 plusmn 14 nm Ge 2007 666 plusmn 38 nm

Habelitz J Structural Bio 2002

A 2002 study in dentin got me interestedhellip

n = 322 wet n = 363 dry

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Collagen in Bone Fibril D-Spacing

bull Internal structure bull Wallace et al Bone 2010 46 1349-1354 bull Wallace et al J Stuct Bio 2011 173 146-152 bull Kemp et al J Stuct Bio 2012 180 428-438 bull Warden et al Endocrinology 2013 154(9)

3178-3187 bull Gallant et al Bone 2014 61 191-200 bull Bart et al Conn Tiss Res 2014 In Press

bull Enzymatic and non-

enzymatic cross-linking bull Hammond et al Bone 2014 60 26-32 bull Diaz Gonzalez et al J Biomech 2014 47(3)

681-686 bull Voytik-Harbin et al In Preparation 2014

D-periodic spacing important structural feature that captures aspects related to

Mouse Femur Mid-diaphysis BBML - All R

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2D Fast Fourier Transform Analysis

Harmonics

Fibril D-Spacing

Erickson B Wallace JM et al Biotechnology Journal 2013

Raw AFM Image 2D FFT Power Spectrum

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2D Fast Fourier Transform Analysis

Harmonics

Fibril D-Spacing

Raw AFM Image 2D FFT Power Spectrum

0

20000

40000

60000

80000

100000

120000

0 001 002 003 004 005 006

Am

plitu

de (

microV2 )

Position (1nm)

D-periodic spacing 0015 (1nm) ~ 67 nm

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ights Reserve

d

D-Spacing Distribution

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Overall Mean 670 plusmn 00 nm

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ights Reserve

d

D-Spacing Distribution

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Overall Mean 670 plusmn 00 nm

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

4 Samples Total of n = 255 fibrils Overall Mean 682 plusmn 12 nm

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Cumulative Distribution Function

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

This population 255 fibrils - Each fibril 1255 = 0392 - Data range 628-716 nm

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CDF vs Histogram 62 63 64 65 66 67 68 69 70 71 72 73 74

0

10

20

30

40

50

60

70

80

90

100

0

5

10

15

20

25

30

35

40

45

50

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

HistoCDF

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d

Changes in D-Spacing with Disease

0

5

10

15

20

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

0

5

10

15

20

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

Wallace et al Bone 2010 46 1349-1354

KS Test plt0001

Control 680 plusmn 26 nm

Estrogen Dep 659 plusmn 31 nm

t-test on mean p=0048

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Changes in D-Spacing with Disease

Wallace et al Bone 2010 46 1349-1354

0

10

20

30

40

50

60

70

80

90

100

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

KS Test plt0001

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Question Model and Tissue Morphology Publication

Tissue Specificity in Mice Male mouse 8 weeks Femur dentin tail tendon

No differences in bone vs dentin both ∆ vs tendon

Wallace et al Langmuir 2011 26(10) 7349-7354

Gender Effects Male and female mice 16 weeks Femur No differences NA

Estrogen Depletion OVX in sheep 5 years + 2 Radius Mean and population shift Wallace et al Bone

2010 46 1349-1354

Estrogen Depletion OVX in female 5 years + 2 Skin (dermis) Significant population shift Erickson et al Biotech

J 2013 8(1) 1174-126

Osteoporosis and Mechanical Loading

OVX in ulnar loading in rats Ulna

No loading effect population shift with OVX

Warden et al Endocrinology 2013 154(9) 3178-3187

Osteogenesis Imperfecta Brtl+ model male mice 2 months Femur

No mean ∆ widening of distribution

Wallace et al J Stuct Bio 2011 173 146-152

Osteogenesis Imperfecta Oimoim model male mice 12 weeks Femur Mean and population shift Bart et al Conn Tiss

Res 2014 In Press

Osteogenesis Imperfecta and Tissue Hydration

Brtl+ model male mice 6 months Tail Tendon Significant population shift Kemp et al J Stuct Bio

2012 180 428-438

Type 2 Diabetes Male ZDSD rats 30 weeks Tibia Significant population shift Hammond et al Bone

2014 60 26-32

Type 2 Diabetes Female ZDSD rats 32 weeks Tail Tendon Significant population shift

Diaz Gonzalez et al J Biomech 2014 47(3)

681-686

Raloxifene (in vivo and in vitro) Female dog treated with Raloxifene Femur Significant population shift Gallant et al Bone

2014 61 191-200 BBML - All R

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d

Issues to Consider Site Selection and Sampling

KS Test plt0001

0023 of bonersquos length

15 m

m =

150

00 micro

m

Wallace et al Langmuir 2011 26(10) 7349-7354

100 microm x 100 microm

35 microm x 35 microm

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ights Reserve

d

Issues to Consider Site Selection and Sampling

60

62

64

66

68

70

72

74

1 (35)

2 (33)

3 (40)

4 (39)

5 (35)

6 (29)

7 (26)

8 (23)

9 (28)

WT (288)

D-P

erio

dic

Gap

Ove

rlap

Spac

ing

(nm

)

Axial Location (Proximal End = 1 Distal End = 9)

( of Fibrils)

91

23

45

87

6

Wallace et al Langmuir 2011 26(10) 7349-7354

288 fibrils for population n=1 for mean comparisons BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

Wet Tendon Dry Tendon Kemp et al J StructBio 2012 180 (3) 428-438

Collagen and bone exist in a hydrated environment in vivo bull Hydration state can impact morphology and mechanics

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ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

Nonsignificant mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet (n=255) 682 plusmn 12 nm

WT Dry (n=257) 686 plusmn 08 nm

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

Brtl DryBrtl Wet

KS Test plt0001

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

WT Case 04 nm mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet 682 plusmn 12 nm

WT Dry 686 plusmn 08 nm

Brtl+ Wet 675 plusmn 14 nm

Brtl+ Dry 689 plusmn 10 nm

Brtl+ Case 14 nm increase with drying (plt0001) bull Entire population shifted upward (plt0001)

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT WetBrtl Wet

KS Test plt0001

Brtl+ distribution shifted downward vs WT when wet

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryBrtl Dry

KS Test plt0001

Brtl+ distribution shifted upward vs WT when dry

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider AFM calibration

bull Absolute distance measurements require accurate piezo calibration

bull AFM systems have large scan ranges (150 microm to lt 1 microm)

bull Scanner non-linearities can introduce substantial error over that range

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

ights Reserve

d

Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

BBML - All R

ights Reserve

d

Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

100 nm 10 80 602 1198 100 nm 7 60 827 1439 100 nm 5 40 814 1427 100 nm 35 20 917 1724 100 nm 2 10 937 1774

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

ights Reserve

d

AFM calibration ndash Take Away Message

Calibration using a scan size and feature size comparable to features of interest is highly recommended

Calibration addresses absolute accuracy not precision (differential sensitivity between measurements)

bull Does not limit ability to differentiate between groups using the same AFM with the same calibration parameters

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ights Reserve

d

AFM Applications in Bone Research Indentation Mechanics

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ights Reserve

d

AFM-Based Indentation

In addition to high resolution imaging AFM can be used to extract nanoscale mechanical data

ApproachWithdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Can

tilev

er D

efle

ctio

n (V

olts

)

1 Ramp into a rigid sample ndash measure the slope

Deflection sensitivity x 119899119899119899119899119881119881

2 Calculate cantilever spring constant

3 Hookersquos law takes deflection (volts) and converts to units of force F = kx

Spring constant k 119873119873119899119899

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Forc

e (n

N)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration Fo

rce

(nN

)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

Tip-Sample Separation (nm)

BBML - All R

ights Reserve

d

AFM-Based Indentation Parameters

Approach Withdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

Peak Force

Area Between Curves Energy Dissipation

Contact Mechanics Fit for Elastic Modulus (E)

Adhesion Force

Indentation Depth (δ)

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

z

r

δ

R

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119877119877 ∙ 12057512057532

Es Sample modulus νs Poissonrsquos ratio R tip radius of curvature δ load point displacement

Relevant Hertz Assumptions bull Strains are small and within the elastic limit bull Indenter is infinitely stiff (only sample deforms) bull Indentation depth ltlt radius of curvature of indenter bull Surfaces are continuous non-conforming (must be flat) bull Load is normal to surface

BBML - All R

ights Reserve

d

Proper Indentation Model is Essential

δ

r

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119903119903 ∙ 12057512057532

δ

α

119865119865 =2120587120587∙

1198641198641199041199041 minus 1205841205841199041199042

∙ tan120572120572 ∙ 1205751205752

Es Sample modulus νs Poissonrsquos ratio R tip radius α opening angle

Hertz Model Spherical Indenter

depth ltlt radius of curvature

Sneddon Model Conical Indenter

depth ge radius of curvature

BBML - All R

ights Reserve

d

Indentation in OI Tendon Study

x

z

yBrtl+ and WT mice 6 month old males

4 tails per group

2-3 fascicles per tail

25-30 fibrils per tail

4-5 locations per fibril

Indent force 20 nN wet (Sneddon) 50 nN dry (Hertz) BBML - A

ll Rights R

eserved

OI Tendon Differential Effects of Dehydration

Dry 1268 plusmn 718 MPa

Wet 305 plusmn 243 MPa

Dry 1737 plusmn 973 MPa

Wet 301 plusmn 202 MPa

Kemp et al J StructBio 2012 180 (3) 428-438

bull Significant stiffening of fibrils with dehydration

bull More pronounced modulus modulus as a function of disease

BBML - All R

ights Reserve

d

OI Tendon Differential Effects of Dehydration

Kemp et al J StructBio 2012 180 (3) 428-438

Diseased phenotype not present when wet phenotype observed when dried

0

10

20

30

40

50

60

0 20 40 60 80 100 120 140 160 180 200

Gro

up S

ampl

es (

)

Indent Modulus (MPa)

WT WetBrtl Wet

0

2

4

6

8

10

12

14

16

18

20

025

050

075

010

0012

5015

0017

5020

0022

5025

0027

5030

0032

5035

0037

5040

0042

5045

0047

5050

00

Gro

up S

ampl

es (

)Indent Modulus (MPa)

WT DryBrtl Dry

555 WT Indents 305 plusmn 243 MPa

614 Brtl+ Indents 301 plusmn 202 MPa

KS Test p=0129 t Test p=0464 464 WT Indents

1268 plusmn 718 MPa 479 Brtl+ Indents 1737 plusmn 973 MPa

KS Test plt0001 T Test plt0001

BBML - All R

ights Reserve

d

Indentation in Diabetic Tendon (Poster 0495)

x

z

y

Control and Diabetic Rat Tail Tendon

4-5 rats per group

2-3 fascicles per rat

~70 fibrilstail

4-5 locationsfibril

20 nN (Sneddon)

BBML - All R

ights Reserve

d

Multiscale Diabetes Study (Poster 0495)

0

5

10

15

20

25

300 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 gt25

Perc

enta

ge o

f Gro

up S

ampl

es

Elastic Modulus (MPa)

CD (1530 indents)ZDSD (913 indents)

CD (n=5) 381 plusmn 149 MPa

ZDSD (n=3) 706 plusmn 263 MPa

0

100

200

300

400

500

600

700

800

900

0 2 4 6 8 10

Mic

rosc

ale

Mod

ulus

(MPa

)

Nanoscale Modulus (MPa)

plt0001

r2=0531 p=0040

bull Increased indentation modulus in diabetic tendon

bull Strong relationship between nanoscale indent modulus and microscale tensile modulus

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

Proper probe selection for intended application bull Cantilever must be stiff relative to sample bull Sharp probes may damage sample easily dull

R or α must be determined for each probe bull Properties provided by manufacturer are unreliable bull Vary from probe to probe can dull during indent process

Spring constant must be determined for each probe

bull Value provided by manufacturer is inaccurate bull Thermal tuning OK for ldquosoftrdquo probes poor performance

for stiffer probes bull Sader method (geometry) added mass reference

cantilevers

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

Veeco Application Technical note Ben Ohler Practical Advice on the Determination of Cantilever Spring Constants

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

bull Sample processing

bull Sample hydration

bull Proper force range

bull Substrate effects for thin samples

bull Proper modulus model and range of data goodness of fit

Bearing in mind these assumptions and limitations AFM can provide true ldquonanoscalerdquo mechanical properties both in terms of forces and deformations

BBML - All R

ights Reserve

d

A Note on Peak Force QNM for Bruker AFMs bull Mechanical properties mapped pixel-by-pixel at same

resolution as the height image (PeakForce QNM)

Height Modulus BBML - All R

ights Reserve

d

A Note on Peak Force QNM

Correct probe stiffness must be used based on sample Pittenger ndash Veeco Product Bulletin

BBML - All R

ights Reserve

d

A Note on Peak Force QNM Extremely sensitive to calibration (details rarely given) Absolute radius and spring constant known

bull especially important from probe to probe bull radius uarr with indent depth ndash must determine R at depth

you expect to image bull Depth may vary as a function of sample (or at different

regions within the sample)

Relative reference material of ldquoknownrdquo modulus used to determine a ratio of 119896119896 119877119877 bull ldquoknownrdquo modulus may come from tension compression

and not indentation bull R is related to depth indentation depth on sample must

be same as on reference BBML - All R

ights Reserve

d

A Note on Peak Force QNM bull Modulus fit on the fly ndash no ability to monitor fit (or range) bull Local sample topography and sample tilt play a role in

measured parameters bull Assuming all modulus values in an image are correct how to

quantify

512 x 512 pixels = 262144 measurements

n=1 n=262144

n from selected ROIs

BBML - All R

ights Reserve

d

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ights Reserve

d

bull Imaging in air on mica ndash substrate effect

bull Same imaging force or indent depth for all

bull For each area were all modulus values pooled

Lamprou et al PLoS ONE 2013 BBML - All R

ights Reserve

d

AFM Applications in Bone Research Other Cool Mechanical Techniques

BBML - All R

ights Reserve

d

ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo Bonds within or between collagen fibrils in bone that rupture to dissipate energy as a toughening mechanism

Thompson et al Nature 2001

Fantner et al Nature Mat 2005

Hansma et al JMNI 2005

Fantner et al Nano Letters 2007 BBML - All R

ights Reserve

d

Other Mechanical Techniques

Jimenez-Palomar J Mech Beh Bio Mat 2012

van der Rijt Macro Bio 2004

Yang J Biomed Mater Res 2007

Yang Biophysical Journal 2008 BBML - All R

ights Reserve

d

Other Cool Techniques bull Piezoresponse Force Microscopy (PFM)

bull Electrochemical AFM (EC-AFM)

bull Force Modulation Microscopy (FMM)

bull Lateral Force Microscopy (LFM)

bull Magnetic Force Microscopy (MFM)

bull Conductive AFM (C-AFM)

bull Force Pulling

bull Cantilever Bending

bull Tensile testing of individual fibrils

bull 3 point bending of fibrils

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ights Reserve

d

Summary Things to rememberhellip

bull Imaging mode imaging medium and probe selection

bull Morphology Measurements bull Sample preparation and hydration state bull Measuring fibril diameter bull Site selection and proper sampling bull Appropriate system calibration

bull Mechanical Measurements

bull Probe selection (stiffness and tip radius) bull Probe calibration (radius angle spring constant) bull Indentation force range and mechanical model bull Hydration in biological samples bull PeakForce QNM

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ights Reserve

d

Acknowledgements BBML

Max Hammond Armando Diaz Gonzalez Zachary Bart Silvia Canelon Creasy Clauser

IU School of Medicine Dr David Burr Dr Matt Allen Dr Stuart Warden Dr Lilian Plotkin

Funding Sources NIHNIDCR 1F32DE018840-01 A1 IUPUI BME Departmental Startup Funds IUPUI Research Support Funds Grant IUPUI Biomechanics and Biomaterials Research Center

NIH NICHD Dr Joan Marini

BBML - All R

ights Reserve

d

Bone Biology and Mechanics Lab (BBML) wwwiupuiedu~bbml Facebook BoneBiologyMechanicsLab

BBML - All R

ights Reserve

d

• Slide Number 1
• General Seminar Outline
• Bonersquos Hierarchical Structure
• Critical Gap in Understanding Collagen
• Methods For Analyzing Collagen Structure
• Methods For Imaging Collagen Structure
• Cryo-TEM
• Atomic Force Microscopy (AFM)
• AFM Image of Pentacene (C22H14)
• AFM Image of Hexabenzocoronene
• Basics of AFM Imaging The Probe
• Basics of AFM Imaging System Components
• van der Waals Potential Energy Curve
• Contact Mode ndash Monitor Deflection
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Peak Force Tapping Mode ndash Monitor Force
• AFM Applications in Bone Research Imaging
• AFM Imaging of Collagen
• Imaging of Osteoclast Activity
• Mineralized collagen deposition in vitro
• Imaging of Canilicular Structure
• AFM Imaging in Bone
• AFM Imaging in Bone
• Removing Mineral in Bone to Expose Collagen
• Removing Mineral in Bone to Expose Collagen
• Demineralization with EDTA
• Demineralization with EDTA
• Demineralization with EDTA
• Issues to Consider Effects of Acid
• Slide Number 32
• Slide Number 33
• Analysis of Collagen Structure in Bone
• Analysis of Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril D-Spacing
• Slide Number 39
• Collagen in Bone Fibril D-Spacing
• Collagen in Bone Fibril D-Spacing
• 2D Fast Fourier Transform Analysis
• 2D Fast Fourier Transform Analysis
• D-Spacing Distribution
• D-Spacing Distribution
• Cumulative Distribution Function
• CDF vs Histogram
• Changes in D-Spacing with Disease
• Changes in D-Spacing with Disease
• Slide Number 50
• Issues to Consider Site Selection and Sampling
• Issues to Consider Site Selection and Sampling
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• AFM calibration ndash Take Away Message
• AFM Applications in Bone ResearchIndentation Mechanics
• AFM-Based Indentation
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Parameters
• Hertz Contact Equation
• Hertz Contact Equation
• Hertz Contact Equation
• Proper Indentation Model is Essential
• Indentation in OI Tendon Study
• OI Tendon Differential Effects of Dehydration
• OI Tendon Differential Effects of Dehydration
• Indentation in Diabetic Tendon (Poster 0495)
• Multiscale Diabetes Study (Poster 0495)
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• A Note on Peak Force QNM for Bruker AFMs
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• Slide Number 83
• Slide Number 84
• AFM Applications in Bone ResearchOther Cool Mechanical Techniques
• ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo
• Other Mechanical Techniques
• Other Cool Techniques
• Summary Things to rememberhellip
• Acknowledgements
• Bone Biology and Mechanics Lab (BBML)

Mineralized collagen deposition in vitro

Collagen deposition from MLO-A5 cells

Barragan-Adjemian CTI 2006 BBML - All R

ights Reserve

d

Imaging of Canilicular Structure

Reilly Annals of BME 2001

Lin J of Microscopy 2010

Average canilicular diameter ~400-500 nm larger than values typically reported from TEM studies

BBML - All R

ights Reserve

d

AFM Imaging in Bone In general we are more interested in collagen in bone

3 microm Diamond Suspension

Unpolished

BBML - All R

ights Reserve

d

AFM Imaging in Bone

The surface is completely mineralizedhellip BBML - All R

ights Reserve

d

Removing Mineral in Bone to Expose Collagen

Dealing with mineral is a challenge to analyzing collagen ndash some mineral must be removed

bull 10 Citric acid (to remove mineral) 65 Sodium Hypochlorite (to remove non-collagenous proteins) Habelitz J Struct Bio 2002

bull 7 or 17 Phosphoric Acid Bozek IEEE 2005 El Feninat J Biomed Mat Res 1998

bull 5 Formic Acid Ge Mat Sci Eng 2007

bull 30 mM EDTA Kindt Nanotechnology 2007

BBML - All R

ights Reserve

d

Removing Mineral in Bone to Expose Collagen

Dealing with mineral is a challenge to analyzing collagen ndash some mineral must be removed

bull 30 mM EDTA Kindt Nanotechnology 2007 BBML - A

ll Rights R

eserved

Demineralization with EDTA EDTA - Ethylenediaminetetraacetic Acid

Chelating agent - sequesters divalent cations (notably Ca2+)

bull Treat with 05M EDTA pH 8 (15-20 minutes) bull Vigorously wash with ultrapure water bull Sonicate to release surface-bound mineral ions bull Repeat 3-4 times

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ights Reserve

d

Demineralization with EDTA

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ights Reserve

d

Demineralization with EDTA

BBML - All R

ights Reserve

d

Issues to Consider Effects of Acid

Immature collagen crosslinks are very sensitive to acid bull Using acids to demineralize bone may inadvertently

destroy crosslinks distort structure

Fixation of tissue may be necessary prior to demineralization bull Fixation (glutaraldehyde or paraformadehyde) may

preserve structural integrity

bull Fixation effects on collagen structure are not fully known

BBML - All R

ights Reserve

d

100 microm x 100 microm

50 microm x 50 microm

25 microm x 25 microm

10 microm x 10 microm

35 microm x 35 microm

BBML - All R

ights Reserve

d

35 microm x 35 microm

BBML - All R

ights Reserve

d

Analysis of Collagen Structure in Bone Spatial organization of newly formed bone following a surgically-induced injury (rat tibial hole)

Baranauskas J Vac Sci Tech 2001 Baranauskas App Surface Sci 2005

ldquohellipstatistical diameter of the fibers is 159 nm in good agreement with the literature values for type I collagen fibersrdquo

BBML - All R

ights Reserve

d

Analysis of Collagen in Bone Fibril Diameter Fibril Diameter easiest to measure property first attempts were a bit crude better with increased resolution

Sasaki J Mat Sci Tech 2002 Thalhammer J Arch Sci 2001

Bozec Ultramicroscopy 2005

BBML - All R

ights Reserve

d

Collagen in Bone Fibril Diameter

Caution must be taken when reporting and interpreting diameters measured from fibrils in bone bull Fibril packing - width may not be fully exposed

Wallace et al Langmuir 2010

BBML - All R

ights Reserve

d

Collagen in Bone Fibril Diameter

Caution must be taken when reporting and interpreting diameters measured from fibrils in bone bull Fibril packing - width may not be fully exposed

Rigozzi ndash J Struct Bio - 2011

BBML - All R

ights Reserve

d

Collagen in Bone Fibril D-Spacing

300 nm 15 nm

N C C N C C N C C N C C

H O H2C CH2 O H

CH2

O OH HHC CH2

CH2

HO

H H

HHN C C

O

H2C CH2

CH2

HN C C

OH

HC CH2

HO

N C C N C C N C C

H O H2C CH2 O

CH2

OH HC CH2

HO

H H

H

CH2 CH2

glycine X - proline Y - hydroxyproline

Hodge and Petruska 1963

Collagen Fibril Axial D-Periodicity BBML - All R

ights Reserve

d

05

101520

0 02 04 06 08 1

Hei

ght (

nm)

Length (microns)

httpremfdartmoutheduimagesindexhtml

Randall Nature and Structure of Collagen 1953

TEM SEM

AFM

D-Spacing = 67 nm

BBML - All R

ights Reserve

d

Collagen in Bone Fibril D-Spacing Many studies have shown observable D-spacing content that near theoretical value

Sasaki 2002 67 plusmn 2 nm Bozek 2005 665 plusmn 14 nm Ge 2007 666 plusmn 38 nm

Habelitz J Structural Bio 2002

A 2002 study in dentin got me interestedhellip

n = 322 wet n = 363 dry

BBML - All R

ights Reserve

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Collagen in Bone Fibril D-Spacing

bull Internal structure bull Wallace et al Bone 2010 46 1349-1354 bull Wallace et al J Stuct Bio 2011 173 146-152 bull Kemp et al J Stuct Bio 2012 180 428-438 bull Warden et al Endocrinology 2013 154(9)

3178-3187 bull Gallant et al Bone 2014 61 191-200 bull Bart et al Conn Tiss Res 2014 In Press

bull Enzymatic and non-

enzymatic cross-linking bull Hammond et al Bone 2014 60 26-32 bull Diaz Gonzalez et al J Biomech 2014 47(3)

681-686 bull Voytik-Harbin et al In Preparation 2014

D-periodic spacing important structural feature that captures aspects related to

Mouse Femur Mid-diaphysis BBML - All R

ights Reserve

d

2D Fast Fourier Transform Analysis

Harmonics

Fibril D-Spacing

Erickson B Wallace JM et al Biotechnology Journal 2013

Raw AFM Image 2D FFT Power Spectrum

BBML - All R

ights Reserve

d

2D Fast Fourier Transform Analysis

Harmonics

Fibril D-Spacing

Raw AFM Image 2D FFT Power Spectrum

0

20000

40000

60000

80000

100000

120000

0 001 002 003 004 005 006

Am

plitu

de (

microV2 )

Position (1nm)

D-periodic spacing 0015 (1nm) ~ 67 nm

BBML - All R

ights Reserve

d

D-Spacing Distribution

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Overall Mean 670 plusmn 00 nm

BBML - All R

ights Reserve

d

D-Spacing Distribution

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Overall Mean 670 plusmn 00 nm

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

4 Samples Total of n = 255 fibrils Overall Mean 682 plusmn 12 nm

BBML - All R

ights Reserve

d

Cumulative Distribution Function

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

This population 255 fibrils - Each fibril 1255 = 0392 - Data range 628-716 nm

BBML - All R

ights Reserve

d

CDF vs Histogram 62 63 64 65 66 67 68 69 70 71 72 73 74

0

10

20

30

40

50

60

70

80

90

100

0

5

10

15

20

25

30

35

40

45

50

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

HistoCDF

BBML - All R

ights Reserve

d

Changes in D-Spacing with Disease

0

5

10

15

20

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

0

5

10

15

20

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

Wallace et al Bone 2010 46 1349-1354

KS Test plt0001

Control 680 plusmn 26 nm

Estrogen Dep 659 plusmn 31 nm

t-test on mean p=0048

BBML - All R

ights Reserve

d

Changes in D-Spacing with Disease

Wallace et al Bone 2010 46 1349-1354

0

10

20

30

40

50

60

70

80

90

100

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

KS Test plt0001

BBML - All R

ights Reserve

d

Question Model and Tissue Morphology Publication

Tissue Specificity in Mice Male mouse 8 weeks Femur dentin tail tendon

No differences in bone vs dentin both ∆ vs tendon

Wallace et al Langmuir 2011 26(10) 7349-7354

Gender Effects Male and female mice 16 weeks Femur No differences NA

Estrogen Depletion OVX in sheep 5 years + 2 Radius Mean and population shift Wallace et al Bone

2010 46 1349-1354

Estrogen Depletion OVX in female 5 years + 2 Skin (dermis) Significant population shift Erickson et al Biotech

J 2013 8(1) 1174-126

Osteoporosis and Mechanical Loading

OVX in ulnar loading in rats Ulna

No loading effect population shift with OVX

Warden et al Endocrinology 2013 154(9) 3178-3187

Osteogenesis Imperfecta Brtl+ model male mice 2 months Femur

No mean ∆ widening of distribution

Wallace et al J Stuct Bio 2011 173 146-152

Osteogenesis Imperfecta Oimoim model male mice 12 weeks Femur Mean and population shift Bart et al Conn Tiss

Res 2014 In Press

Osteogenesis Imperfecta and Tissue Hydration

Brtl+ model male mice 6 months Tail Tendon Significant population shift Kemp et al J Stuct Bio

2012 180 428-438

Type 2 Diabetes Male ZDSD rats 30 weeks Tibia Significant population shift Hammond et al Bone

2014 60 26-32

Type 2 Diabetes Female ZDSD rats 32 weeks Tail Tendon Significant population shift

Diaz Gonzalez et al J Biomech 2014 47(3)

681-686

Raloxifene (in vivo and in vitro) Female dog treated with Raloxifene Femur Significant population shift Gallant et al Bone

2014 61 191-200 BBML - All R

ights Reserve

d

Issues to Consider Site Selection and Sampling

KS Test plt0001

0023 of bonersquos length

15 m

m =

150

00 micro

m

Wallace et al Langmuir 2011 26(10) 7349-7354

100 microm x 100 microm

35 microm x 35 microm

BBML - All R

ights Reserve

d

Issues to Consider Site Selection and Sampling

60

62

64

66

68

70

72

74

1 (35)

2 (33)

3 (40)

4 (39)

5 (35)

6 (29)

7 (26)

8 (23)

9 (28)

WT (288)

D-P

erio

dic

Gap

Ove

rlap

Spac

ing

(nm

)

Axial Location (Proximal End = 1 Distal End = 9)

( of Fibrils)

91

23

45

87

6

Wallace et al Langmuir 2011 26(10) 7349-7354

288 fibrils for population n=1 for mean comparisons BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

Wet Tendon Dry Tendon Kemp et al J StructBio 2012 180 (3) 428-438

Collagen and bone exist in a hydrated environment in vivo bull Hydration state can impact morphology and mechanics

BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

Nonsignificant mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet (n=255) 682 plusmn 12 nm

WT Dry (n=257) 686 plusmn 08 nm

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

Brtl DryBrtl Wet

KS Test plt0001

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

WT Case 04 nm mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet 682 plusmn 12 nm

WT Dry 686 plusmn 08 nm

Brtl+ Wet 675 plusmn 14 nm

Brtl+ Dry 689 plusmn 10 nm

Brtl+ Case 14 nm increase with drying (plt0001) bull Entire population shifted upward (plt0001)

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT WetBrtl Wet

KS Test plt0001

Brtl+ distribution shifted downward vs WT when wet

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryBrtl Dry

KS Test plt0001

Brtl+ distribution shifted upward vs WT when dry

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider AFM calibration

bull Absolute distance measurements require accurate piezo calibration

bull AFM systems have large scan ranges (150 microm to lt 1 microm)

bull Scanner non-linearities can introduce substantial error over that range

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

ights Reserve

d

Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

BBML - All R

ights Reserve

d

Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

100 nm 10 80 602 1198 100 nm 7 60 827 1439 100 nm 5 40 814 1427 100 nm 35 20 917 1724 100 nm 2 10 937 1774

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

ights Reserve

d

AFM calibration ndash Take Away Message

Calibration using a scan size and feature size comparable to features of interest is highly recommended

Calibration addresses absolute accuracy not precision (differential sensitivity between measurements)

bull Does not limit ability to differentiate between groups using the same AFM with the same calibration parameters

BBML - All R

ights Reserve

d

AFM Applications in Bone Research Indentation Mechanics

BBML - All R

ights Reserve

d

AFM-Based Indentation

In addition to high resolution imaging AFM can be used to extract nanoscale mechanical data

ApproachWithdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Can

tilev

er D

efle

ctio

n (V

olts

)

1 Ramp into a rigid sample ndash measure the slope

Deflection sensitivity x 119899119899119899119899119881119881

2 Calculate cantilever spring constant

3 Hookersquos law takes deflection (volts) and converts to units of force F = kx

Spring constant k 119873119873119899119899

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Forc

e (n

N)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

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ights Reserve

d

AFM-Based Indentation Cantilever Calibration Fo

rce

(nN

)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

Tip-Sample Separation (nm)

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ights Reserve

d

AFM-Based Indentation Parameters

Approach Withdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

Peak Force

Area Between Curves Energy Dissipation

Contact Mechanics Fit for Elastic Modulus (E)

Adhesion Force

Indentation Depth (δ)

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

z

r

δ

R

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119877119877 ∙ 12057512057532

Es Sample modulus νs Poissonrsquos ratio R tip radius of curvature δ load point displacement

Relevant Hertz Assumptions bull Strains are small and within the elastic limit bull Indenter is infinitely stiff (only sample deforms) bull Indentation depth ltlt radius of curvature of indenter bull Surfaces are continuous non-conforming (must be flat) bull Load is normal to surface

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ights Reserve

d

Proper Indentation Model is Essential

δ

r

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119903119903 ∙ 12057512057532

δ

α

119865119865 =2120587120587∙

1198641198641199041199041 minus 1205841205841199041199042

∙ tan120572120572 ∙ 1205751205752

Es Sample modulus νs Poissonrsquos ratio R tip radius α opening angle

Hertz Model Spherical Indenter

depth ltlt radius of curvature

Sneddon Model Conical Indenter

depth ge radius of curvature

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ights Reserve

d

Indentation in OI Tendon Study

x

z

yBrtl+ and WT mice 6 month old males

4 tails per group

2-3 fascicles per tail

25-30 fibrils per tail

4-5 locations per fibril

Indent force 20 nN wet (Sneddon) 50 nN dry (Hertz) BBML - A

ll Rights R

eserved

OI Tendon Differential Effects of Dehydration

Dry 1268 plusmn 718 MPa

Wet 305 plusmn 243 MPa

Dry 1737 plusmn 973 MPa

Wet 301 plusmn 202 MPa

Kemp et al J StructBio 2012 180 (3) 428-438

bull Significant stiffening of fibrils with dehydration

bull More pronounced modulus modulus as a function of disease

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ights Reserve

d

OI Tendon Differential Effects of Dehydration

Kemp et al J StructBio 2012 180 (3) 428-438

Diseased phenotype not present when wet phenotype observed when dried

0

10

20

30

40

50

60

0 20 40 60 80 100 120 140 160 180 200

Gro

up S

ampl

es (

)

Indent Modulus (MPa)

WT WetBrtl Wet

0

2

4

6

8

10

12

14

16

18

20

025

050

075

010

0012

5015

0017

5020

0022

5025

0027

5030

0032

5035

0037

5040

0042

5045

0047

5050

00

Gro

up S

ampl

es (

)Indent Modulus (MPa)

WT DryBrtl Dry

555 WT Indents 305 plusmn 243 MPa

614 Brtl+ Indents 301 plusmn 202 MPa

KS Test p=0129 t Test p=0464 464 WT Indents

1268 plusmn 718 MPa 479 Brtl+ Indents 1737 plusmn 973 MPa

KS Test plt0001 T Test plt0001

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ights Reserve

d

Indentation in Diabetic Tendon (Poster 0495)

x

z

y

Control and Diabetic Rat Tail Tendon

4-5 rats per group

2-3 fascicles per rat

~70 fibrilstail

4-5 locationsfibril

20 nN (Sneddon)

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ights Reserve

d

Multiscale Diabetes Study (Poster 0495)

0

5

10

15

20

25

300 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 gt25

Perc

enta

ge o

f Gro

up S

ampl

es

Elastic Modulus (MPa)

CD (1530 indents)ZDSD (913 indents)

CD (n=5) 381 plusmn 149 MPa

ZDSD (n=3) 706 plusmn 263 MPa

0

100

200

300

400

500

600

700

800

900

0 2 4 6 8 10

Mic

rosc

ale

Mod

ulus

(MPa

)

Nanoscale Modulus (MPa)

plt0001

r2=0531 p=0040

bull Increased indentation modulus in diabetic tendon

bull Strong relationship between nanoscale indent modulus and microscale tensile modulus

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ights Reserve

d

Indentation Limitations and Weaknesses

Proper probe selection for intended application bull Cantilever must be stiff relative to sample bull Sharp probes may damage sample easily dull

R or α must be determined for each probe bull Properties provided by manufacturer are unreliable bull Vary from probe to probe can dull during indent process

Spring constant must be determined for each probe

bull Value provided by manufacturer is inaccurate bull Thermal tuning OK for ldquosoftrdquo probes poor performance

for stiffer probes bull Sader method (geometry) added mass reference

cantilevers

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ights Reserve

d

Indentation Limitations and Weaknesses

Veeco Application Technical note Ben Ohler Practical Advice on the Determination of Cantilever Spring Constants

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d

Indentation Limitations and Weaknesses

bull Sample processing

bull Sample hydration

bull Proper force range

bull Substrate effects for thin samples

bull Proper modulus model and range of data goodness of fit

Bearing in mind these assumptions and limitations AFM can provide true ldquonanoscalerdquo mechanical properties both in terms of forces and deformations

BBML - All R

ights Reserve

d

A Note on Peak Force QNM for Bruker AFMs bull Mechanical properties mapped pixel-by-pixel at same

resolution as the height image (PeakForce QNM)

Height Modulus BBML - All R

ights Reserve

d

A Note on Peak Force QNM

Correct probe stiffness must be used based on sample Pittenger ndash Veeco Product Bulletin

BBML - All R

ights Reserve

d

A Note on Peak Force QNM Extremely sensitive to calibration (details rarely given) Absolute radius and spring constant known

bull especially important from probe to probe bull radius uarr with indent depth ndash must determine R at depth

you expect to image bull Depth may vary as a function of sample (or at different

regions within the sample)

Relative reference material of ldquoknownrdquo modulus used to determine a ratio of 119896119896 119877119877 bull ldquoknownrdquo modulus may come from tension compression

and not indentation bull R is related to depth indentation depth on sample must

be same as on reference BBML - All R

ights Reserve

d

A Note on Peak Force QNM bull Modulus fit on the fly ndash no ability to monitor fit (or range) bull Local sample topography and sample tilt play a role in

measured parameters bull Assuming all modulus values in an image are correct how to

quantify

512 x 512 pixels = 262144 measurements

n=1 n=262144

n from selected ROIs

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ights Reserve

d

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d

bull Imaging in air on mica ndash substrate effect

bull Same imaging force or indent depth for all

bull For each area were all modulus values pooled

Lamprou et al PLoS ONE 2013 BBML - All R

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d

AFM Applications in Bone Research Other Cool Mechanical Techniques

BBML - All R

ights Reserve

d

ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo Bonds within or between collagen fibrils in bone that rupture to dissipate energy as a toughening mechanism

Thompson et al Nature 2001

Fantner et al Nature Mat 2005

Hansma et al JMNI 2005

Fantner et al Nano Letters 2007 BBML - All R

ights Reserve

d

Other Mechanical Techniques

Jimenez-Palomar J Mech Beh Bio Mat 2012

van der Rijt Macro Bio 2004

Yang J Biomed Mater Res 2007

Yang Biophysical Journal 2008 BBML - All R

ights Reserve

d

Other Cool Techniques bull Piezoresponse Force Microscopy (PFM)

bull Electrochemical AFM (EC-AFM)

bull Force Modulation Microscopy (FMM)

bull Lateral Force Microscopy (LFM)

bull Magnetic Force Microscopy (MFM)

bull Conductive AFM (C-AFM)

bull Force Pulling

bull Cantilever Bending

bull Tensile testing of individual fibrils

bull 3 point bending of fibrils

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ights Reserve

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Summary Things to rememberhellip

bull Imaging mode imaging medium and probe selection

bull Morphology Measurements bull Sample preparation and hydration state bull Measuring fibril diameter bull Site selection and proper sampling bull Appropriate system calibration

bull Mechanical Measurements

bull Probe selection (stiffness and tip radius) bull Probe calibration (radius angle spring constant) bull Indentation force range and mechanical model bull Hydration in biological samples bull PeakForce QNM

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d

Acknowledgements BBML

Max Hammond Armando Diaz Gonzalez Zachary Bart Silvia Canelon Creasy Clauser

IU School of Medicine Dr David Burr Dr Matt Allen Dr Stuart Warden Dr Lilian Plotkin

Funding Sources NIHNIDCR 1F32DE018840-01 A1 IUPUI BME Departmental Startup Funds IUPUI Research Support Funds Grant IUPUI Biomechanics and Biomaterials Research Center

NIH NICHD Dr Joan Marini

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ights Reserve

d

Bone Biology and Mechanics Lab (BBML) wwwiupuiedu~bbml Facebook BoneBiologyMechanicsLab

BBML - All R

ights Reserve

d

• Slide Number 1
• General Seminar Outline
• Bonersquos Hierarchical Structure
• Critical Gap in Understanding Collagen
• Methods For Analyzing Collagen Structure
• Methods For Imaging Collagen Structure
• Cryo-TEM
• Atomic Force Microscopy (AFM)
• AFM Image of Pentacene (C22H14)
• AFM Image of Hexabenzocoronene
• Basics of AFM Imaging The Probe
• Basics of AFM Imaging System Components
• van der Waals Potential Energy Curve
• Contact Mode ndash Monitor Deflection
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Peak Force Tapping Mode ndash Monitor Force
• AFM Applications in Bone Research Imaging
• AFM Imaging of Collagen
• Imaging of Osteoclast Activity
• Mineralized collagen deposition in vitro
• Imaging of Canilicular Structure
• AFM Imaging in Bone
• AFM Imaging in Bone
• Removing Mineral in Bone to Expose Collagen
• Removing Mineral in Bone to Expose Collagen
• Demineralization with EDTA
• Demineralization with EDTA
• Demineralization with EDTA
• Issues to Consider Effects of Acid
• Slide Number 32
• Slide Number 33
• Analysis of Collagen Structure in Bone
• Analysis of Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril D-Spacing
• Slide Number 39
• Collagen in Bone Fibril D-Spacing
• Collagen in Bone Fibril D-Spacing
• 2D Fast Fourier Transform Analysis
• 2D Fast Fourier Transform Analysis
• D-Spacing Distribution
• D-Spacing Distribution
• Cumulative Distribution Function
• CDF vs Histogram
• Changes in D-Spacing with Disease
• Changes in D-Spacing with Disease
• Slide Number 50
• Issues to Consider Site Selection and Sampling
• Issues to Consider Site Selection and Sampling
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• AFM calibration ndash Take Away Message
• AFM Applications in Bone ResearchIndentation Mechanics
• AFM-Based Indentation
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Parameters
• Hertz Contact Equation
• Hertz Contact Equation
• Hertz Contact Equation
• Proper Indentation Model is Essential
• Indentation in OI Tendon Study
• OI Tendon Differential Effects of Dehydration
• OI Tendon Differential Effects of Dehydration
• Indentation in Diabetic Tendon (Poster 0495)
• Multiscale Diabetes Study (Poster 0495)
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• A Note on Peak Force QNM for Bruker AFMs
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• Slide Number 83
• Slide Number 84
• AFM Applications in Bone ResearchOther Cool Mechanical Techniques
• ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo
• Other Mechanical Techniques
• Other Cool Techniques
• Summary Things to rememberhellip
• Acknowledgements
• Bone Biology and Mechanics Lab (BBML)

Imaging of Canilicular Structure

Reilly Annals of BME 2001

Lin J of Microscopy 2010

Average canilicular diameter ~400-500 nm larger than values typically reported from TEM studies

BBML - All R

ights Reserve

d

AFM Imaging in Bone In general we are more interested in collagen in bone

3 microm Diamond Suspension

Unpolished

BBML - All R

ights Reserve

d

AFM Imaging in Bone

The surface is completely mineralizedhellip BBML - All R

ights Reserve

d

Removing Mineral in Bone to Expose Collagen

Dealing with mineral is a challenge to analyzing collagen ndash some mineral must be removed

bull 10 Citric acid (to remove mineral) 65 Sodium Hypochlorite (to remove non-collagenous proteins) Habelitz J Struct Bio 2002

bull 7 or 17 Phosphoric Acid Bozek IEEE 2005 El Feninat J Biomed Mat Res 1998

bull 5 Formic Acid Ge Mat Sci Eng 2007

bull 30 mM EDTA Kindt Nanotechnology 2007

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ights Reserve

d

Removing Mineral in Bone to Expose Collagen

Dealing with mineral is a challenge to analyzing collagen ndash some mineral must be removed

bull 30 mM EDTA Kindt Nanotechnology 2007 BBML - A

ll Rights R

eserved

Demineralization with EDTA EDTA - Ethylenediaminetetraacetic Acid

Chelating agent - sequesters divalent cations (notably Ca2+)

bull Treat with 05M EDTA pH 8 (15-20 minutes) bull Vigorously wash with ultrapure water bull Sonicate to release surface-bound mineral ions bull Repeat 3-4 times

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d

Demineralization with EDTA

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d

Demineralization with EDTA

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d

Issues to Consider Effects of Acid

Immature collagen crosslinks are very sensitive to acid bull Using acids to demineralize bone may inadvertently

destroy crosslinks distort structure

Fixation of tissue may be necessary prior to demineralization bull Fixation (glutaraldehyde or paraformadehyde) may

preserve structural integrity

bull Fixation effects on collagen structure are not fully known

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d

100 microm x 100 microm

50 microm x 50 microm

25 microm x 25 microm

10 microm x 10 microm

35 microm x 35 microm

BBML - All R

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d

35 microm x 35 microm

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ights Reserve

d

Analysis of Collagen Structure in Bone Spatial organization of newly formed bone following a surgically-induced injury (rat tibial hole)

Baranauskas J Vac Sci Tech 2001 Baranauskas App Surface Sci 2005

ldquohellipstatistical diameter of the fibers is 159 nm in good agreement with the literature values for type I collagen fibersrdquo

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ights Reserve

d

Analysis of Collagen in Bone Fibril Diameter Fibril Diameter easiest to measure property first attempts were a bit crude better with increased resolution

Sasaki J Mat Sci Tech 2002 Thalhammer J Arch Sci 2001

Bozec Ultramicroscopy 2005

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Collagen in Bone Fibril Diameter

Caution must be taken when reporting and interpreting diameters measured from fibrils in bone bull Fibril packing - width may not be fully exposed

Wallace et al Langmuir 2010

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Collagen in Bone Fibril Diameter

Caution must be taken when reporting and interpreting diameters measured from fibrils in bone bull Fibril packing - width may not be fully exposed

Rigozzi ndash J Struct Bio - 2011

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d

Collagen in Bone Fibril D-Spacing

300 nm 15 nm

N C C N C C N C C N C C

H O H2C CH2 O H

CH2

O OH HHC CH2

CH2

HO

H H

HHN C C

O

H2C CH2

CH2

HN C C

OH

HC CH2

HO

N C C N C C N C C

H O H2C CH2 O

CH2

OH HC CH2

HO

H H

H

CH2 CH2

glycine X - proline Y - hydroxyproline

Hodge and Petruska 1963

Collagen Fibril Axial D-Periodicity BBML - All R

ights Reserve

d

05

101520

0 02 04 06 08 1

Hei

ght (

nm)

Length (microns)

httpremfdartmoutheduimagesindexhtml

Randall Nature and Structure of Collagen 1953

TEM SEM

AFM

D-Spacing = 67 nm

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ights Reserve

d

Collagen in Bone Fibril D-Spacing Many studies have shown observable D-spacing content that near theoretical value

Sasaki 2002 67 plusmn 2 nm Bozek 2005 665 plusmn 14 nm Ge 2007 666 plusmn 38 nm

Habelitz J Structural Bio 2002

A 2002 study in dentin got me interestedhellip

n = 322 wet n = 363 dry

BBML - All R

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Collagen in Bone Fibril D-Spacing

bull Internal structure bull Wallace et al Bone 2010 46 1349-1354 bull Wallace et al J Stuct Bio 2011 173 146-152 bull Kemp et al J Stuct Bio 2012 180 428-438 bull Warden et al Endocrinology 2013 154(9)

3178-3187 bull Gallant et al Bone 2014 61 191-200 bull Bart et al Conn Tiss Res 2014 In Press

bull Enzymatic and non-

enzymatic cross-linking bull Hammond et al Bone 2014 60 26-32 bull Diaz Gonzalez et al J Biomech 2014 47(3)

681-686 bull Voytik-Harbin et al In Preparation 2014

D-periodic spacing important structural feature that captures aspects related to

Mouse Femur Mid-diaphysis BBML - All R

ights Reserve

d

2D Fast Fourier Transform Analysis

Harmonics

Fibril D-Spacing

Erickson B Wallace JM et al Biotechnology Journal 2013

Raw AFM Image 2D FFT Power Spectrum

BBML - All R

ights Reserve

d

2D Fast Fourier Transform Analysis

Harmonics

Fibril D-Spacing

Raw AFM Image 2D FFT Power Spectrum

0

20000

40000

60000

80000

100000

120000

0 001 002 003 004 005 006

Am

plitu

de (

microV2 )

Position (1nm)

D-periodic spacing 0015 (1nm) ~ 67 nm

BBML - All R

ights Reserve

d

D-Spacing Distribution

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Overall Mean 670 plusmn 00 nm

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ights Reserve

d

D-Spacing Distribution

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Overall Mean 670 plusmn 00 nm

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

4 Samples Total of n = 255 fibrils Overall Mean 682 plusmn 12 nm

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d

Cumulative Distribution Function

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

This population 255 fibrils - Each fibril 1255 = 0392 - Data range 628-716 nm

BBML - All R

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d

CDF vs Histogram 62 63 64 65 66 67 68 69 70 71 72 73 74

0

10

20

30

40

50

60

70

80

90

100

0

5

10

15

20

25

30

35

40

45

50

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

HistoCDF

BBML - All R

ights Reserve

d

Changes in D-Spacing with Disease

0

5

10

15

20

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

0

5

10

15

20

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

Wallace et al Bone 2010 46 1349-1354

KS Test plt0001

Control 680 plusmn 26 nm

Estrogen Dep 659 plusmn 31 nm

t-test on mean p=0048

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Changes in D-Spacing with Disease

Wallace et al Bone 2010 46 1349-1354

0

10

20

30

40

50

60

70

80

90

100

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

KS Test plt0001

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ights Reserve

d

Question Model and Tissue Morphology Publication

Tissue Specificity in Mice Male mouse 8 weeks Femur dentin tail tendon

No differences in bone vs dentin both ∆ vs tendon

Wallace et al Langmuir 2011 26(10) 7349-7354

Gender Effects Male and female mice 16 weeks Femur No differences NA

Estrogen Depletion OVX in sheep 5 years + 2 Radius Mean and population shift Wallace et al Bone

2010 46 1349-1354

Estrogen Depletion OVX in female 5 years + 2 Skin (dermis) Significant population shift Erickson et al Biotech

J 2013 8(1) 1174-126

Osteoporosis and Mechanical Loading

OVX in ulnar loading in rats Ulna

No loading effect population shift with OVX

Warden et al Endocrinology 2013 154(9) 3178-3187

Osteogenesis Imperfecta Brtl+ model male mice 2 months Femur

No mean ∆ widening of distribution

Wallace et al J Stuct Bio 2011 173 146-152

Osteogenesis Imperfecta Oimoim model male mice 12 weeks Femur Mean and population shift Bart et al Conn Tiss

Res 2014 In Press

Osteogenesis Imperfecta and Tissue Hydration

Brtl+ model male mice 6 months Tail Tendon Significant population shift Kemp et al J Stuct Bio

2012 180 428-438

Type 2 Diabetes Male ZDSD rats 30 weeks Tibia Significant population shift Hammond et al Bone

2014 60 26-32

Type 2 Diabetes Female ZDSD rats 32 weeks Tail Tendon Significant population shift

Diaz Gonzalez et al J Biomech 2014 47(3)

681-686

Raloxifene (in vivo and in vitro) Female dog treated with Raloxifene Femur Significant population shift Gallant et al Bone

2014 61 191-200 BBML - All R

ights Reserve

d

Issues to Consider Site Selection and Sampling

KS Test plt0001

0023 of bonersquos length

15 m

m =

150

00 micro

m

Wallace et al Langmuir 2011 26(10) 7349-7354

100 microm x 100 microm

35 microm x 35 microm

BBML - All R

ights Reserve

d

Issues to Consider Site Selection and Sampling

60

62

64

66

68

70

72

74

1 (35)

2 (33)

3 (40)

4 (39)

5 (35)

6 (29)

7 (26)

8 (23)

9 (28)

WT (288)

D-P

erio

dic

Gap

Ove

rlap

Spac

ing

(nm

)

Axial Location (Proximal End = 1 Distal End = 9)

( of Fibrils)

91

23

45

87

6

Wallace et al Langmuir 2011 26(10) 7349-7354

288 fibrils for population n=1 for mean comparisons BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

Wet Tendon Dry Tendon Kemp et al J StructBio 2012 180 (3) 428-438

Collagen and bone exist in a hydrated environment in vivo bull Hydration state can impact morphology and mechanics

BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

Nonsignificant mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet (n=255) 682 plusmn 12 nm

WT Dry (n=257) 686 plusmn 08 nm

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

Brtl DryBrtl Wet

KS Test plt0001

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

WT Case 04 nm mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet 682 plusmn 12 nm

WT Dry 686 plusmn 08 nm

Brtl+ Wet 675 plusmn 14 nm

Brtl+ Dry 689 plusmn 10 nm

Brtl+ Case 14 nm increase with drying (plt0001) bull Entire population shifted upward (plt0001)

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT WetBrtl Wet

KS Test plt0001

Brtl+ distribution shifted downward vs WT when wet

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryBrtl Dry

KS Test plt0001

Brtl+ distribution shifted upward vs WT when dry

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider AFM calibration

bull Absolute distance measurements require accurate piezo calibration

bull AFM systems have large scan ranges (150 microm to lt 1 microm)

bull Scanner non-linearities can introduce substantial error over that range

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

ights Reserve

d

Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

BBML - All R

ights Reserve

d

Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

100 nm 10 80 602 1198 100 nm 7 60 827 1439 100 nm 5 40 814 1427 100 nm 35 20 917 1724 100 nm 2 10 937 1774

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

ights Reserve

d

AFM calibration ndash Take Away Message

Calibration using a scan size and feature size comparable to features of interest is highly recommended

Calibration addresses absolute accuracy not precision (differential sensitivity between measurements)

bull Does not limit ability to differentiate between groups using the same AFM with the same calibration parameters

BBML - All R

ights Reserve

d

AFM Applications in Bone Research Indentation Mechanics

BBML - All R

ights Reserve

d

AFM-Based Indentation

In addition to high resolution imaging AFM can be used to extract nanoscale mechanical data

ApproachWithdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Can

tilev

er D

efle

ctio

n (V

olts

)

1 Ramp into a rigid sample ndash measure the slope

Deflection sensitivity x 119899119899119899119899119881119881

2 Calculate cantilever spring constant

3 Hookersquos law takes deflection (volts) and converts to units of force F = kx

Spring constant k 119873119873119899119899

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Forc

e (n

N)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration Fo

rce

(nN

)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

Tip-Sample Separation (nm)

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ights Reserve

d

AFM-Based Indentation Parameters

Approach Withdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

Peak Force

Area Between Curves Energy Dissipation

Contact Mechanics Fit for Elastic Modulus (E)

Adhesion Force

Indentation Depth (δ)

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

z

r

δ

R

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119877119877 ∙ 12057512057532

Es Sample modulus νs Poissonrsquos ratio R tip radius of curvature δ load point displacement

Relevant Hertz Assumptions bull Strains are small and within the elastic limit bull Indenter is infinitely stiff (only sample deforms) bull Indentation depth ltlt radius of curvature of indenter bull Surfaces are continuous non-conforming (must be flat) bull Load is normal to surface

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ights Reserve

d

Proper Indentation Model is Essential

δ

r

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119903119903 ∙ 12057512057532

δ

α

119865119865 =2120587120587∙

1198641198641199041199041 minus 1205841205841199041199042

∙ tan120572120572 ∙ 1205751205752

Es Sample modulus νs Poissonrsquos ratio R tip radius α opening angle

Hertz Model Spherical Indenter

depth ltlt radius of curvature

Sneddon Model Conical Indenter

depth ge radius of curvature

BBML - All R

ights Reserve

d

Indentation in OI Tendon Study

x

z

yBrtl+ and WT mice 6 month old males

4 tails per group

2-3 fascicles per tail

25-30 fibrils per tail

4-5 locations per fibril

Indent force 20 nN wet (Sneddon) 50 nN dry (Hertz) BBML - A

ll Rights R

eserved

OI Tendon Differential Effects of Dehydration

Dry 1268 plusmn 718 MPa

Wet 305 plusmn 243 MPa

Dry 1737 plusmn 973 MPa

Wet 301 plusmn 202 MPa

Kemp et al J StructBio 2012 180 (3) 428-438

bull Significant stiffening of fibrils with dehydration

bull More pronounced modulus modulus as a function of disease

BBML - All R

ights Reserve

d

OI Tendon Differential Effects of Dehydration

Kemp et al J StructBio 2012 180 (3) 428-438

Diseased phenotype not present when wet phenotype observed when dried

0

10

20

30

40

50

60

0 20 40 60 80 100 120 140 160 180 200

Gro

up S

ampl

es (

)

Indent Modulus (MPa)

WT WetBrtl Wet

0

2

4

6

8

10

12

14

16

18

20

025

050

075

010

0012

5015

0017

5020

0022

5025

0027

5030

0032

5035

0037

5040

0042

5045

0047

5050

00

Gro

up S

ampl

es (

)Indent Modulus (MPa)

WT DryBrtl Dry

555 WT Indents 305 plusmn 243 MPa

614 Brtl+ Indents 301 plusmn 202 MPa

KS Test p=0129 t Test p=0464 464 WT Indents

1268 plusmn 718 MPa 479 Brtl+ Indents 1737 plusmn 973 MPa

KS Test plt0001 T Test plt0001

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ights Reserve

d

Indentation in Diabetic Tendon (Poster 0495)

x

z

y

Control and Diabetic Rat Tail Tendon

4-5 rats per group

2-3 fascicles per rat

~70 fibrilstail

4-5 locationsfibril

20 nN (Sneddon)

BBML - All R

ights Reserve

d

Multiscale Diabetes Study (Poster 0495)

0

5

10

15

20

25

300 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 gt25

Perc

enta

ge o

f Gro

up S

ampl

es

Elastic Modulus (MPa)

CD (1530 indents)ZDSD (913 indents)

CD (n=5) 381 plusmn 149 MPa

ZDSD (n=3) 706 plusmn 263 MPa

0

100

200

300

400

500

600

700

800

900

0 2 4 6 8 10

Mic

rosc

ale

Mod

ulus

(MPa

)

Nanoscale Modulus (MPa)

plt0001

r2=0531 p=0040

bull Increased indentation modulus in diabetic tendon

bull Strong relationship between nanoscale indent modulus and microscale tensile modulus

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Indentation Limitations and Weaknesses

Proper probe selection for intended application bull Cantilever must be stiff relative to sample bull Sharp probes may damage sample easily dull

R or α must be determined for each probe bull Properties provided by manufacturer are unreliable bull Vary from probe to probe can dull during indent process

Spring constant must be determined for each probe

bull Value provided by manufacturer is inaccurate bull Thermal tuning OK for ldquosoftrdquo probes poor performance

for stiffer probes bull Sader method (geometry) added mass reference

cantilevers

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d

Indentation Limitations and Weaknesses

Veeco Application Technical note Ben Ohler Practical Advice on the Determination of Cantilever Spring Constants

BBML - All R

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d

Indentation Limitations and Weaknesses

bull Sample processing

bull Sample hydration

bull Proper force range

bull Substrate effects for thin samples

bull Proper modulus model and range of data goodness of fit

Bearing in mind these assumptions and limitations AFM can provide true ldquonanoscalerdquo mechanical properties both in terms of forces and deformations

BBML - All R

ights Reserve

d

A Note on Peak Force QNM for Bruker AFMs bull Mechanical properties mapped pixel-by-pixel at same

resolution as the height image (PeakForce QNM)

Height Modulus BBML - All R

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d

A Note on Peak Force QNM

Correct probe stiffness must be used based on sample Pittenger ndash Veeco Product Bulletin

BBML - All R

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d

A Note on Peak Force QNM Extremely sensitive to calibration (details rarely given) Absolute radius and spring constant known

bull especially important from probe to probe bull radius uarr with indent depth ndash must determine R at depth

you expect to image bull Depth may vary as a function of sample (or at different

regions within the sample)

Relative reference material of ldquoknownrdquo modulus used to determine a ratio of 119896119896 119877119877 bull ldquoknownrdquo modulus may come from tension compression

and not indentation bull R is related to depth indentation depth on sample must

be same as on reference BBML - All R

ights Reserve

d

A Note on Peak Force QNM bull Modulus fit on the fly ndash no ability to monitor fit (or range) bull Local sample topography and sample tilt play a role in

measured parameters bull Assuming all modulus values in an image are correct how to

quantify

512 x 512 pixels = 262144 measurements

n=1 n=262144

n from selected ROIs

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d

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d

bull Imaging in air on mica ndash substrate effect

bull Same imaging force or indent depth for all

bull For each area were all modulus values pooled

Lamprou et al PLoS ONE 2013 BBML - All R

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d

AFM Applications in Bone Research Other Cool Mechanical Techniques

BBML - All R

ights Reserve

d

ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo Bonds within or between collagen fibrils in bone that rupture to dissipate energy as a toughening mechanism

Thompson et al Nature 2001

Fantner et al Nature Mat 2005

Hansma et al JMNI 2005

Fantner et al Nano Letters 2007 BBML - All R

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d

Other Mechanical Techniques

Jimenez-Palomar J Mech Beh Bio Mat 2012

van der Rijt Macro Bio 2004

Yang J Biomed Mater Res 2007

Yang Biophysical Journal 2008 BBML - All R

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d

Other Cool Techniques bull Piezoresponse Force Microscopy (PFM)

bull Electrochemical AFM (EC-AFM)

bull Force Modulation Microscopy (FMM)

bull Lateral Force Microscopy (LFM)

bull Magnetic Force Microscopy (MFM)

bull Conductive AFM (C-AFM)

bull Force Pulling

bull Cantilever Bending

bull Tensile testing of individual fibrils

bull 3 point bending of fibrils

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d

Summary Things to rememberhellip

bull Imaging mode imaging medium and probe selection

bull Morphology Measurements bull Sample preparation and hydration state bull Measuring fibril diameter bull Site selection and proper sampling bull Appropriate system calibration

bull Mechanical Measurements

bull Probe selection (stiffness and tip radius) bull Probe calibration (radius angle spring constant) bull Indentation force range and mechanical model bull Hydration in biological samples bull PeakForce QNM

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Acknowledgements BBML

Max Hammond Armando Diaz Gonzalez Zachary Bart Silvia Canelon Creasy Clauser

IU School of Medicine Dr David Burr Dr Matt Allen Dr Stuart Warden Dr Lilian Plotkin

Funding Sources NIHNIDCR 1F32DE018840-01 A1 IUPUI BME Departmental Startup Funds IUPUI Research Support Funds Grant IUPUI Biomechanics and Biomaterials Research Center

NIH NICHD Dr Joan Marini

BBML - All R

ights Reserve

d

Bone Biology and Mechanics Lab (BBML) wwwiupuiedu~bbml Facebook BoneBiologyMechanicsLab

BBML - All R

ights Reserve

d

• Slide Number 1
• General Seminar Outline
• Bonersquos Hierarchical Structure
• Critical Gap in Understanding Collagen
• Methods For Analyzing Collagen Structure
• Methods For Imaging Collagen Structure
• Cryo-TEM
• Atomic Force Microscopy (AFM)
• AFM Image of Pentacene (C22H14)
• AFM Image of Hexabenzocoronene
• Basics of AFM Imaging The Probe
• Basics of AFM Imaging System Components
• van der Waals Potential Energy Curve
• Contact Mode ndash Monitor Deflection
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Peak Force Tapping Mode ndash Monitor Force
• AFM Applications in Bone Research Imaging
• AFM Imaging of Collagen
• Imaging of Osteoclast Activity
• Mineralized collagen deposition in vitro
• Imaging of Canilicular Structure
• AFM Imaging in Bone
• AFM Imaging in Bone
• Removing Mineral in Bone to Expose Collagen
• Removing Mineral in Bone to Expose Collagen
• Demineralization with EDTA
• Demineralization with EDTA
• Demineralization with EDTA
• Issues to Consider Effects of Acid
• Slide Number 32
• Slide Number 33
• Analysis of Collagen Structure in Bone
• Analysis of Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril D-Spacing
• Slide Number 39
• Collagen in Bone Fibril D-Spacing
• Collagen in Bone Fibril D-Spacing
• 2D Fast Fourier Transform Analysis
• 2D Fast Fourier Transform Analysis
• D-Spacing Distribution
• D-Spacing Distribution
• Cumulative Distribution Function
• CDF vs Histogram
• Changes in D-Spacing with Disease
• Changes in D-Spacing with Disease
• Slide Number 50
• Issues to Consider Site Selection and Sampling
• Issues to Consider Site Selection and Sampling
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• AFM calibration ndash Take Away Message
• AFM Applications in Bone ResearchIndentation Mechanics
• AFM-Based Indentation
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Parameters
• Hertz Contact Equation
• Hertz Contact Equation
• Hertz Contact Equation
• Proper Indentation Model is Essential
• Indentation in OI Tendon Study
• OI Tendon Differential Effects of Dehydration
• OI Tendon Differential Effects of Dehydration
• Indentation in Diabetic Tendon (Poster 0495)
• Multiscale Diabetes Study (Poster 0495)
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• A Note on Peak Force QNM for Bruker AFMs
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• Slide Number 83
• Slide Number 84
• AFM Applications in Bone ResearchOther Cool Mechanical Techniques
• ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo
• Other Mechanical Techniques
• Other Cool Techniques
• Summary Things to rememberhellip
• Acknowledgements
• Bone Biology and Mechanics Lab (BBML)

AFM Imaging in Bone In general we are more interested in collagen in bone

3 microm Diamond Suspension

Unpolished

BBML - All R

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d

AFM Imaging in Bone

The surface is completely mineralizedhellip BBML - All R

ights Reserve

d

Removing Mineral in Bone to Expose Collagen

Dealing with mineral is a challenge to analyzing collagen ndash some mineral must be removed

bull 10 Citric acid (to remove mineral) 65 Sodium Hypochlorite (to remove non-collagenous proteins) Habelitz J Struct Bio 2002

bull 7 or 17 Phosphoric Acid Bozek IEEE 2005 El Feninat J Biomed Mat Res 1998

bull 5 Formic Acid Ge Mat Sci Eng 2007

bull 30 mM EDTA Kindt Nanotechnology 2007

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ights Reserve

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Removing Mineral in Bone to Expose Collagen

Dealing with mineral is a challenge to analyzing collagen ndash some mineral must be removed

bull 30 mM EDTA Kindt Nanotechnology 2007 BBML - A

ll Rights R

eserved

Demineralization with EDTA EDTA - Ethylenediaminetetraacetic Acid

Chelating agent - sequesters divalent cations (notably Ca2+)

bull Treat with 05M EDTA pH 8 (15-20 minutes) bull Vigorously wash with ultrapure water bull Sonicate to release surface-bound mineral ions bull Repeat 3-4 times

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d

Demineralization with EDTA

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d

Demineralization with EDTA

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ights Reserve

d

Issues to Consider Effects of Acid

Immature collagen crosslinks are very sensitive to acid bull Using acids to demineralize bone may inadvertently

destroy crosslinks distort structure

Fixation of tissue may be necessary prior to demineralization bull Fixation (glutaraldehyde or paraformadehyde) may

preserve structural integrity

bull Fixation effects on collagen structure are not fully known

BBML - All R

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d

100 microm x 100 microm

50 microm x 50 microm

25 microm x 25 microm

10 microm x 10 microm

35 microm x 35 microm

BBML - All R

ights Reserve

d

35 microm x 35 microm

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ights Reserve

d

Analysis of Collagen Structure in Bone Spatial organization of newly formed bone following a surgically-induced injury (rat tibial hole)

Baranauskas J Vac Sci Tech 2001 Baranauskas App Surface Sci 2005

ldquohellipstatistical diameter of the fibers is 159 nm in good agreement with the literature values for type I collagen fibersrdquo

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ights Reserve

d

Analysis of Collagen in Bone Fibril Diameter Fibril Diameter easiest to measure property first attempts were a bit crude better with increased resolution

Sasaki J Mat Sci Tech 2002 Thalhammer J Arch Sci 2001

Bozec Ultramicroscopy 2005

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Collagen in Bone Fibril Diameter

Caution must be taken when reporting and interpreting diameters measured from fibrils in bone bull Fibril packing - width may not be fully exposed

Wallace et al Langmuir 2010

BBML - All R

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d

Collagen in Bone Fibril Diameter

Caution must be taken when reporting and interpreting diameters measured from fibrils in bone bull Fibril packing - width may not be fully exposed

Rigozzi ndash J Struct Bio - 2011

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Collagen in Bone Fibril D-Spacing

300 nm 15 nm

N C C N C C N C C N C C

H O H2C CH2 O H

CH2

O OH HHC CH2

CH2

HO

H H

HHN C C

O

H2C CH2

CH2

HN C C

OH

HC CH2

HO

N C C N C C N C C

H O H2C CH2 O

CH2

OH HC CH2

HO

H H

H

CH2 CH2

glycine X - proline Y - hydroxyproline

Hodge and Petruska 1963

Collagen Fibril Axial D-Periodicity BBML - All R

ights Reserve

d

05

101520

0 02 04 06 08 1

Hei

ght (

nm)

Length (microns)

httpremfdartmoutheduimagesindexhtml

Randall Nature and Structure of Collagen 1953

TEM SEM

AFM

D-Spacing = 67 nm

BBML - All R

ights Reserve

d

Collagen in Bone Fibril D-Spacing Many studies have shown observable D-spacing content that near theoretical value

Sasaki 2002 67 plusmn 2 nm Bozek 2005 665 plusmn 14 nm Ge 2007 666 plusmn 38 nm

Habelitz J Structural Bio 2002

A 2002 study in dentin got me interestedhellip

n = 322 wet n = 363 dry

BBML - All R

ights Reserve

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Collagen in Bone Fibril D-Spacing

bull Internal structure bull Wallace et al Bone 2010 46 1349-1354 bull Wallace et al J Stuct Bio 2011 173 146-152 bull Kemp et al J Stuct Bio 2012 180 428-438 bull Warden et al Endocrinology 2013 154(9)

3178-3187 bull Gallant et al Bone 2014 61 191-200 bull Bart et al Conn Tiss Res 2014 In Press

bull Enzymatic and non-

enzymatic cross-linking bull Hammond et al Bone 2014 60 26-32 bull Diaz Gonzalez et al J Biomech 2014 47(3)

681-686 bull Voytik-Harbin et al In Preparation 2014

D-periodic spacing important structural feature that captures aspects related to

Mouse Femur Mid-diaphysis BBML - All R

ights Reserve

d

2D Fast Fourier Transform Analysis

Harmonics

Fibril D-Spacing

Erickson B Wallace JM et al Biotechnology Journal 2013

Raw AFM Image 2D FFT Power Spectrum

BBML - All R

ights Reserve

d

2D Fast Fourier Transform Analysis

Harmonics

Fibril D-Spacing

Raw AFM Image 2D FFT Power Spectrum

0

20000

40000

60000

80000

100000

120000

0 001 002 003 004 005 006

Am

plitu

de (

microV2 )

Position (1nm)

D-periodic spacing 0015 (1nm) ~ 67 nm

BBML - All R

ights Reserve

d

D-Spacing Distribution

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Overall Mean 670 plusmn 00 nm

BBML - All R

ights Reserve

d

D-Spacing Distribution

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Overall Mean 670 plusmn 00 nm

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

4 Samples Total of n = 255 fibrils Overall Mean 682 plusmn 12 nm

BBML - All R

ights Reserve

d

Cumulative Distribution Function

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

This population 255 fibrils - Each fibril 1255 = 0392 - Data range 628-716 nm

BBML - All R

ights Reserve

d

CDF vs Histogram 62 63 64 65 66 67 68 69 70 71 72 73 74

0

10

20

30

40

50

60

70

80

90

100

0

5

10

15

20

25

30

35

40

45

50

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

HistoCDF

BBML - All R

ights Reserve

d

Changes in D-Spacing with Disease

0

5

10

15

20

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

0

5

10

15

20

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

Wallace et al Bone 2010 46 1349-1354

KS Test plt0001

Control 680 plusmn 26 nm

Estrogen Dep 659 plusmn 31 nm

t-test on mean p=0048

BBML - All R

ights Reserve

d

Changes in D-Spacing with Disease

Wallace et al Bone 2010 46 1349-1354

0

10

20

30

40

50

60

70

80

90

100

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

KS Test plt0001

BBML - All R

ights Reserve

d

Question Model and Tissue Morphology Publication

Tissue Specificity in Mice Male mouse 8 weeks Femur dentin tail tendon

No differences in bone vs dentin both ∆ vs tendon

Wallace et al Langmuir 2011 26(10) 7349-7354

Gender Effects Male and female mice 16 weeks Femur No differences NA

Estrogen Depletion OVX in sheep 5 years + 2 Radius Mean and population shift Wallace et al Bone

2010 46 1349-1354

Estrogen Depletion OVX in female 5 years + 2 Skin (dermis) Significant population shift Erickson et al Biotech

J 2013 8(1) 1174-126

Osteoporosis and Mechanical Loading

OVX in ulnar loading in rats Ulna

No loading effect population shift with OVX

Warden et al Endocrinology 2013 154(9) 3178-3187

Osteogenesis Imperfecta Brtl+ model male mice 2 months Femur

No mean ∆ widening of distribution

Wallace et al J Stuct Bio 2011 173 146-152

Osteogenesis Imperfecta Oimoim model male mice 12 weeks Femur Mean and population shift Bart et al Conn Tiss

Res 2014 In Press

Osteogenesis Imperfecta and Tissue Hydration

Brtl+ model male mice 6 months Tail Tendon Significant population shift Kemp et al J Stuct Bio

2012 180 428-438

Type 2 Diabetes Male ZDSD rats 30 weeks Tibia Significant population shift Hammond et al Bone

2014 60 26-32

Type 2 Diabetes Female ZDSD rats 32 weeks Tail Tendon Significant population shift

Diaz Gonzalez et al J Biomech 2014 47(3)

681-686

Raloxifene (in vivo and in vitro) Female dog treated with Raloxifene Femur Significant population shift Gallant et al Bone

2014 61 191-200 BBML - All R

ights Reserve

d

Issues to Consider Site Selection and Sampling

KS Test plt0001

0023 of bonersquos length

15 m

m =

150

00 micro

m

Wallace et al Langmuir 2011 26(10) 7349-7354

100 microm x 100 microm

35 microm x 35 microm

BBML - All R

ights Reserve

d

Issues to Consider Site Selection and Sampling

60

62

64

66

68

70

72

74

1 (35)

2 (33)

3 (40)

4 (39)

5 (35)

6 (29)

7 (26)

8 (23)

9 (28)

WT (288)

D-P

erio

dic

Gap

Ove

rlap

Spac

ing

(nm

)

Axial Location (Proximal End = 1 Distal End = 9)

( of Fibrils)

91

23

45

87

6

Wallace et al Langmuir 2011 26(10) 7349-7354

288 fibrils for population n=1 for mean comparisons BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

Wet Tendon Dry Tendon Kemp et al J StructBio 2012 180 (3) 428-438

Collagen and bone exist in a hydrated environment in vivo bull Hydration state can impact morphology and mechanics

BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

Nonsignificant mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet (n=255) 682 plusmn 12 nm

WT Dry (n=257) 686 plusmn 08 nm

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

Brtl DryBrtl Wet

KS Test plt0001

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

WT Case 04 nm mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet 682 plusmn 12 nm

WT Dry 686 plusmn 08 nm

Brtl+ Wet 675 plusmn 14 nm

Brtl+ Dry 689 plusmn 10 nm

Brtl+ Case 14 nm increase with drying (plt0001) bull Entire population shifted upward (plt0001)

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT WetBrtl Wet

KS Test plt0001

Brtl+ distribution shifted downward vs WT when wet

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryBrtl Dry

KS Test plt0001

Brtl+ distribution shifted upward vs WT when dry

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider AFM calibration

bull Absolute distance measurements require accurate piezo calibration

bull AFM systems have large scan ranges (150 microm to lt 1 microm)

bull Scanner non-linearities can introduce substantial error over that range

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

ights Reserve

d

Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

BBML - All R

ights Reserve

d

Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

100 nm 10 80 602 1198 100 nm 7 60 827 1439 100 nm 5 40 814 1427 100 nm 35 20 917 1724 100 nm 2 10 937 1774

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

ights Reserve

d

AFM calibration ndash Take Away Message

Calibration using a scan size and feature size comparable to features of interest is highly recommended

Calibration addresses absolute accuracy not precision (differential sensitivity between measurements)

bull Does not limit ability to differentiate between groups using the same AFM with the same calibration parameters

BBML - All R

ights Reserve

d

AFM Applications in Bone Research Indentation Mechanics

BBML - All R

ights Reserve

d

AFM-Based Indentation

In addition to high resolution imaging AFM can be used to extract nanoscale mechanical data

ApproachWithdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Can

tilev

er D

efle

ctio

n (V

olts

)

1 Ramp into a rigid sample ndash measure the slope

Deflection sensitivity x 119899119899119899119899119881119881

2 Calculate cantilever spring constant

3 Hookersquos law takes deflection (volts) and converts to units of force F = kx

Spring constant k 119873119873119899119899

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Forc

e (n

N)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration Fo

rce

(nN

)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

Tip-Sample Separation (nm)

BBML - All R

ights Reserve

d

AFM-Based Indentation Parameters

Approach Withdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

Peak Force

Area Between Curves Energy Dissipation

Contact Mechanics Fit for Elastic Modulus (E)

Adhesion Force

Indentation Depth (δ)

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

z

r

δ

R

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119877119877 ∙ 12057512057532

Es Sample modulus νs Poissonrsquos ratio R tip radius of curvature δ load point displacement

Relevant Hertz Assumptions bull Strains are small and within the elastic limit bull Indenter is infinitely stiff (only sample deforms) bull Indentation depth ltlt radius of curvature of indenter bull Surfaces are continuous non-conforming (must be flat) bull Load is normal to surface

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ights Reserve

d

Proper Indentation Model is Essential

δ

r

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119903119903 ∙ 12057512057532

δ

α

119865119865 =2120587120587∙

1198641198641199041199041 minus 1205841205841199041199042

∙ tan120572120572 ∙ 1205751205752

Es Sample modulus νs Poissonrsquos ratio R tip radius α opening angle

Hertz Model Spherical Indenter

depth ltlt radius of curvature

Sneddon Model Conical Indenter

depth ge radius of curvature

BBML - All R

ights Reserve

d

Indentation in OI Tendon Study

x

z

yBrtl+ and WT mice 6 month old males

4 tails per group

2-3 fascicles per tail

25-30 fibrils per tail

4-5 locations per fibril

Indent force 20 nN wet (Sneddon) 50 nN dry (Hertz) BBML - A

ll Rights R

eserved

OI Tendon Differential Effects of Dehydration

Dry 1268 plusmn 718 MPa

Wet 305 plusmn 243 MPa

Dry 1737 plusmn 973 MPa

Wet 301 plusmn 202 MPa

Kemp et al J StructBio 2012 180 (3) 428-438

bull Significant stiffening of fibrils with dehydration

bull More pronounced modulus modulus as a function of disease

BBML - All R

ights Reserve

d

OI Tendon Differential Effects of Dehydration

Kemp et al J StructBio 2012 180 (3) 428-438

Diseased phenotype not present when wet phenotype observed when dried

0

10

20

30

40

50

60

0 20 40 60 80 100 120 140 160 180 200

Gro

up S

ampl

es (

)

Indent Modulus (MPa)

WT WetBrtl Wet

0

2

4

6

8

10

12

14

16

18

20

025

050

075

010

0012

5015

0017

5020

0022

5025

0027

5030

0032

5035

0037

5040

0042

5045

0047

5050

00

Gro

up S

ampl

es (

)Indent Modulus (MPa)

WT DryBrtl Dry

555 WT Indents 305 plusmn 243 MPa

614 Brtl+ Indents 301 plusmn 202 MPa

KS Test p=0129 t Test p=0464 464 WT Indents

1268 plusmn 718 MPa 479 Brtl+ Indents 1737 plusmn 973 MPa

KS Test plt0001 T Test plt0001

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ights Reserve

d

Indentation in Diabetic Tendon (Poster 0495)

x

z

y

Control and Diabetic Rat Tail Tendon

4-5 rats per group

2-3 fascicles per rat

~70 fibrilstail

4-5 locationsfibril

20 nN (Sneddon)

BBML - All R

ights Reserve

d

Multiscale Diabetes Study (Poster 0495)

0

5

10

15

20

25

300 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 gt25

Perc

enta

ge o

f Gro

up S

ampl

es

Elastic Modulus (MPa)

CD (1530 indents)ZDSD (913 indents)

CD (n=5) 381 plusmn 149 MPa

ZDSD (n=3) 706 plusmn 263 MPa

0

100

200

300

400

500

600

700

800

900

0 2 4 6 8 10

Mic

rosc

ale

Mod

ulus

(MPa

)

Nanoscale Modulus (MPa)

plt0001

r2=0531 p=0040

bull Increased indentation modulus in diabetic tendon

bull Strong relationship between nanoscale indent modulus and microscale tensile modulus

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ights Reserve

d

Indentation Limitations and Weaknesses

Proper probe selection for intended application bull Cantilever must be stiff relative to sample bull Sharp probes may damage sample easily dull

R or α must be determined for each probe bull Properties provided by manufacturer are unreliable bull Vary from probe to probe can dull during indent process

Spring constant must be determined for each probe

bull Value provided by manufacturer is inaccurate bull Thermal tuning OK for ldquosoftrdquo probes poor performance

for stiffer probes bull Sader method (geometry) added mass reference

cantilevers

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

Veeco Application Technical note Ben Ohler Practical Advice on the Determination of Cantilever Spring Constants

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

bull Sample processing

bull Sample hydration

bull Proper force range

bull Substrate effects for thin samples

bull Proper modulus model and range of data goodness of fit

Bearing in mind these assumptions and limitations AFM can provide true ldquonanoscalerdquo mechanical properties both in terms of forces and deformations

BBML - All R

ights Reserve

d

A Note on Peak Force QNM for Bruker AFMs bull Mechanical properties mapped pixel-by-pixel at same

resolution as the height image (PeakForce QNM)

Height Modulus BBML - All R

ights Reserve

d

A Note on Peak Force QNM

Correct probe stiffness must be used based on sample Pittenger ndash Veeco Product Bulletin

BBML - All R

ights Reserve

d

A Note on Peak Force QNM Extremely sensitive to calibration (details rarely given) Absolute radius and spring constant known

bull especially important from probe to probe bull radius uarr with indent depth ndash must determine R at depth

you expect to image bull Depth may vary as a function of sample (or at different

regions within the sample)

Relative reference material of ldquoknownrdquo modulus used to determine a ratio of 119896119896 119877119877 bull ldquoknownrdquo modulus may come from tension compression

and not indentation bull R is related to depth indentation depth on sample must

be same as on reference BBML - All R

ights Reserve

d

A Note on Peak Force QNM bull Modulus fit on the fly ndash no ability to monitor fit (or range) bull Local sample topography and sample tilt play a role in

measured parameters bull Assuming all modulus values in an image are correct how to

quantify

512 x 512 pixels = 262144 measurements

n=1 n=262144

n from selected ROIs

BBML - All R

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d

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d

bull Imaging in air on mica ndash substrate effect

bull Same imaging force or indent depth for all

bull For each area were all modulus values pooled

Lamprou et al PLoS ONE 2013 BBML - All R

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d

AFM Applications in Bone Research Other Cool Mechanical Techniques

BBML - All R

ights Reserve

d

ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo Bonds within or between collagen fibrils in bone that rupture to dissipate energy as a toughening mechanism

Thompson et al Nature 2001

Fantner et al Nature Mat 2005

Hansma et al JMNI 2005

Fantner et al Nano Letters 2007 BBML - All R

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d

Other Mechanical Techniques

Jimenez-Palomar J Mech Beh Bio Mat 2012

van der Rijt Macro Bio 2004

Yang J Biomed Mater Res 2007

Yang Biophysical Journal 2008 BBML - All R

ights Reserve

d

Other Cool Techniques bull Piezoresponse Force Microscopy (PFM)

bull Electrochemical AFM (EC-AFM)

bull Force Modulation Microscopy (FMM)

bull Lateral Force Microscopy (LFM)

bull Magnetic Force Microscopy (MFM)

bull Conductive AFM (C-AFM)

bull Force Pulling

bull Cantilever Bending

bull Tensile testing of individual fibrils

bull 3 point bending of fibrils

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d

Summary Things to rememberhellip

bull Imaging mode imaging medium and probe selection

bull Morphology Measurements bull Sample preparation and hydration state bull Measuring fibril diameter bull Site selection and proper sampling bull Appropriate system calibration

bull Mechanical Measurements

bull Probe selection (stiffness and tip radius) bull Probe calibration (radius angle spring constant) bull Indentation force range and mechanical model bull Hydration in biological samples bull PeakForce QNM

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Acknowledgements BBML

Max Hammond Armando Diaz Gonzalez Zachary Bart Silvia Canelon Creasy Clauser

IU School of Medicine Dr David Burr Dr Matt Allen Dr Stuart Warden Dr Lilian Plotkin

Funding Sources NIHNIDCR 1F32DE018840-01 A1 IUPUI BME Departmental Startup Funds IUPUI Research Support Funds Grant IUPUI Biomechanics and Biomaterials Research Center

NIH NICHD Dr Joan Marini

BBML - All R

ights Reserve

d

Bone Biology and Mechanics Lab (BBML) wwwiupuiedu~bbml Facebook BoneBiologyMechanicsLab

BBML - All R

ights Reserve

d

• Slide Number 1
• General Seminar Outline
• Bonersquos Hierarchical Structure
• Critical Gap in Understanding Collagen
• Methods For Analyzing Collagen Structure
• Methods For Imaging Collagen Structure
• Cryo-TEM
• Atomic Force Microscopy (AFM)
• AFM Image of Pentacene (C22H14)
• AFM Image of Hexabenzocoronene
• Basics of AFM Imaging The Probe
• Basics of AFM Imaging System Components
• van der Waals Potential Energy Curve
• Contact Mode ndash Monitor Deflection
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Peak Force Tapping Mode ndash Monitor Force
• AFM Applications in Bone Research Imaging
• AFM Imaging of Collagen
• Imaging of Osteoclast Activity
• Mineralized collagen deposition in vitro
• Imaging of Canilicular Structure
• AFM Imaging in Bone
• AFM Imaging in Bone
• Removing Mineral in Bone to Expose Collagen
• Removing Mineral in Bone to Expose Collagen
• Demineralization with EDTA
• Demineralization with EDTA
• Demineralization with EDTA
• Issues to Consider Effects of Acid
• Slide Number 32
• Slide Number 33
• Analysis of Collagen Structure in Bone
• Analysis of Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril D-Spacing
• Slide Number 39
• Collagen in Bone Fibril D-Spacing
• Collagen in Bone Fibril D-Spacing
• 2D Fast Fourier Transform Analysis
• 2D Fast Fourier Transform Analysis
• D-Spacing Distribution
• D-Spacing Distribution
• Cumulative Distribution Function
• CDF vs Histogram
• Changes in D-Spacing with Disease
• Changes in D-Spacing with Disease
• Slide Number 50
• Issues to Consider Site Selection and Sampling
• Issues to Consider Site Selection and Sampling
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• AFM calibration ndash Take Away Message
• AFM Applications in Bone ResearchIndentation Mechanics
• AFM-Based Indentation
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Parameters
• Hertz Contact Equation
• Hertz Contact Equation
• Hertz Contact Equation
• Proper Indentation Model is Essential
• Indentation in OI Tendon Study
• OI Tendon Differential Effects of Dehydration
• OI Tendon Differential Effects of Dehydration
• Indentation in Diabetic Tendon (Poster 0495)
• Multiscale Diabetes Study (Poster 0495)
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• A Note on Peak Force QNM for Bruker AFMs
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• Slide Number 83
• Slide Number 84
• AFM Applications in Bone ResearchOther Cool Mechanical Techniques
• ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo
• Other Mechanical Techniques
• Other Cool Techniques
• Summary Things to rememberhellip
• Acknowledgements
• Bone Biology and Mechanics Lab (BBML)

AFM Imaging in Bone

The surface is completely mineralizedhellip BBML - All R

ights Reserve

d

Removing Mineral in Bone to Expose Collagen

Dealing with mineral is a challenge to analyzing collagen ndash some mineral must be removed

bull 10 Citric acid (to remove mineral) 65 Sodium Hypochlorite (to remove non-collagenous proteins) Habelitz J Struct Bio 2002

bull 7 or 17 Phosphoric Acid Bozek IEEE 2005 El Feninat J Biomed Mat Res 1998

bull 5 Formic Acid Ge Mat Sci Eng 2007

bull 30 mM EDTA Kindt Nanotechnology 2007

BBML - All R

ights Reserve

d

Removing Mineral in Bone to Expose Collagen

Dealing with mineral is a challenge to analyzing collagen ndash some mineral must be removed

bull 30 mM EDTA Kindt Nanotechnology 2007 BBML - A

ll Rights R

eserved

Demineralization with EDTA EDTA - Ethylenediaminetetraacetic Acid

Chelating agent - sequesters divalent cations (notably Ca2+)

bull Treat with 05M EDTA pH 8 (15-20 minutes) bull Vigorously wash with ultrapure water bull Sonicate to release surface-bound mineral ions bull Repeat 3-4 times

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d

Demineralization with EDTA

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ights Reserve

d

Demineralization with EDTA

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ights Reserve

d

Issues to Consider Effects of Acid

Immature collagen crosslinks are very sensitive to acid bull Using acids to demineralize bone may inadvertently

destroy crosslinks distort structure

Fixation of tissue may be necessary prior to demineralization bull Fixation (glutaraldehyde or paraformadehyde) may

preserve structural integrity

bull Fixation effects on collagen structure are not fully known

BBML - All R

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d

100 microm x 100 microm

50 microm x 50 microm

25 microm x 25 microm

10 microm x 10 microm

35 microm x 35 microm

BBML - All R

ights Reserve

d

35 microm x 35 microm

BBML - All R

ights Reserve

d

Analysis of Collagen Structure in Bone Spatial organization of newly formed bone following a surgically-induced injury (rat tibial hole)

Baranauskas J Vac Sci Tech 2001 Baranauskas App Surface Sci 2005

ldquohellipstatistical diameter of the fibers is 159 nm in good agreement with the literature values for type I collagen fibersrdquo

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ights Reserve

d

Analysis of Collagen in Bone Fibril Diameter Fibril Diameter easiest to measure property first attempts were a bit crude better with increased resolution

Sasaki J Mat Sci Tech 2002 Thalhammer J Arch Sci 2001

Bozec Ultramicroscopy 2005

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ights Reserve

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Collagen in Bone Fibril Diameter

Caution must be taken when reporting and interpreting diameters measured from fibrils in bone bull Fibril packing - width may not be fully exposed

Wallace et al Langmuir 2010

BBML - All R

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Collagen in Bone Fibril Diameter

Caution must be taken when reporting and interpreting diameters measured from fibrils in bone bull Fibril packing - width may not be fully exposed

Rigozzi ndash J Struct Bio - 2011

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ights Reserve

d

Collagen in Bone Fibril D-Spacing

300 nm 15 nm

N C C N C C N C C N C C

H O H2C CH2 O H

CH2

O OH HHC CH2

CH2

HO

H H

HHN C C

O

H2C CH2

CH2

HN C C

OH

HC CH2

HO

N C C N C C N C C

H O H2C CH2 O

CH2

OH HC CH2

HO

H H

H

CH2 CH2

glycine X - proline Y - hydroxyproline

Hodge and Petruska 1963

Collagen Fibril Axial D-Periodicity BBML - All R

ights Reserve

d

05

101520

0 02 04 06 08 1

Hei

ght (

nm)

Length (microns)

httpremfdartmoutheduimagesindexhtml

Randall Nature and Structure of Collagen 1953

TEM SEM

AFM

D-Spacing = 67 nm

BBML - All R

ights Reserve

d

Collagen in Bone Fibril D-Spacing Many studies have shown observable D-spacing content that near theoretical value

Sasaki 2002 67 plusmn 2 nm Bozek 2005 665 plusmn 14 nm Ge 2007 666 plusmn 38 nm

Habelitz J Structural Bio 2002

A 2002 study in dentin got me interestedhellip

n = 322 wet n = 363 dry

BBML - All R

ights Reserve

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Collagen in Bone Fibril D-Spacing

bull Internal structure bull Wallace et al Bone 2010 46 1349-1354 bull Wallace et al J Stuct Bio 2011 173 146-152 bull Kemp et al J Stuct Bio 2012 180 428-438 bull Warden et al Endocrinology 2013 154(9)

3178-3187 bull Gallant et al Bone 2014 61 191-200 bull Bart et al Conn Tiss Res 2014 In Press

bull Enzymatic and non-

enzymatic cross-linking bull Hammond et al Bone 2014 60 26-32 bull Diaz Gonzalez et al J Biomech 2014 47(3)

681-686 bull Voytik-Harbin et al In Preparation 2014

D-periodic spacing important structural feature that captures aspects related to

Mouse Femur Mid-diaphysis BBML - All R

ights Reserve

d

2D Fast Fourier Transform Analysis

Harmonics

Fibril D-Spacing

Erickson B Wallace JM et al Biotechnology Journal 2013

Raw AFM Image 2D FFT Power Spectrum

BBML - All R

ights Reserve

d

2D Fast Fourier Transform Analysis

Harmonics

Fibril D-Spacing

Raw AFM Image 2D FFT Power Spectrum

0

20000

40000

60000

80000

100000

120000

0 001 002 003 004 005 006

Am

plitu

de (

microV2 )

Position (1nm)

D-periodic spacing 0015 (1nm) ~ 67 nm

BBML - All R

ights Reserve

d

D-Spacing Distribution

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Overall Mean 670 plusmn 00 nm

BBML - All R

ights Reserve

d

D-Spacing Distribution

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Overall Mean 670 plusmn 00 nm

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

4 Samples Total of n = 255 fibrils Overall Mean 682 plusmn 12 nm

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ights Reserve

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Cumulative Distribution Function

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

This population 255 fibrils - Each fibril 1255 = 0392 - Data range 628-716 nm

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ights Reserve

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CDF vs Histogram 62 63 64 65 66 67 68 69 70 71 72 73 74

0

10

20

30

40

50

60

70

80

90

100

0

5

10

15

20

25

30

35

40

45

50

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

HistoCDF

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ights Reserve

d

Changes in D-Spacing with Disease

0

5

10

15

20

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

0

5

10

15

20

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

Wallace et al Bone 2010 46 1349-1354

KS Test plt0001

Control 680 plusmn 26 nm

Estrogen Dep 659 plusmn 31 nm

t-test on mean p=0048

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Changes in D-Spacing with Disease

Wallace et al Bone 2010 46 1349-1354

0

10

20

30

40

50

60

70

80

90

100

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

KS Test plt0001

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Question Model and Tissue Morphology Publication

Tissue Specificity in Mice Male mouse 8 weeks Femur dentin tail tendon

No differences in bone vs dentin both ∆ vs tendon

Wallace et al Langmuir 2011 26(10) 7349-7354

Gender Effects Male and female mice 16 weeks Femur No differences NA

Estrogen Depletion OVX in sheep 5 years + 2 Radius Mean and population shift Wallace et al Bone

2010 46 1349-1354

Estrogen Depletion OVX in female 5 years + 2 Skin (dermis) Significant population shift Erickson et al Biotech

J 2013 8(1) 1174-126

Osteoporosis and Mechanical Loading

OVX in ulnar loading in rats Ulna

No loading effect population shift with OVX

Warden et al Endocrinology 2013 154(9) 3178-3187

Osteogenesis Imperfecta Brtl+ model male mice 2 months Femur

No mean ∆ widening of distribution

Wallace et al J Stuct Bio 2011 173 146-152

Osteogenesis Imperfecta Oimoim model male mice 12 weeks Femur Mean and population shift Bart et al Conn Tiss

Res 2014 In Press

Osteogenesis Imperfecta and Tissue Hydration

Brtl+ model male mice 6 months Tail Tendon Significant population shift Kemp et al J Stuct Bio

2012 180 428-438

Type 2 Diabetes Male ZDSD rats 30 weeks Tibia Significant population shift Hammond et al Bone

2014 60 26-32

Type 2 Diabetes Female ZDSD rats 32 weeks Tail Tendon Significant population shift

Diaz Gonzalez et al J Biomech 2014 47(3)

681-686

Raloxifene (in vivo and in vitro) Female dog treated with Raloxifene Femur Significant population shift Gallant et al Bone

2014 61 191-200 BBML - All R

ights Reserve

d

Issues to Consider Site Selection and Sampling

KS Test plt0001

0023 of bonersquos length

15 m

m =

150

00 micro

m

Wallace et al Langmuir 2011 26(10) 7349-7354

100 microm x 100 microm

35 microm x 35 microm

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ights Reserve

d

Issues to Consider Site Selection and Sampling

60

62

64

66

68

70

72

74

1 (35)

2 (33)

3 (40)

4 (39)

5 (35)

6 (29)

7 (26)

8 (23)

9 (28)

WT (288)

D-P

erio

dic

Gap

Ove

rlap

Spac

ing

(nm

)

Axial Location (Proximal End = 1 Distal End = 9)

( of Fibrils)

91

23

45

87

6

Wallace et al Langmuir 2011 26(10) 7349-7354

288 fibrils for population n=1 for mean comparisons BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

Wet Tendon Dry Tendon Kemp et al J StructBio 2012 180 (3) 428-438

Collagen and bone exist in a hydrated environment in vivo bull Hydration state can impact morphology and mechanics

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Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

Nonsignificant mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet (n=255) 682 plusmn 12 nm

WT Dry (n=257) 686 plusmn 08 nm

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

Brtl DryBrtl Wet

KS Test plt0001

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

WT Case 04 nm mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet 682 plusmn 12 nm

WT Dry 686 plusmn 08 nm

Brtl+ Wet 675 plusmn 14 nm

Brtl+ Dry 689 plusmn 10 nm

Brtl+ Case 14 nm increase with drying (plt0001) bull Entire population shifted upward (plt0001)

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

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Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT WetBrtl Wet

KS Test plt0001

Brtl+ distribution shifted downward vs WT when wet

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryBrtl Dry

KS Test plt0001

Brtl+ distribution shifted upward vs WT when dry

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider AFM calibration

bull Absolute distance measurements require accurate piezo calibration

bull AFM systems have large scan ranges (150 microm to lt 1 microm)

bull Scanner non-linearities can introduce substantial error over that range

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

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d

Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

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Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

100 nm 10 80 602 1198 100 nm 7 60 827 1439 100 nm 5 40 814 1427 100 nm 35 20 917 1724 100 nm 2 10 937 1774

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

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d

AFM calibration ndash Take Away Message

Calibration using a scan size and feature size comparable to features of interest is highly recommended

Calibration addresses absolute accuracy not precision (differential sensitivity between measurements)

bull Does not limit ability to differentiate between groups using the same AFM with the same calibration parameters

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d

AFM Applications in Bone Research Indentation Mechanics

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ights Reserve

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AFM-Based Indentation

In addition to high resolution imaging AFM can be used to extract nanoscale mechanical data

ApproachWithdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

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ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Can

tilev

er D

efle

ctio

n (V

olts

)

1 Ramp into a rigid sample ndash measure the slope

Deflection sensitivity x 119899119899119899119899119881119881

2 Calculate cantilever spring constant

3 Hookersquos law takes deflection (volts) and converts to units of force F = kx

Spring constant k 119873119873119899119899

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ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Forc

e (n

N)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

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ights Reserve

d

AFM-Based Indentation Cantilever Calibration Fo

rce

(nN

)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

Tip-Sample Separation (nm)

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ights Reserve

d

AFM-Based Indentation Parameters

Approach Withdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

Peak Force

Area Between Curves Energy Dissipation

Contact Mechanics Fit for Elastic Modulus (E)

Adhesion Force

Indentation Depth (δ)

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

z

r

δ

R

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119877119877 ∙ 12057512057532

Es Sample modulus νs Poissonrsquos ratio R tip radius of curvature δ load point displacement

Relevant Hertz Assumptions bull Strains are small and within the elastic limit bull Indenter is infinitely stiff (only sample deforms) bull Indentation depth ltlt radius of curvature of indenter bull Surfaces are continuous non-conforming (must be flat) bull Load is normal to surface

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ights Reserve

d

Proper Indentation Model is Essential

δ

r

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119903119903 ∙ 12057512057532

δ

α

119865119865 =2120587120587∙

1198641198641199041199041 minus 1205841205841199041199042

∙ tan120572120572 ∙ 1205751205752

Es Sample modulus νs Poissonrsquos ratio R tip radius α opening angle

Hertz Model Spherical Indenter

depth ltlt radius of curvature

Sneddon Model Conical Indenter

depth ge radius of curvature

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ights Reserve

d

Indentation in OI Tendon Study

x

z

yBrtl+ and WT mice 6 month old males

4 tails per group

2-3 fascicles per tail

25-30 fibrils per tail

4-5 locations per fibril

Indent force 20 nN wet (Sneddon) 50 nN dry (Hertz) BBML - A

ll Rights R

eserved

OI Tendon Differential Effects of Dehydration

Dry 1268 plusmn 718 MPa

Wet 305 plusmn 243 MPa

Dry 1737 plusmn 973 MPa

Wet 301 plusmn 202 MPa

Kemp et al J StructBio 2012 180 (3) 428-438

bull Significant stiffening of fibrils with dehydration

bull More pronounced modulus modulus as a function of disease

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ights Reserve

d

OI Tendon Differential Effects of Dehydration

Kemp et al J StructBio 2012 180 (3) 428-438

Diseased phenotype not present when wet phenotype observed when dried

0

10

20

30

40

50

60

0 20 40 60 80 100 120 140 160 180 200

Gro

up S

ampl

es (

)

Indent Modulus (MPa)

WT WetBrtl Wet

0

2

4

6

8

10

12

14

16

18

20

025

050

075

010

0012

5015

0017

5020

0022

5025

0027

5030

0032

5035

0037

5040

0042

5045

0047

5050

00

Gro

up S

ampl

es (

)Indent Modulus (MPa)

WT DryBrtl Dry

555 WT Indents 305 plusmn 243 MPa

614 Brtl+ Indents 301 plusmn 202 MPa

KS Test p=0129 t Test p=0464 464 WT Indents

1268 plusmn 718 MPa 479 Brtl+ Indents 1737 plusmn 973 MPa

KS Test plt0001 T Test plt0001

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ights Reserve

d

Indentation in Diabetic Tendon (Poster 0495)

x

z

y

Control and Diabetic Rat Tail Tendon

4-5 rats per group

2-3 fascicles per rat

~70 fibrilstail

4-5 locationsfibril

20 nN (Sneddon)

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d

Multiscale Diabetes Study (Poster 0495)

0

5

10

15

20

25

300 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 gt25

Perc

enta

ge o

f Gro

up S

ampl

es

Elastic Modulus (MPa)

CD (1530 indents)ZDSD (913 indents)

CD (n=5) 381 plusmn 149 MPa

ZDSD (n=3) 706 plusmn 263 MPa

0

100

200

300

400

500

600

700

800

900

0 2 4 6 8 10

Mic

rosc

ale

Mod

ulus

(MPa

)

Nanoscale Modulus (MPa)

plt0001

r2=0531 p=0040

bull Increased indentation modulus in diabetic tendon

bull Strong relationship between nanoscale indent modulus and microscale tensile modulus

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Indentation Limitations and Weaknesses

Proper probe selection for intended application bull Cantilever must be stiff relative to sample bull Sharp probes may damage sample easily dull

R or α must be determined for each probe bull Properties provided by manufacturer are unreliable bull Vary from probe to probe can dull during indent process

Spring constant must be determined for each probe

bull Value provided by manufacturer is inaccurate bull Thermal tuning OK for ldquosoftrdquo probes poor performance

for stiffer probes bull Sader method (geometry) added mass reference

cantilevers

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d

Indentation Limitations and Weaknesses

Veeco Application Technical note Ben Ohler Practical Advice on the Determination of Cantilever Spring Constants

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Indentation Limitations and Weaknesses

bull Sample processing

bull Sample hydration

bull Proper force range

bull Substrate effects for thin samples

bull Proper modulus model and range of data goodness of fit

Bearing in mind these assumptions and limitations AFM can provide true ldquonanoscalerdquo mechanical properties both in terms of forces and deformations

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ights Reserve

d

A Note on Peak Force QNM for Bruker AFMs bull Mechanical properties mapped pixel-by-pixel at same

resolution as the height image (PeakForce QNM)

Height Modulus BBML - All R

ights Reserve

d

A Note on Peak Force QNM

Correct probe stiffness must be used based on sample Pittenger ndash Veeco Product Bulletin

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d

A Note on Peak Force QNM Extremely sensitive to calibration (details rarely given) Absolute radius and spring constant known

bull especially important from probe to probe bull radius uarr with indent depth ndash must determine R at depth

you expect to image bull Depth may vary as a function of sample (or at different

regions within the sample)

Relative reference material of ldquoknownrdquo modulus used to determine a ratio of 119896119896 119877119877 bull ldquoknownrdquo modulus may come from tension compression

and not indentation bull R is related to depth indentation depth on sample must

be same as on reference BBML - All R

ights Reserve

d

A Note on Peak Force QNM bull Modulus fit on the fly ndash no ability to monitor fit (or range) bull Local sample topography and sample tilt play a role in

measured parameters bull Assuming all modulus values in an image are correct how to

quantify

512 x 512 pixels = 262144 measurements

n=1 n=262144

n from selected ROIs

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bull Imaging in air on mica ndash substrate effect

bull Same imaging force or indent depth for all

bull For each area were all modulus values pooled

Lamprou et al PLoS ONE 2013 BBML - All R

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d

AFM Applications in Bone Research Other Cool Mechanical Techniques

BBML - All R

ights Reserve

d

ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo Bonds within or between collagen fibrils in bone that rupture to dissipate energy as a toughening mechanism

Thompson et al Nature 2001

Fantner et al Nature Mat 2005

Hansma et al JMNI 2005

Fantner et al Nano Letters 2007 BBML - All R

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Other Mechanical Techniques

Jimenez-Palomar J Mech Beh Bio Mat 2012

van der Rijt Macro Bio 2004

Yang J Biomed Mater Res 2007

Yang Biophysical Journal 2008 BBML - All R

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d

Other Cool Techniques bull Piezoresponse Force Microscopy (PFM)

bull Electrochemical AFM (EC-AFM)

bull Force Modulation Microscopy (FMM)

bull Lateral Force Microscopy (LFM)

bull Magnetic Force Microscopy (MFM)

bull Conductive AFM (C-AFM)

bull Force Pulling

bull Cantilever Bending

bull Tensile testing of individual fibrils

bull 3 point bending of fibrils

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Summary Things to rememberhellip

bull Imaging mode imaging medium and probe selection

bull Morphology Measurements bull Sample preparation and hydration state bull Measuring fibril diameter bull Site selection and proper sampling bull Appropriate system calibration

bull Mechanical Measurements

bull Probe selection (stiffness and tip radius) bull Probe calibration (radius angle spring constant) bull Indentation force range and mechanical model bull Hydration in biological samples bull PeakForce QNM

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Acknowledgements BBML

Max Hammond Armando Diaz Gonzalez Zachary Bart Silvia Canelon Creasy Clauser

IU School of Medicine Dr David Burr Dr Matt Allen Dr Stuart Warden Dr Lilian Plotkin

Funding Sources NIHNIDCR 1F32DE018840-01 A1 IUPUI BME Departmental Startup Funds IUPUI Research Support Funds Grant IUPUI Biomechanics and Biomaterials Research Center

NIH NICHD Dr Joan Marini

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ights Reserve

d

Bone Biology and Mechanics Lab (BBML) wwwiupuiedu~bbml Facebook BoneBiologyMechanicsLab

BBML - All R

ights Reserve

d

• Slide Number 1
• General Seminar Outline
• Bonersquos Hierarchical Structure
• Critical Gap in Understanding Collagen
• Methods For Analyzing Collagen Structure
• Methods For Imaging Collagen Structure
• Cryo-TEM
• Atomic Force Microscopy (AFM)
• AFM Image of Pentacene (C22H14)
• AFM Image of Hexabenzocoronene
• Basics of AFM Imaging The Probe
• Basics of AFM Imaging System Components
• van der Waals Potential Energy Curve
• Contact Mode ndash Monitor Deflection
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Peak Force Tapping Mode ndash Monitor Force
• AFM Applications in Bone Research Imaging
• AFM Imaging of Collagen
• Imaging of Osteoclast Activity
• Mineralized collagen deposition in vitro
• Imaging of Canilicular Structure
• AFM Imaging in Bone
• AFM Imaging in Bone
• Removing Mineral in Bone to Expose Collagen
• Removing Mineral in Bone to Expose Collagen
• Demineralization with EDTA
• Demineralization with EDTA
• Demineralization with EDTA
• Issues to Consider Effects of Acid
• Slide Number 32
• Slide Number 33
• Analysis of Collagen Structure in Bone
• Analysis of Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril D-Spacing
• Slide Number 39
• Collagen in Bone Fibril D-Spacing
• Collagen in Bone Fibril D-Spacing
• 2D Fast Fourier Transform Analysis
• 2D Fast Fourier Transform Analysis
• D-Spacing Distribution
• D-Spacing Distribution
• Cumulative Distribution Function
• CDF vs Histogram
• Changes in D-Spacing with Disease
• Changes in D-Spacing with Disease
• Slide Number 50
• Issues to Consider Site Selection and Sampling
• Issues to Consider Site Selection and Sampling
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• AFM calibration ndash Take Away Message
• AFM Applications in Bone ResearchIndentation Mechanics
• AFM-Based Indentation
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Parameters
• Hertz Contact Equation
• Hertz Contact Equation
• Hertz Contact Equation
• Proper Indentation Model is Essential
• Indentation in OI Tendon Study
• OI Tendon Differential Effects of Dehydration
• OI Tendon Differential Effects of Dehydration
• Indentation in Diabetic Tendon (Poster 0495)
• Multiscale Diabetes Study (Poster 0495)
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• A Note on Peak Force QNM for Bruker AFMs
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• Slide Number 83
• Slide Number 84
• AFM Applications in Bone ResearchOther Cool Mechanical Techniques
• ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo
• Other Mechanical Techniques
• Other Cool Techniques
• Summary Things to rememberhellip
• Acknowledgements
• Bone Biology and Mechanics Lab (BBML)

Removing Mineral in Bone to Expose Collagen

Dealing with mineral is a challenge to analyzing collagen ndash some mineral must be removed

bull 10 Citric acid (to remove mineral) 65 Sodium Hypochlorite (to remove non-collagenous proteins) Habelitz J Struct Bio 2002

bull 7 or 17 Phosphoric Acid Bozek IEEE 2005 El Feninat J Biomed Mat Res 1998

bull 5 Formic Acid Ge Mat Sci Eng 2007

bull 30 mM EDTA Kindt Nanotechnology 2007

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d

Removing Mineral in Bone to Expose Collagen

Dealing with mineral is a challenge to analyzing collagen ndash some mineral must be removed

bull 30 mM EDTA Kindt Nanotechnology 2007 BBML - A

ll Rights R

eserved

Demineralization with EDTA EDTA - Ethylenediaminetetraacetic Acid

Chelating agent - sequesters divalent cations (notably Ca2+)

bull Treat with 05M EDTA pH 8 (15-20 minutes) bull Vigorously wash with ultrapure water bull Sonicate to release surface-bound mineral ions bull Repeat 3-4 times

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d

Demineralization with EDTA

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d

Demineralization with EDTA

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d

Issues to Consider Effects of Acid

Immature collagen crosslinks are very sensitive to acid bull Using acids to demineralize bone may inadvertently

destroy crosslinks distort structure

Fixation of tissue may be necessary prior to demineralization bull Fixation (glutaraldehyde or paraformadehyde) may

preserve structural integrity

bull Fixation effects on collagen structure are not fully known

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d

100 microm x 100 microm

50 microm x 50 microm

25 microm x 25 microm

10 microm x 10 microm

35 microm x 35 microm

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d

35 microm x 35 microm

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d

Analysis of Collagen Structure in Bone Spatial organization of newly formed bone following a surgically-induced injury (rat tibial hole)

Baranauskas J Vac Sci Tech 2001 Baranauskas App Surface Sci 2005

ldquohellipstatistical diameter of the fibers is 159 nm in good agreement with the literature values for type I collagen fibersrdquo

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d

Analysis of Collagen in Bone Fibril Diameter Fibril Diameter easiest to measure property first attempts were a bit crude better with increased resolution

Sasaki J Mat Sci Tech 2002 Thalhammer J Arch Sci 2001

Bozec Ultramicroscopy 2005

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Collagen in Bone Fibril Diameter

Caution must be taken when reporting and interpreting diameters measured from fibrils in bone bull Fibril packing - width may not be fully exposed

Wallace et al Langmuir 2010

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Collagen in Bone Fibril Diameter

Caution must be taken when reporting and interpreting diameters measured from fibrils in bone bull Fibril packing - width may not be fully exposed

Rigozzi ndash J Struct Bio - 2011

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d

Collagen in Bone Fibril D-Spacing

300 nm 15 nm

N C C N C C N C C N C C

H O H2C CH2 O H

CH2

O OH HHC CH2

CH2

HO

H H

HHN C C

O

H2C CH2

CH2

HN C C

OH

HC CH2

HO

N C C N C C N C C

H O H2C CH2 O

CH2

OH HC CH2

HO

H H

H

CH2 CH2

glycine X - proline Y - hydroxyproline

Hodge and Petruska 1963

Collagen Fibril Axial D-Periodicity BBML - All R

ights Reserve

d

05

101520

0 02 04 06 08 1

Hei

ght (

nm)

Length (microns)

httpremfdartmoutheduimagesindexhtml

Randall Nature and Structure of Collagen 1953

TEM SEM

AFM

D-Spacing = 67 nm

BBML - All R

ights Reserve

d

Collagen in Bone Fibril D-Spacing Many studies have shown observable D-spacing content that near theoretical value

Sasaki 2002 67 plusmn 2 nm Bozek 2005 665 plusmn 14 nm Ge 2007 666 plusmn 38 nm

Habelitz J Structural Bio 2002

A 2002 study in dentin got me interestedhellip

n = 322 wet n = 363 dry

BBML - All R

ights Reserve

d

Collagen in Bone Fibril D-Spacing

bull Internal structure bull Wallace et al Bone 2010 46 1349-1354 bull Wallace et al J Stuct Bio 2011 173 146-152 bull Kemp et al J Stuct Bio 2012 180 428-438 bull Warden et al Endocrinology 2013 154(9)

3178-3187 bull Gallant et al Bone 2014 61 191-200 bull Bart et al Conn Tiss Res 2014 In Press

bull Enzymatic and non-

enzymatic cross-linking bull Hammond et al Bone 2014 60 26-32 bull Diaz Gonzalez et al J Biomech 2014 47(3)

681-686 bull Voytik-Harbin et al In Preparation 2014

D-periodic spacing important structural feature that captures aspects related to

Mouse Femur Mid-diaphysis BBML - All R

ights Reserve

d

2D Fast Fourier Transform Analysis

Harmonics

Fibril D-Spacing

Erickson B Wallace JM et al Biotechnology Journal 2013

Raw AFM Image 2D FFT Power Spectrum

BBML - All R

ights Reserve

d

2D Fast Fourier Transform Analysis

Harmonics

Fibril D-Spacing

Raw AFM Image 2D FFT Power Spectrum

0

20000

40000

60000

80000

100000

120000

0 001 002 003 004 005 006

Am

plitu

de (

microV2 )

Position (1nm)

D-periodic spacing 0015 (1nm) ~ 67 nm

BBML - All R

ights Reserve

d

D-Spacing Distribution

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Overall Mean 670 plusmn 00 nm

BBML - All R

ights Reserve

d

D-Spacing Distribution

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Overall Mean 670 plusmn 00 nm

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

4 Samples Total of n = 255 fibrils Overall Mean 682 plusmn 12 nm

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ights Reserve

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Cumulative Distribution Function

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

This population 255 fibrils - Each fibril 1255 = 0392 - Data range 628-716 nm

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ights Reserve

d

CDF vs Histogram 62 63 64 65 66 67 68 69 70 71 72 73 74

0

10

20

30

40

50

60

70

80

90

100

0

5

10

15

20

25

30

35

40

45

50

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

HistoCDF

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ights Reserve

d

Changes in D-Spacing with Disease

0

5

10

15

20

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

0

5

10

15

20

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

Wallace et al Bone 2010 46 1349-1354

KS Test plt0001

Control 680 plusmn 26 nm

Estrogen Dep 659 plusmn 31 nm

t-test on mean p=0048

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ights Reserve

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Changes in D-Spacing with Disease

Wallace et al Bone 2010 46 1349-1354

0

10

20

30

40

50

60

70

80

90

100

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

KS Test plt0001

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ights Reserve

d

Question Model and Tissue Morphology Publication

Tissue Specificity in Mice Male mouse 8 weeks Femur dentin tail tendon

No differences in bone vs dentin both ∆ vs tendon

Wallace et al Langmuir 2011 26(10) 7349-7354

Gender Effects Male and female mice 16 weeks Femur No differences NA

Estrogen Depletion OVX in sheep 5 years + 2 Radius Mean and population shift Wallace et al Bone

2010 46 1349-1354

Estrogen Depletion OVX in female 5 years + 2 Skin (dermis) Significant population shift Erickson et al Biotech

J 2013 8(1) 1174-126

Osteoporosis and Mechanical Loading

OVX in ulnar loading in rats Ulna

No loading effect population shift with OVX

Warden et al Endocrinology 2013 154(9) 3178-3187

Osteogenesis Imperfecta Brtl+ model male mice 2 months Femur

No mean ∆ widening of distribution

Wallace et al J Stuct Bio 2011 173 146-152

Osteogenesis Imperfecta Oimoim model male mice 12 weeks Femur Mean and population shift Bart et al Conn Tiss

Res 2014 In Press

Osteogenesis Imperfecta and Tissue Hydration

Brtl+ model male mice 6 months Tail Tendon Significant population shift Kemp et al J Stuct Bio

2012 180 428-438

Type 2 Diabetes Male ZDSD rats 30 weeks Tibia Significant population shift Hammond et al Bone

2014 60 26-32

Type 2 Diabetes Female ZDSD rats 32 weeks Tail Tendon Significant population shift

Diaz Gonzalez et al J Biomech 2014 47(3)

681-686

Raloxifene (in vivo and in vitro) Female dog treated with Raloxifene Femur Significant population shift Gallant et al Bone

2014 61 191-200 BBML - All R

ights Reserve

d

Issues to Consider Site Selection and Sampling

KS Test plt0001

0023 of bonersquos length

15 m

m =

150

00 micro

m

Wallace et al Langmuir 2011 26(10) 7349-7354

100 microm x 100 microm

35 microm x 35 microm

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ights Reserve

d

Issues to Consider Site Selection and Sampling

60

62

64

66

68

70

72

74

1 (35)

2 (33)

3 (40)

4 (39)

5 (35)

6 (29)

7 (26)

8 (23)

9 (28)

WT (288)

D-P

erio

dic

Gap

Ove

rlap

Spac

ing

(nm

)

Axial Location (Proximal End = 1 Distal End = 9)

( of Fibrils)

91

23

45

87

6

Wallace et al Langmuir 2011 26(10) 7349-7354

288 fibrils for population n=1 for mean comparisons BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

Wet Tendon Dry Tendon Kemp et al J StructBio 2012 180 (3) 428-438

Collagen and bone exist in a hydrated environment in vivo bull Hydration state can impact morphology and mechanics

BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

Nonsignificant mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet (n=255) 682 plusmn 12 nm

WT Dry (n=257) 686 plusmn 08 nm

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

Brtl DryBrtl Wet

KS Test plt0001

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

WT Case 04 nm mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet 682 plusmn 12 nm

WT Dry 686 plusmn 08 nm

Brtl+ Wet 675 plusmn 14 nm

Brtl+ Dry 689 plusmn 10 nm

Brtl+ Case 14 nm increase with drying (plt0001) bull Entire population shifted upward (plt0001)

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT WetBrtl Wet

KS Test plt0001

Brtl+ distribution shifted downward vs WT when wet

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryBrtl Dry

KS Test plt0001

Brtl+ distribution shifted upward vs WT when dry

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider AFM calibration

bull Absolute distance measurements require accurate piezo calibration

bull AFM systems have large scan ranges (150 microm to lt 1 microm)

bull Scanner non-linearities can introduce substantial error over that range

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

ights Reserve

d

Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

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ights Reserve

d

Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

100 nm 10 80 602 1198 100 nm 7 60 827 1439 100 nm 5 40 814 1427 100 nm 35 20 917 1724 100 nm 2 10 937 1774

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

ights Reserve

d

AFM calibration ndash Take Away Message

Calibration using a scan size and feature size comparable to features of interest is highly recommended

Calibration addresses absolute accuracy not precision (differential sensitivity between measurements)

bull Does not limit ability to differentiate between groups using the same AFM with the same calibration parameters

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ights Reserve

d

AFM Applications in Bone Research Indentation Mechanics

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ights Reserve

d

AFM-Based Indentation

In addition to high resolution imaging AFM can be used to extract nanoscale mechanical data

ApproachWithdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

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ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Can

tilev

er D

efle

ctio

n (V

olts

)

1 Ramp into a rigid sample ndash measure the slope

Deflection sensitivity x 119899119899119899119899119881119881

2 Calculate cantilever spring constant

3 Hookersquos law takes deflection (volts) and converts to units of force F = kx

Spring constant k 119873119873119899119899

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Forc

e (n

N)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration Fo

rce

(nN

)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

Tip-Sample Separation (nm)

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ights Reserve

d

AFM-Based Indentation Parameters

Approach Withdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

Peak Force

Area Between Curves Energy Dissipation

Contact Mechanics Fit for Elastic Modulus (E)

Adhesion Force

Indentation Depth (δ)

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

z

r

δ

R

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119877119877 ∙ 12057512057532

Es Sample modulus νs Poissonrsquos ratio R tip radius of curvature δ load point displacement

Relevant Hertz Assumptions bull Strains are small and within the elastic limit bull Indenter is infinitely stiff (only sample deforms) bull Indentation depth ltlt radius of curvature of indenter bull Surfaces are continuous non-conforming (must be flat) bull Load is normal to surface

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ights Reserve

d

Proper Indentation Model is Essential

δ

r

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119903119903 ∙ 12057512057532

δ

α

119865119865 =2120587120587∙

1198641198641199041199041 minus 1205841205841199041199042

∙ tan120572120572 ∙ 1205751205752

Es Sample modulus νs Poissonrsquos ratio R tip radius α opening angle

Hertz Model Spherical Indenter

depth ltlt radius of curvature

Sneddon Model Conical Indenter

depth ge radius of curvature

BBML - All R

ights Reserve

d

Indentation in OI Tendon Study

x

z

yBrtl+ and WT mice 6 month old males

4 tails per group

2-3 fascicles per tail

25-30 fibrils per tail

4-5 locations per fibril

Indent force 20 nN wet (Sneddon) 50 nN dry (Hertz) BBML - A

ll Rights R

eserved

OI Tendon Differential Effects of Dehydration

Dry 1268 plusmn 718 MPa

Wet 305 plusmn 243 MPa

Dry 1737 plusmn 973 MPa

Wet 301 plusmn 202 MPa

Kemp et al J StructBio 2012 180 (3) 428-438

bull Significant stiffening of fibrils with dehydration

bull More pronounced modulus modulus as a function of disease

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d

OI Tendon Differential Effects of Dehydration

Kemp et al J StructBio 2012 180 (3) 428-438

Diseased phenotype not present when wet phenotype observed when dried

0

10

20

30

40

50

60

0 20 40 60 80 100 120 140 160 180 200

Gro

up S

ampl

es (

)

Indent Modulus (MPa)

WT WetBrtl Wet

0

2

4

6

8

10

12

14

16

18

20

025

050

075

010

0012

5015

0017

5020

0022

5025

0027

5030

0032

5035

0037

5040

0042

5045

0047

5050

00

Gro

up S

ampl

es (

)Indent Modulus (MPa)

WT DryBrtl Dry

555 WT Indents 305 plusmn 243 MPa

614 Brtl+ Indents 301 plusmn 202 MPa

KS Test p=0129 t Test p=0464 464 WT Indents

1268 plusmn 718 MPa 479 Brtl+ Indents 1737 plusmn 973 MPa

KS Test plt0001 T Test plt0001

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ights Reserve

d

Indentation in Diabetic Tendon (Poster 0495)

x

z

y

Control and Diabetic Rat Tail Tendon

4-5 rats per group

2-3 fascicles per rat

~70 fibrilstail

4-5 locationsfibril

20 nN (Sneddon)

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ights Reserve

d

Multiscale Diabetes Study (Poster 0495)

0

5

10

15

20

25

300 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 gt25

Perc

enta

ge o

f Gro

up S

ampl

es

Elastic Modulus (MPa)

CD (1530 indents)ZDSD (913 indents)

CD (n=5) 381 plusmn 149 MPa

ZDSD (n=3) 706 plusmn 263 MPa

0

100

200

300

400

500

600

700

800

900

0 2 4 6 8 10

Mic

rosc

ale

Mod

ulus

(MPa

)

Nanoscale Modulus (MPa)

plt0001

r2=0531 p=0040

bull Increased indentation modulus in diabetic tendon

bull Strong relationship between nanoscale indent modulus and microscale tensile modulus

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d

Indentation Limitations and Weaknesses

Proper probe selection for intended application bull Cantilever must be stiff relative to sample bull Sharp probes may damage sample easily dull

R or α must be determined for each probe bull Properties provided by manufacturer are unreliable bull Vary from probe to probe can dull during indent process

Spring constant must be determined for each probe

bull Value provided by manufacturer is inaccurate bull Thermal tuning OK for ldquosoftrdquo probes poor performance

for stiffer probes bull Sader method (geometry) added mass reference

cantilevers

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d

Indentation Limitations and Weaknesses

Veeco Application Technical note Ben Ohler Practical Advice on the Determination of Cantilever Spring Constants

BBML - All R

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d

Indentation Limitations and Weaknesses

bull Sample processing

bull Sample hydration

bull Proper force range

bull Substrate effects for thin samples

bull Proper modulus model and range of data goodness of fit

Bearing in mind these assumptions and limitations AFM can provide true ldquonanoscalerdquo mechanical properties both in terms of forces and deformations

BBML - All R

ights Reserve

d

A Note on Peak Force QNM for Bruker AFMs bull Mechanical properties mapped pixel-by-pixel at same

resolution as the height image (PeakForce QNM)

Height Modulus BBML - All R

ights Reserve

d

A Note on Peak Force QNM

Correct probe stiffness must be used based on sample Pittenger ndash Veeco Product Bulletin

BBML - All R

ights Reserve

d

A Note on Peak Force QNM Extremely sensitive to calibration (details rarely given) Absolute radius and spring constant known

bull especially important from probe to probe bull radius uarr with indent depth ndash must determine R at depth

you expect to image bull Depth may vary as a function of sample (or at different

regions within the sample)

Relative reference material of ldquoknownrdquo modulus used to determine a ratio of 119896119896 119877119877 bull ldquoknownrdquo modulus may come from tension compression

and not indentation bull R is related to depth indentation depth on sample must

be same as on reference BBML - All R

ights Reserve

d

A Note on Peak Force QNM bull Modulus fit on the fly ndash no ability to monitor fit (or range) bull Local sample topography and sample tilt play a role in

measured parameters bull Assuming all modulus values in an image are correct how to

quantify

512 x 512 pixels = 262144 measurements

n=1 n=262144

n from selected ROIs

BBML - All R

ights Reserve

d

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ights Reserve

d

bull Imaging in air on mica ndash substrate effect

bull Same imaging force or indent depth for all

bull For each area were all modulus values pooled

Lamprou et al PLoS ONE 2013 BBML - All R

ights Reserve

d

AFM Applications in Bone Research Other Cool Mechanical Techniques

BBML - All R

ights Reserve

d

ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo Bonds within or between collagen fibrils in bone that rupture to dissipate energy as a toughening mechanism

Thompson et al Nature 2001

Fantner et al Nature Mat 2005

Hansma et al JMNI 2005

Fantner et al Nano Letters 2007 BBML - All R

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d

Other Mechanical Techniques

Jimenez-Palomar J Mech Beh Bio Mat 2012

van der Rijt Macro Bio 2004

Yang J Biomed Mater Res 2007

Yang Biophysical Journal 2008 BBML - All R

ights Reserve

d

Other Cool Techniques bull Piezoresponse Force Microscopy (PFM)

bull Electrochemical AFM (EC-AFM)

bull Force Modulation Microscopy (FMM)

bull Lateral Force Microscopy (LFM)

bull Magnetic Force Microscopy (MFM)

bull Conductive AFM (C-AFM)

bull Force Pulling

bull Cantilever Bending

bull Tensile testing of individual fibrils

bull 3 point bending of fibrils

BBML - All R

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d

Summary Things to rememberhellip

bull Imaging mode imaging medium and probe selection

bull Morphology Measurements bull Sample preparation and hydration state bull Measuring fibril diameter bull Site selection and proper sampling bull Appropriate system calibration

bull Mechanical Measurements

bull Probe selection (stiffness and tip radius) bull Probe calibration (radius angle spring constant) bull Indentation force range and mechanical model bull Hydration in biological samples bull PeakForce QNM

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d

Acknowledgements BBML

Max Hammond Armando Diaz Gonzalez Zachary Bart Silvia Canelon Creasy Clauser

IU School of Medicine Dr David Burr Dr Matt Allen Dr Stuart Warden Dr Lilian Plotkin

Funding Sources NIHNIDCR 1F32DE018840-01 A1 IUPUI BME Departmental Startup Funds IUPUI Research Support Funds Grant IUPUI Biomechanics and Biomaterials Research Center

NIH NICHD Dr Joan Marini

BBML - All R

ights Reserve

d

Bone Biology and Mechanics Lab (BBML) wwwiupuiedu~bbml Facebook BoneBiologyMechanicsLab

BBML - All R

ights Reserve

d

• Slide Number 1
• General Seminar Outline
• Bonersquos Hierarchical Structure
• Critical Gap in Understanding Collagen
• Methods For Analyzing Collagen Structure
• Methods For Imaging Collagen Structure
• Cryo-TEM
• Atomic Force Microscopy (AFM)
• AFM Image of Pentacene (C22H14)
• AFM Image of Hexabenzocoronene
• Basics of AFM Imaging The Probe
• Basics of AFM Imaging System Components
• van der Waals Potential Energy Curve
• Contact Mode ndash Monitor Deflection
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Peak Force Tapping Mode ndash Monitor Force
• AFM Applications in Bone Research Imaging
• AFM Imaging of Collagen
• Imaging of Osteoclast Activity
• Mineralized collagen deposition in vitro
• Imaging of Canilicular Structure
• AFM Imaging in Bone
• AFM Imaging in Bone
• Removing Mineral in Bone to Expose Collagen
• Removing Mineral in Bone to Expose Collagen
• Demineralization with EDTA
• Demineralization with EDTA
• Demineralization with EDTA
• Issues to Consider Effects of Acid
• Slide Number 32
• Slide Number 33
• Analysis of Collagen Structure in Bone
• Analysis of Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril D-Spacing
• Slide Number 39
• Collagen in Bone Fibril D-Spacing
• Collagen in Bone Fibril D-Spacing
• 2D Fast Fourier Transform Analysis
• 2D Fast Fourier Transform Analysis
• D-Spacing Distribution
• D-Spacing Distribution
• Cumulative Distribution Function
• CDF vs Histogram
• Changes in D-Spacing with Disease
• Changes in D-Spacing with Disease
• Slide Number 50
• Issues to Consider Site Selection and Sampling
• Issues to Consider Site Selection and Sampling
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• AFM calibration ndash Take Away Message
• AFM Applications in Bone ResearchIndentation Mechanics
• AFM-Based Indentation
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Parameters
• Hertz Contact Equation
• Hertz Contact Equation
• Hertz Contact Equation
• Proper Indentation Model is Essential
• Indentation in OI Tendon Study
• OI Tendon Differential Effects of Dehydration
• OI Tendon Differential Effects of Dehydration
• Indentation in Diabetic Tendon (Poster 0495)
• Multiscale Diabetes Study (Poster 0495)
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• A Note on Peak Force QNM for Bruker AFMs
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• Slide Number 83
• Slide Number 84
• AFM Applications in Bone ResearchOther Cool Mechanical Techniques
• ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo
• Other Mechanical Techniques
• Other Cool Techniques
• Summary Things to rememberhellip
• Acknowledgements
• Bone Biology and Mechanics Lab (BBML)

Removing Mineral in Bone to Expose Collagen

Dealing with mineral is a challenge to analyzing collagen ndash some mineral must be removed

bull 30 mM EDTA Kindt Nanotechnology 2007 BBML - A

ll Rights R

eserved

Demineralization with EDTA EDTA - Ethylenediaminetetraacetic Acid

Chelating agent - sequesters divalent cations (notably Ca2+)

bull Treat with 05M EDTA pH 8 (15-20 minutes) bull Vigorously wash with ultrapure water bull Sonicate to release surface-bound mineral ions bull Repeat 3-4 times

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Demineralization with EDTA

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Demineralization with EDTA

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Issues to Consider Effects of Acid

Immature collagen crosslinks are very sensitive to acid bull Using acids to demineralize bone may inadvertently

destroy crosslinks distort structure

Fixation of tissue may be necessary prior to demineralization bull Fixation (glutaraldehyde or paraformadehyde) may

preserve structural integrity

bull Fixation effects on collagen structure are not fully known

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100 microm x 100 microm

50 microm x 50 microm

25 microm x 25 microm

10 microm x 10 microm

35 microm x 35 microm

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35 microm x 35 microm

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ights Reserve

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Analysis of Collagen Structure in Bone Spatial organization of newly formed bone following a surgically-induced injury (rat tibial hole)

Baranauskas J Vac Sci Tech 2001 Baranauskas App Surface Sci 2005

ldquohellipstatistical diameter of the fibers is 159 nm in good agreement with the literature values for type I collagen fibersrdquo

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Analysis of Collagen in Bone Fibril Diameter Fibril Diameter easiest to measure property first attempts were a bit crude better with increased resolution

Sasaki J Mat Sci Tech 2002 Thalhammer J Arch Sci 2001

Bozec Ultramicroscopy 2005

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Collagen in Bone Fibril Diameter

Caution must be taken when reporting and interpreting diameters measured from fibrils in bone bull Fibril packing - width may not be fully exposed

Wallace et al Langmuir 2010

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Collagen in Bone Fibril Diameter

Caution must be taken when reporting and interpreting diameters measured from fibrils in bone bull Fibril packing - width may not be fully exposed

Rigozzi ndash J Struct Bio - 2011

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Collagen in Bone Fibril D-Spacing

300 nm 15 nm

N C C N C C N C C N C C

H O H2C CH2 O H

CH2

O OH HHC CH2

CH2

HO

H H

HHN C C

O

H2C CH2

CH2

HN C C

OH

HC CH2

HO

N C C N C C N C C

H O H2C CH2 O

CH2

OH HC CH2

HO

H H

H

CH2 CH2

glycine X - proline Y - hydroxyproline

Hodge and Petruska 1963

Collagen Fibril Axial D-Periodicity BBML - All R

ights Reserve

d

05

101520

0 02 04 06 08 1

Hei

ght (

nm)

Length (microns)

httpremfdartmoutheduimagesindexhtml

Randall Nature and Structure of Collagen 1953

TEM SEM

AFM

D-Spacing = 67 nm

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Collagen in Bone Fibril D-Spacing Many studies have shown observable D-spacing content that near theoretical value

Sasaki 2002 67 plusmn 2 nm Bozek 2005 665 plusmn 14 nm Ge 2007 666 plusmn 38 nm

Habelitz J Structural Bio 2002

A 2002 study in dentin got me interestedhellip

n = 322 wet n = 363 dry

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Collagen in Bone Fibril D-Spacing

bull Internal structure bull Wallace et al Bone 2010 46 1349-1354 bull Wallace et al J Stuct Bio 2011 173 146-152 bull Kemp et al J Stuct Bio 2012 180 428-438 bull Warden et al Endocrinology 2013 154(9)

3178-3187 bull Gallant et al Bone 2014 61 191-200 bull Bart et al Conn Tiss Res 2014 In Press

bull Enzymatic and non-

enzymatic cross-linking bull Hammond et al Bone 2014 60 26-32 bull Diaz Gonzalez et al J Biomech 2014 47(3)

681-686 bull Voytik-Harbin et al In Preparation 2014

D-periodic spacing important structural feature that captures aspects related to

Mouse Femur Mid-diaphysis BBML - All R

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2D Fast Fourier Transform Analysis

Harmonics

Fibril D-Spacing

Erickson B Wallace JM et al Biotechnology Journal 2013

Raw AFM Image 2D FFT Power Spectrum

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2D Fast Fourier Transform Analysis

Harmonics

Fibril D-Spacing

Raw AFM Image 2D FFT Power Spectrum

0

20000

40000

60000

80000

100000

120000

0 001 002 003 004 005 006

Am

plitu

de (

microV2 )

Position (1nm)

D-periodic spacing 0015 (1nm) ~ 67 nm

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ights Reserve

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D-Spacing Distribution

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Overall Mean 670 plusmn 00 nm

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ights Reserve

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D-Spacing Distribution

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Overall Mean 670 plusmn 00 nm

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

4 Samples Total of n = 255 fibrils Overall Mean 682 plusmn 12 nm

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Cumulative Distribution Function

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

This population 255 fibrils - Each fibril 1255 = 0392 - Data range 628-716 nm

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CDF vs Histogram 62 63 64 65 66 67 68 69 70 71 72 73 74

0

10

20

30

40

50

60

70

80

90

100

0

5

10

15

20

25

30

35

40

45

50

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

HistoCDF

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d

Changes in D-Spacing with Disease

0

5

10

15

20

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

0

5

10

15

20

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

Wallace et al Bone 2010 46 1349-1354

KS Test plt0001

Control 680 plusmn 26 nm

Estrogen Dep 659 plusmn 31 nm

t-test on mean p=0048

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Changes in D-Spacing with Disease

Wallace et al Bone 2010 46 1349-1354

0

10

20

30

40

50

60

70

80

90

100

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

KS Test plt0001

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Question Model and Tissue Morphology Publication

Tissue Specificity in Mice Male mouse 8 weeks Femur dentin tail tendon

No differences in bone vs dentin both ∆ vs tendon

Wallace et al Langmuir 2011 26(10) 7349-7354

Gender Effects Male and female mice 16 weeks Femur No differences NA

Estrogen Depletion OVX in sheep 5 years + 2 Radius Mean and population shift Wallace et al Bone

2010 46 1349-1354

Estrogen Depletion OVX in female 5 years + 2 Skin (dermis) Significant population shift Erickson et al Biotech

J 2013 8(1) 1174-126

Osteoporosis and Mechanical Loading

OVX in ulnar loading in rats Ulna

No loading effect population shift with OVX

Warden et al Endocrinology 2013 154(9) 3178-3187

Osteogenesis Imperfecta Brtl+ model male mice 2 months Femur

No mean ∆ widening of distribution

Wallace et al J Stuct Bio 2011 173 146-152

Osteogenesis Imperfecta Oimoim model male mice 12 weeks Femur Mean and population shift Bart et al Conn Tiss

Res 2014 In Press

Osteogenesis Imperfecta and Tissue Hydration

Brtl+ model male mice 6 months Tail Tendon Significant population shift Kemp et al J Stuct Bio

2012 180 428-438

Type 2 Diabetes Male ZDSD rats 30 weeks Tibia Significant population shift Hammond et al Bone

2014 60 26-32

Type 2 Diabetes Female ZDSD rats 32 weeks Tail Tendon Significant population shift

Diaz Gonzalez et al J Biomech 2014 47(3)

681-686

Raloxifene (in vivo and in vitro) Female dog treated with Raloxifene Femur Significant population shift Gallant et al Bone

2014 61 191-200 BBML - All R

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Issues to Consider Site Selection and Sampling

KS Test plt0001

0023 of bonersquos length

15 m

m =

150

00 micro

m

Wallace et al Langmuir 2011 26(10) 7349-7354

100 microm x 100 microm

35 microm x 35 microm

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Issues to Consider Site Selection and Sampling

60

62

64

66

68

70

72

74

1 (35)

2 (33)

3 (40)

4 (39)

5 (35)

6 (29)

7 (26)

8 (23)

9 (28)

WT (288)

D-P

erio

dic

Gap

Ove

rlap

Spac

ing

(nm

)

Axial Location (Proximal End = 1 Distal End = 9)

( of Fibrils)

91

23

45

87

6

Wallace et al Langmuir 2011 26(10) 7349-7354

288 fibrils for population n=1 for mean comparisons BBML - All R

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Issues to Consider Effects of Hydration

Wet Tendon Dry Tendon Kemp et al J StructBio 2012 180 (3) 428-438

Collagen and bone exist in a hydrated environment in vivo bull Hydration state can impact morphology and mechanics

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Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

Nonsignificant mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet (n=255) 682 plusmn 12 nm

WT Dry (n=257) 686 plusmn 08 nm

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

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Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

Brtl DryBrtl Wet

KS Test plt0001

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

WT Case 04 nm mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet 682 plusmn 12 nm

WT Dry 686 plusmn 08 nm

Brtl+ Wet 675 plusmn 14 nm

Brtl+ Dry 689 plusmn 10 nm

Brtl+ Case 14 nm increase with drying (plt0001) bull Entire population shifted upward (plt0001)

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

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Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT WetBrtl Wet

KS Test plt0001

Brtl+ distribution shifted downward vs WT when wet

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryBrtl Dry

KS Test plt0001

Brtl+ distribution shifted upward vs WT when dry

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

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Issues to Consider AFM calibration

bull Absolute distance measurements require accurate piezo calibration

bull AFM systems have large scan ranges (150 microm to lt 1 microm)

bull Scanner non-linearities can introduce substantial error over that range

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

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Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

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Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

100 nm 10 80 602 1198 100 nm 7 60 827 1439 100 nm 5 40 814 1427 100 nm 35 20 917 1724 100 nm 2 10 937 1774

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

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AFM calibration ndash Take Away Message

Calibration using a scan size and feature size comparable to features of interest is highly recommended

Calibration addresses absolute accuracy not precision (differential sensitivity between measurements)

bull Does not limit ability to differentiate between groups using the same AFM with the same calibration parameters

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AFM Applications in Bone Research Indentation Mechanics

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AFM-Based Indentation

In addition to high resolution imaging AFM can be used to extract nanoscale mechanical data

ApproachWithdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

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AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Can

tilev

er D

efle

ctio

n (V

olts

)

1 Ramp into a rigid sample ndash measure the slope

Deflection sensitivity x 119899119899119899119899119881119881

2 Calculate cantilever spring constant

3 Hookersquos law takes deflection (volts) and converts to units of force F = kx

Spring constant k 119873119873119899119899

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ights Reserve

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AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Forc

e (n

N)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

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AFM-Based Indentation Cantilever Calibration Fo

rce

(nN

)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

Tip-Sample Separation (nm)

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AFM-Based Indentation Parameters

Approach Withdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

Peak Force

Area Between Curves Energy Dissipation

Contact Mechanics Fit for Elastic Modulus (E)

Adhesion Force

Indentation Depth (δ)

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ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

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ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

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ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

z

r

δ

R

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119877119877 ∙ 12057512057532

Es Sample modulus νs Poissonrsquos ratio R tip radius of curvature δ load point displacement

Relevant Hertz Assumptions bull Strains are small and within the elastic limit bull Indenter is infinitely stiff (only sample deforms) bull Indentation depth ltlt radius of curvature of indenter bull Surfaces are continuous non-conforming (must be flat) bull Load is normal to surface

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d

Proper Indentation Model is Essential

δ

r

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119903119903 ∙ 12057512057532

δ

α

119865119865 =2120587120587∙

1198641198641199041199041 minus 1205841205841199041199042

∙ tan120572120572 ∙ 1205751205752

Es Sample modulus νs Poissonrsquos ratio R tip radius α opening angle

Hertz Model Spherical Indenter

depth ltlt radius of curvature

Sneddon Model Conical Indenter

depth ge radius of curvature

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ights Reserve

d

Indentation in OI Tendon Study

x

z

yBrtl+ and WT mice 6 month old males

4 tails per group

2-3 fascicles per tail

25-30 fibrils per tail

4-5 locations per fibril

Indent force 20 nN wet (Sneddon) 50 nN dry (Hertz) BBML - A

ll Rights R

eserved

OI Tendon Differential Effects of Dehydration

Dry 1268 plusmn 718 MPa

Wet 305 plusmn 243 MPa

Dry 1737 plusmn 973 MPa

Wet 301 plusmn 202 MPa

Kemp et al J StructBio 2012 180 (3) 428-438

bull Significant stiffening of fibrils with dehydration

bull More pronounced modulus modulus as a function of disease

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OI Tendon Differential Effects of Dehydration

Kemp et al J StructBio 2012 180 (3) 428-438

Diseased phenotype not present when wet phenotype observed when dried

0

10

20

30

40

50

60

0 20 40 60 80 100 120 140 160 180 200

Gro

up S

ampl

es (

)

Indent Modulus (MPa)

WT WetBrtl Wet

0

2

4

6

8

10

12

14

16

18

20

025

050

075

010

0012

5015

0017

5020

0022

5025

0027

5030

0032

5035

0037

5040

0042

5045

0047

5050

00

Gro

up S

ampl

es (

)Indent Modulus (MPa)

WT DryBrtl Dry

555 WT Indents 305 plusmn 243 MPa

614 Brtl+ Indents 301 plusmn 202 MPa

KS Test p=0129 t Test p=0464 464 WT Indents

1268 plusmn 718 MPa 479 Brtl+ Indents 1737 plusmn 973 MPa

KS Test plt0001 T Test plt0001

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Indentation in Diabetic Tendon (Poster 0495)

x

z

y

Control and Diabetic Rat Tail Tendon

4-5 rats per group

2-3 fascicles per rat

~70 fibrilstail

4-5 locationsfibril

20 nN (Sneddon)

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d

Multiscale Diabetes Study (Poster 0495)

0

5

10

15

20

25

300 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 gt25

Perc

enta

ge o

f Gro

up S

ampl

es

Elastic Modulus (MPa)

CD (1530 indents)ZDSD (913 indents)

CD (n=5) 381 plusmn 149 MPa

ZDSD (n=3) 706 plusmn 263 MPa

0

100

200

300

400

500

600

700

800

900

0 2 4 6 8 10

Mic

rosc

ale

Mod

ulus

(MPa

)

Nanoscale Modulus (MPa)

plt0001

r2=0531 p=0040

bull Increased indentation modulus in diabetic tendon

bull Strong relationship between nanoscale indent modulus and microscale tensile modulus

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Indentation Limitations and Weaknesses

Proper probe selection for intended application bull Cantilever must be stiff relative to sample bull Sharp probes may damage sample easily dull

R or α must be determined for each probe bull Properties provided by manufacturer are unreliable bull Vary from probe to probe can dull during indent process

Spring constant must be determined for each probe

bull Value provided by manufacturer is inaccurate bull Thermal tuning OK for ldquosoftrdquo probes poor performance

for stiffer probes bull Sader method (geometry) added mass reference

cantilevers

BBML - All R

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d

Indentation Limitations and Weaknesses

Veeco Application Technical note Ben Ohler Practical Advice on the Determination of Cantilever Spring Constants

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d

Indentation Limitations and Weaknesses

bull Sample processing

bull Sample hydration

bull Proper force range

bull Substrate effects for thin samples

bull Proper modulus model and range of data goodness of fit

Bearing in mind these assumptions and limitations AFM can provide true ldquonanoscalerdquo mechanical properties both in terms of forces and deformations

BBML - All R

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d

A Note on Peak Force QNM for Bruker AFMs bull Mechanical properties mapped pixel-by-pixel at same

resolution as the height image (PeakForce QNM)

Height Modulus BBML - All R

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d

A Note on Peak Force QNM

Correct probe stiffness must be used based on sample Pittenger ndash Veeco Product Bulletin

BBML - All R

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d

A Note on Peak Force QNM Extremely sensitive to calibration (details rarely given) Absolute radius and spring constant known

bull especially important from probe to probe bull radius uarr with indent depth ndash must determine R at depth

you expect to image bull Depth may vary as a function of sample (or at different

regions within the sample)

Relative reference material of ldquoknownrdquo modulus used to determine a ratio of 119896119896 119877119877 bull ldquoknownrdquo modulus may come from tension compression

and not indentation bull R is related to depth indentation depth on sample must

be same as on reference BBML - All R

ights Reserve

d

A Note on Peak Force QNM bull Modulus fit on the fly ndash no ability to monitor fit (or range) bull Local sample topography and sample tilt play a role in

measured parameters bull Assuming all modulus values in an image are correct how to

quantify

512 x 512 pixels = 262144 measurements

n=1 n=262144

n from selected ROIs

BBML - All R

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bull Imaging in air on mica ndash substrate effect

bull Same imaging force or indent depth for all

bull For each area were all modulus values pooled

Lamprou et al PLoS ONE 2013 BBML - All R

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d

AFM Applications in Bone Research Other Cool Mechanical Techniques

BBML - All R

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d

ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo Bonds within or between collagen fibrils in bone that rupture to dissipate energy as a toughening mechanism

Thompson et al Nature 2001

Fantner et al Nature Mat 2005

Hansma et al JMNI 2005

Fantner et al Nano Letters 2007 BBML - All R

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Other Mechanical Techniques

Jimenez-Palomar J Mech Beh Bio Mat 2012

van der Rijt Macro Bio 2004

Yang J Biomed Mater Res 2007

Yang Biophysical Journal 2008 BBML - All R

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d

Other Cool Techniques bull Piezoresponse Force Microscopy (PFM)

bull Electrochemical AFM (EC-AFM)

bull Force Modulation Microscopy (FMM)

bull Lateral Force Microscopy (LFM)

bull Magnetic Force Microscopy (MFM)

bull Conductive AFM (C-AFM)

bull Force Pulling

bull Cantilever Bending

bull Tensile testing of individual fibrils

bull 3 point bending of fibrils

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Summary Things to rememberhellip

bull Imaging mode imaging medium and probe selection

bull Morphology Measurements bull Sample preparation and hydration state bull Measuring fibril diameter bull Site selection and proper sampling bull Appropriate system calibration

bull Mechanical Measurements

bull Probe selection (stiffness and tip radius) bull Probe calibration (radius angle spring constant) bull Indentation force range and mechanical model bull Hydration in biological samples bull PeakForce QNM

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Acknowledgements BBML

Max Hammond Armando Diaz Gonzalez Zachary Bart Silvia Canelon Creasy Clauser

IU School of Medicine Dr David Burr Dr Matt Allen Dr Stuart Warden Dr Lilian Plotkin

Funding Sources NIHNIDCR 1F32DE018840-01 A1 IUPUI BME Departmental Startup Funds IUPUI Research Support Funds Grant IUPUI Biomechanics and Biomaterials Research Center

NIH NICHD Dr Joan Marini

BBML - All R

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d

Bone Biology and Mechanics Lab (BBML) wwwiupuiedu~bbml Facebook BoneBiologyMechanicsLab

BBML - All R

ights Reserve

d

• Slide Number 1
• General Seminar Outline
• Bonersquos Hierarchical Structure
• Critical Gap in Understanding Collagen
• Methods For Analyzing Collagen Structure
• Methods For Imaging Collagen Structure
• Cryo-TEM
• Atomic Force Microscopy (AFM)
• AFM Image of Pentacene (C22H14)
• AFM Image of Hexabenzocoronene
• Basics of AFM Imaging The Probe
• Basics of AFM Imaging System Components
• van der Waals Potential Energy Curve
• Contact Mode ndash Monitor Deflection
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Peak Force Tapping Mode ndash Monitor Force
• AFM Applications in Bone Research Imaging
• AFM Imaging of Collagen
• Imaging of Osteoclast Activity
• Mineralized collagen deposition in vitro
• Imaging of Canilicular Structure
• AFM Imaging in Bone
• AFM Imaging in Bone
• Removing Mineral in Bone to Expose Collagen
• Removing Mineral in Bone to Expose Collagen
• Demineralization with EDTA
• Demineralization with EDTA
• Demineralization with EDTA
• Issues to Consider Effects of Acid
• Slide Number 32
• Slide Number 33
• Analysis of Collagen Structure in Bone
• Analysis of Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril D-Spacing
• Slide Number 39
• Collagen in Bone Fibril D-Spacing
• Collagen in Bone Fibril D-Spacing
• 2D Fast Fourier Transform Analysis
• 2D Fast Fourier Transform Analysis
• D-Spacing Distribution
• D-Spacing Distribution
• Cumulative Distribution Function
• CDF vs Histogram
• Changes in D-Spacing with Disease
• Changes in D-Spacing with Disease
• Slide Number 50
• Issues to Consider Site Selection and Sampling
• Issues to Consider Site Selection and Sampling
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• AFM calibration ndash Take Away Message
• AFM Applications in Bone ResearchIndentation Mechanics
• AFM-Based Indentation
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Parameters
• Hertz Contact Equation
• Hertz Contact Equation
• Hertz Contact Equation
• Proper Indentation Model is Essential
• Indentation in OI Tendon Study
• OI Tendon Differential Effects of Dehydration
• OI Tendon Differential Effects of Dehydration
• Indentation in Diabetic Tendon (Poster 0495)
• Multiscale Diabetes Study (Poster 0495)
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• A Note on Peak Force QNM for Bruker AFMs
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• Slide Number 83
• Slide Number 84
• AFM Applications in Bone ResearchOther Cool Mechanical Techniques
• ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo
• Other Mechanical Techniques
• Other Cool Techniques
• Summary Things to rememberhellip
• Acknowledgements
• Bone Biology and Mechanics Lab (BBML)

Demineralization with EDTA EDTA - Ethylenediaminetetraacetic Acid

Chelating agent - sequesters divalent cations (notably Ca2+)

bull Treat with 05M EDTA pH 8 (15-20 minutes) bull Vigorously wash with ultrapure water bull Sonicate to release surface-bound mineral ions bull Repeat 3-4 times

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Demineralization with EDTA

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Demineralization with EDTA

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d

Issues to Consider Effects of Acid

Immature collagen crosslinks are very sensitive to acid bull Using acids to demineralize bone may inadvertently

destroy crosslinks distort structure

Fixation of tissue may be necessary prior to demineralization bull Fixation (glutaraldehyde or paraformadehyde) may

preserve structural integrity

bull Fixation effects on collagen structure are not fully known

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d

100 microm x 100 microm

50 microm x 50 microm

25 microm x 25 microm

10 microm x 10 microm

35 microm x 35 microm

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d

35 microm x 35 microm

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ights Reserve

d

Analysis of Collagen Structure in Bone Spatial organization of newly formed bone following a surgically-induced injury (rat tibial hole)

Baranauskas J Vac Sci Tech 2001 Baranauskas App Surface Sci 2005

ldquohellipstatistical diameter of the fibers is 159 nm in good agreement with the literature values for type I collagen fibersrdquo

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d

Analysis of Collagen in Bone Fibril Diameter Fibril Diameter easiest to measure property first attempts were a bit crude better with increased resolution

Sasaki J Mat Sci Tech 2002 Thalhammer J Arch Sci 2001

Bozec Ultramicroscopy 2005

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Collagen in Bone Fibril Diameter

Caution must be taken when reporting and interpreting diameters measured from fibrils in bone bull Fibril packing - width may not be fully exposed

Wallace et al Langmuir 2010

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Collagen in Bone Fibril Diameter

Caution must be taken when reporting and interpreting diameters measured from fibrils in bone bull Fibril packing - width may not be fully exposed

Rigozzi ndash J Struct Bio - 2011

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Collagen in Bone Fibril D-Spacing

300 nm 15 nm

N C C N C C N C C N C C

H O H2C CH2 O H

CH2

O OH HHC CH2

CH2

HO

H H

HHN C C

O

H2C CH2

CH2

HN C C

OH

HC CH2

HO

N C C N C C N C C

H O H2C CH2 O

CH2

OH HC CH2

HO

H H

H

CH2 CH2

glycine X - proline Y - hydroxyproline

Hodge and Petruska 1963

Collagen Fibril Axial D-Periodicity BBML - All R

ights Reserve

d

05

101520

0 02 04 06 08 1

Hei

ght (

nm)

Length (microns)

httpremfdartmoutheduimagesindexhtml

Randall Nature and Structure of Collagen 1953

TEM SEM

AFM

D-Spacing = 67 nm

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d

Collagen in Bone Fibril D-Spacing Many studies have shown observable D-spacing content that near theoretical value

Sasaki 2002 67 plusmn 2 nm Bozek 2005 665 plusmn 14 nm Ge 2007 666 plusmn 38 nm

Habelitz J Structural Bio 2002

A 2002 study in dentin got me interestedhellip

n = 322 wet n = 363 dry

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d

Collagen in Bone Fibril D-Spacing

bull Internal structure bull Wallace et al Bone 2010 46 1349-1354 bull Wallace et al J Stuct Bio 2011 173 146-152 bull Kemp et al J Stuct Bio 2012 180 428-438 bull Warden et al Endocrinology 2013 154(9)

3178-3187 bull Gallant et al Bone 2014 61 191-200 bull Bart et al Conn Tiss Res 2014 In Press

bull Enzymatic and non-

enzymatic cross-linking bull Hammond et al Bone 2014 60 26-32 bull Diaz Gonzalez et al J Biomech 2014 47(3)

681-686 bull Voytik-Harbin et al In Preparation 2014

D-periodic spacing important structural feature that captures aspects related to

Mouse Femur Mid-diaphysis BBML - All R

ights Reserve

d

2D Fast Fourier Transform Analysis

Harmonics

Fibril D-Spacing

Erickson B Wallace JM et al Biotechnology Journal 2013

Raw AFM Image 2D FFT Power Spectrum

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ights Reserve

d

2D Fast Fourier Transform Analysis

Harmonics

Fibril D-Spacing

Raw AFM Image 2D FFT Power Spectrum

0

20000

40000

60000

80000

100000

120000

0 001 002 003 004 005 006

Am

plitu

de (

microV2 )

Position (1nm)

D-periodic spacing 0015 (1nm) ~ 67 nm

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ights Reserve

d

D-Spacing Distribution

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Overall Mean 670 plusmn 00 nm

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ights Reserve

d

D-Spacing Distribution

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Overall Mean 670 plusmn 00 nm

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

4 Samples Total of n = 255 fibrils Overall Mean 682 plusmn 12 nm

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d

Cumulative Distribution Function

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

This population 255 fibrils - Each fibril 1255 = 0392 - Data range 628-716 nm

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d

CDF vs Histogram 62 63 64 65 66 67 68 69 70 71 72 73 74

0

10

20

30

40

50

60

70

80

90

100

0

5

10

15

20

25

30

35

40

45

50

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

HistoCDF

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ights Reserve

d

Changes in D-Spacing with Disease

0

5

10

15

20

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

0

5

10

15

20

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

Wallace et al Bone 2010 46 1349-1354

KS Test plt0001

Control 680 plusmn 26 nm

Estrogen Dep 659 plusmn 31 nm

t-test on mean p=0048

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Changes in D-Spacing with Disease

Wallace et al Bone 2010 46 1349-1354

0

10

20

30

40

50

60

70

80

90

100

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

KS Test plt0001

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Question Model and Tissue Morphology Publication

Tissue Specificity in Mice Male mouse 8 weeks Femur dentin tail tendon

No differences in bone vs dentin both ∆ vs tendon

Wallace et al Langmuir 2011 26(10) 7349-7354

Gender Effects Male and female mice 16 weeks Femur No differences NA

Estrogen Depletion OVX in sheep 5 years + 2 Radius Mean and population shift Wallace et al Bone

2010 46 1349-1354

Estrogen Depletion OVX in female 5 years + 2 Skin (dermis) Significant population shift Erickson et al Biotech

J 2013 8(1) 1174-126

Osteoporosis and Mechanical Loading

OVX in ulnar loading in rats Ulna

No loading effect population shift with OVX

Warden et al Endocrinology 2013 154(9) 3178-3187

Osteogenesis Imperfecta Brtl+ model male mice 2 months Femur

No mean ∆ widening of distribution

Wallace et al J Stuct Bio 2011 173 146-152

Osteogenesis Imperfecta Oimoim model male mice 12 weeks Femur Mean and population shift Bart et al Conn Tiss

Res 2014 In Press

Osteogenesis Imperfecta and Tissue Hydration

Brtl+ model male mice 6 months Tail Tendon Significant population shift Kemp et al J Stuct Bio

2012 180 428-438

Type 2 Diabetes Male ZDSD rats 30 weeks Tibia Significant population shift Hammond et al Bone

2014 60 26-32

Type 2 Diabetes Female ZDSD rats 32 weeks Tail Tendon Significant population shift

Diaz Gonzalez et al J Biomech 2014 47(3)

681-686

Raloxifene (in vivo and in vitro) Female dog treated with Raloxifene Femur Significant population shift Gallant et al Bone

2014 61 191-200 BBML - All R

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d

Issues to Consider Site Selection and Sampling

KS Test plt0001

0023 of bonersquos length

15 m

m =

150

00 micro

m

Wallace et al Langmuir 2011 26(10) 7349-7354

100 microm x 100 microm

35 microm x 35 microm

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Issues to Consider Site Selection and Sampling

60

62

64

66

68

70

72

74

1 (35)

2 (33)

3 (40)

4 (39)

5 (35)

6 (29)

7 (26)

8 (23)

9 (28)

WT (288)

D-P

erio

dic

Gap

Ove

rlap

Spac

ing

(nm

)

Axial Location (Proximal End = 1 Distal End = 9)

( of Fibrils)

91

23

45

87

6

Wallace et al Langmuir 2011 26(10) 7349-7354

288 fibrils for population n=1 for mean comparisons BBML - All R

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Issues to Consider Effects of Hydration

Wet Tendon Dry Tendon Kemp et al J StructBio 2012 180 (3) 428-438

Collagen and bone exist in a hydrated environment in vivo bull Hydration state can impact morphology and mechanics

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Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

Nonsignificant mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet (n=255) 682 plusmn 12 nm

WT Dry (n=257) 686 plusmn 08 nm

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

Brtl DryBrtl Wet

KS Test plt0001

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

WT Case 04 nm mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet 682 plusmn 12 nm

WT Dry 686 plusmn 08 nm

Brtl+ Wet 675 plusmn 14 nm

Brtl+ Dry 689 plusmn 10 nm

Brtl+ Case 14 nm increase with drying (plt0001) bull Entire population shifted upward (plt0001)

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT WetBrtl Wet

KS Test plt0001

Brtl+ distribution shifted downward vs WT when wet

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryBrtl Dry

KS Test plt0001

Brtl+ distribution shifted upward vs WT when dry

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider AFM calibration

bull Absolute distance measurements require accurate piezo calibration

bull AFM systems have large scan ranges (150 microm to lt 1 microm)

bull Scanner non-linearities can introduce substantial error over that range

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

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Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

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Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

100 nm 10 80 602 1198 100 nm 7 60 827 1439 100 nm 5 40 814 1427 100 nm 35 20 917 1724 100 nm 2 10 937 1774

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

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d

AFM calibration ndash Take Away Message

Calibration using a scan size and feature size comparable to features of interest is highly recommended

Calibration addresses absolute accuracy not precision (differential sensitivity between measurements)

bull Does not limit ability to differentiate between groups using the same AFM with the same calibration parameters

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AFM Applications in Bone Research Indentation Mechanics

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AFM-Based Indentation

In addition to high resolution imaging AFM can be used to extract nanoscale mechanical data

ApproachWithdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

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ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Can

tilev

er D

efle

ctio

n (V

olts

)

1 Ramp into a rigid sample ndash measure the slope

Deflection sensitivity x 119899119899119899119899119881119881

2 Calculate cantilever spring constant

3 Hookersquos law takes deflection (volts) and converts to units of force F = kx

Spring constant k 119873119873119899119899

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ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Forc

e (n

N)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

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ights Reserve

d

AFM-Based Indentation Cantilever Calibration Fo

rce

(nN

)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

Tip-Sample Separation (nm)

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ights Reserve

d

AFM-Based Indentation Parameters

Approach Withdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

Peak Force

Area Between Curves Energy Dissipation

Contact Mechanics Fit for Elastic Modulus (E)

Adhesion Force

Indentation Depth (δ)

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

z

r

δ

R

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119877119877 ∙ 12057512057532

Es Sample modulus νs Poissonrsquos ratio R tip radius of curvature δ load point displacement

Relevant Hertz Assumptions bull Strains are small and within the elastic limit bull Indenter is infinitely stiff (only sample deforms) bull Indentation depth ltlt radius of curvature of indenter bull Surfaces are continuous non-conforming (must be flat) bull Load is normal to surface

BBML - All R

ights Reserve

d

Proper Indentation Model is Essential

δ

r

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119903119903 ∙ 12057512057532

δ

α

119865119865 =2120587120587∙

1198641198641199041199041 minus 1205841205841199041199042

∙ tan120572120572 ∙ 1205751205752

Es Sample modulus νs Poissonrsquos ratio R tip radius α opening angle

Hertz Model Spherical Indenter

depth ltlt radius of curvature

Sneddon Model Conical Indenter

depth ge radius of curvature

BBML - All R

ights Reserve

d

Indentation in OI Tendon Study

x

z

yBrtl+ and WT mice 6 month old males

4 tails per group

2-3 fascicles per tail

25-30 fibrils per tail

4-5 locations per fibril

Indent force 20 nN wet (Sneddon) 50 nN dry (Hertz) BBML - A

ll Rights R

eserved

OI Tendon Differential Effects of Dehydration

Dry 1268 plusmn 718 MPa

Wet 305 plusmn 243 MPa

Dry 1737 plusmn 973 MPa

Wet 301 plusmn 202 MPa

Kemp et al J StructBio 2012 180 (3) 428-438

bull Significant stiffening of fibrils with dehydration

bull More pronounced modulus modulus as a function of disease

BBML - All R

ights Reserve

d

OI Tendon Differential Effects of Dehydration

Kemp et al J StructBio 2012 180 (3) 428-438

Diseased phenotype not present when wet phenotype observed when dried

0

10

20

30

40

50

60

0 20 40 60 80 100 120 140 160 180 200

Gro

up S

ampl

es (

)

Indent Modulus (MPa)

WT WetBrtl Wet

0

2

4

6

8

10

12

14

16

18

20

025

050

075

010

0012

5015

0017

5020

0022

5025

0027

5030

0032

5035

0037

5040

0042

5045

0047

5050

00

Gro

up S

ampl

es (

)Indent Modulus (MPa)

WT DryBrtl Dry

555 WT Indents 305 plusmn 243 MPa

614 Brtl+ Indents 301 plusmn 202 MPa

KS Test p=0129 t Test p=0464 464 WT Indents

1268 plusmn 718 MPa 479 Brtl+ Indents 1737 plusmn 973 MPa

KS Test plt0001 T Test plt0001

BBML - All R

ights Reserve

d

Indentation in Diabetic Tendon (Poster 0495)

x

z

y

Control and Diabetic Rat Tail Tendon

4-5 rats per group

2-3 fascicles per rat

~70 fibrilstail

4-5 locationsfibril

20 nN (Sneddon)

BBML - All R

ights Reserve

d

Multiscale Diabetes Study (Poster 0495)

0

5

10

15

20

25

300 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 gt25

Perc

enta

ge o

f Gro

up S

ampl

es

Elastic Modulus (MPa)

CD (1530 indents)ZDSD (913 indents)

CD (n=5) 381 plusmn 149 MPa

ZDSD (n=3) 706 plusmn 263 MPa

0

100

200

300

400

500

600

700

800

900

0 2 4 6 8 10

Mic

rosc

ale

Mod

ulus

(MPa

)

Nanoscale Modulus (MPa)

plt0001

r2=0531 p=0040

bull Increased indentation modulus in diabetic tendon

bull Strong relationship between nanoscale indent modulus and microscale tensile modulus

BBML - All R

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d

Indentation Limitations and Weaknesses

Proper probe selection for intended application bull Cantilever must be stiff relative to sample bull Sharp probes may damage sample easily dull

R or α must be determined for each probe bull Properties provided by manufacturer are unreliable bull Vary from probe to probe can dull during indent process

Spring constant must be determined for each probe

bull Value provided by manufacturer is inaccurate bull Thermal tuning OK for ldquosoftrdquo probes poor performance

for stiffer probes bull Sader method (geometry) added mass reference

cantilevers

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

Veeco Application Technical note Ben Ohler Practical Advice on the Determination of Cantilever Spring Constants

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

bull Sample processing

bull Sample hydration

bull Proper force range

bull Substrate effects for thin samples

bull Proper modulus model and range of data goodness of fit

Bearing in mind these assumptions and limitations AFM can provide true ldquonanoscalerdquo mechanical properties both in terms of forces and deformations

BBML - All R

ights Reserve

d

A Note on Peak Force QNM for Bruker AFMs bull Mechanical properties mapped pixel-by-pixel at same

resolution as the height image (PeakForce QNM)

Height Modulus BBML - All R

ights Reserve

d

A Note on Peak Force QNM

Correct probe stiffness must be used based on sample Pittenger ndash Veeco Product Bulletin

BBML - All R

ights Reserve

d

A Note on Peak Force QNM Extremely sensitive to calibration (details rarely given) Absolute radius and spring constant known

bull especially important from probe to probe bull radius uarr with indent depth ndash must determine R at depth

you expect to image bull Depth may vary as a function of sample (or at different

regions within the sample)

Relative reference material of ldquoknownrdquo modulus used to determine a ratio of 119896119896 119877119877 bull ldquoknownrdquo modulus may come from tension compression

and not indentation bull R is related to depth indentation depth on sample must

be same as on reference BBML - All R

ights Reserve

d

A Note on Peak Force QNM bull Modulus fit on the fly ndash no ability to monitor fit (or range) bull Local sample topography and sample tilt play a role in

measured parameters bull Assuming all modulus values in an image are correct how to

quantify

512 x 512 pixels = 262144 measurements

n=1 n=262144

n from selected ROIs

BBML - All R

ights Reserve

d

BBML - All R

ights Reserve

d

bull Imaging in air on mica ndash substrate effect

bull Same imaging force or indent depth for all

bull For each area were all modulus values pooled

Lamprou et al PLoS ONE 2013 BBML - All R

ights Reserve

d

AFM Applications in Bone Research Other Cool Mechanical Techniques

BBML - All R

ights Reserve

d

ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo Bonds within or between collagen fibrils in bone that rupture to dissipate energy as a toughening mechanism

Thompson et al Nature 2001

Fantner et al Nature Mat 2005

Hansma et al JMNI 2005

Fantner et al Nano Letters 2007 BBML - All R

ights Reserve

d

Other Mechanical Techniques

Jimenez-Palomar J Mech Beh Bio Mat 2012

van der Rijt Macro Bio 2004

Yang J Biomed Mater Res 2007

Yang Biophysical Journal 2008 BBML - All R

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Other Cool Techniques bull Piezoresponse Force Microscopy (PFM)

bull Electrochemical AFM (EC-AFM)

bull Force Modulation Microscopy (FMM)

bull Lateral Force Microscopy (LFM)

bull Magnetic Force Microscopy (MFM)

bull Conductive AFM (C-AFM)

bull Force Pulling

bull Cantilever Bending

bull Tensile testing of individual fibrils

bull 3 point bending of fibrils

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Summary Things to rememberhellip

bull Imaging mode imaging medium and probe selection

bull Morphology Measurements bull Sample preparation and hydration state bull Measuring fibril diameter bull Site selection and proper sampling bull Appropriate system calibration

bull Mechanical Measurements

bull Probe selection (stiffness and tip radius) bull Probe calibration (radius angle spring constant) bull Indentation force range and mechanical model bull Hydration in biological samples bull PeakForce QNM

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Acknowledgements BBML

Max Hammond Armando Diaz Gonzalez Zachary Bart Silvia Canelon Creasy Clauser

IU School of Medicine Dr David Burr Dr Matt Allen Dr Stuart Warden Dr Lilian Plotkin

Funding Sources NIHNIDCR 1F32DE018840-01 A1 IUPUI BME Departmental Startup Funds IUPUI Research Support Funds Grant IUPUI Biomechanics and Biomaterials Research Center

NIH NICHD Dr Joan Marini

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Bone Biology and Mechanics Lab (BBML) wwwiupuiedu~bbml Facebook BoneBiologyMechanicsLab

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• Slide Number 1
• General Seminar Outline
• Bonersquos Hierarchical Structure
• Critical Gap in Understanding Collagen
• Methods For Analyzing Collagen Structure
• Methods For Imaging Collagen Structure
• Cryo-TEM
• Atomic Force Microscopy (AFM)
• AFM Image of Pentacene (C22H14)
• AFM Image of Hexabenzocoronene
• Basics of AFM Imaging The Probe
• Basics of AFM Imaging System Components
• van der Waals Potential Energy Curve
• Contact Mode ndash Monitor Deflection
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Peak Force Tapping Mode ndash Monitor Force
• AFM Applications in Bone Research Imaging
• AFM Imaging of Collagen
• Imaging of Osteoclast Activity
• Mineralized collagen deposition in vitro
• Imaging of Canilicular Structure
• AFM Imaging in Bone
• AFM Imaging in Bone
• Removing Mineral in Bone to Expose Collagen
• Removing Mineral in Bone to Expose Collagen
• Demineralization with EDTA
• Demineralization with EDTA
• Demineralization with EDTA
• Issues to Consider Effects of Acid
• Slide Number 32
• Slide Number 33
• Analysis of Collagen Structure in Bone
• Analysis of Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril D-Spacing
• Slide Number 39
• Collagen in Bone Fibril D-Spacing
• Collagen in Bone Fibril D-Spacing
• 2D Fast Fourier Transform Analysis
• 2D Fast Fourier Transform Analysis
• D-Spacing Distribution
• D-Spacing Distribution
• Cumulative Distribution Function
• CDF vs Histogram
• Changes in D-Spacing with Disease
• Changes in D-Spacing with Disease
• Slide Number 50
• Issues to Consider Site Selection and Sampling
• Issues to Consider Site Selection and Sampling
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• AFM calibration ndash Take Away Message
• AFM Applications in Bone ResearchIndentation Mechanics
• AFM-Based Indentation
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Parameters
• Hertz Contact Equation
• Hertz Contact Equation
• Hertz Contact Equation
• Proper Indentation Model is Essential
• Indentation in OI Tendon Study
• OI Tendon Differential Effects of Dehydration
• OI Tendon Differential Effects of Dehydration
• Indentation in Diabetic Tendon (Poster 0495)
• Multiscale Diabetes Study (Poster 0495)
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• A Note on Peak Force QNM for Bruker AFMs
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• Slide Number 83
• Slide Number 84
• AFM Applications in Bone ResearchOther Cool Mechanical Techniques
• ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo
• Other Mechanical Techniques
• Other Cool Techniques
• Summary Things to rememberhellip
• Acknowledgements
• Bone Biology and Mechanics Lab (BBML)

Demineralization with EDTA

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Demineralization with EDTA

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Issues to Consider Effects of Acid

Immature collagen crosslinks are very sensitive to acid bull Using acids to demineralize bone may inadvertently

destroy crosslinks distort structure

Fixation of tissue may be necessary prior to demineralization bull Fixation (glutaraldehyde or paraformadehyde) may

preserve structural integrity

bull Fixation effects on collagen structure are not fully known

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100 microm x 100 microm

50 microm x 50 microm

25 microm x 25 microm

10 microm x 10 microm

35 microm x 35 microm

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35 microm x 35 microm

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Analysis of Collagen Structure in Bone Spatial organization of newly formed bone following a surgically-induced injury (rat tibial hole)

Baranauskas J Vac Sci Tech 2001 Baranauskas App Surface Sci 2005

ldquohellipstatistical diameter of the fibers is 159 nm in good agreement with the literature values for type I collagen fibersrdquo

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Analysis of Collagen in Bone Fibril Diameter Fibril Diameter easiest to measure property first attempts were a bit crude better with increased resolution

Sasaki J Mat Sci Tech 2002 Thalhammer J Arch Sci 2001

Bozec Ultramicroscopy 2005

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Collagen in Bone Fibril Diameter

Caution must be taken when reporting and interpreting diameters measured from fibrils in bone bull Fibril packing - width may not be fully exposed

Wallace et al Langmuir 2010

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Collagen in Bone Fibril Diameter

Caution must be taken when reporting and interpreting diameters measured from fibrils in bone bull Fibril packing - width may not be fully exposed

Rigozzi ndash J Struct Bio - 2011

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Collagen in Bone Fibril D-Spacing

300 nm 15 nm

N C C N C C N C C N C C

H O H2C CH2 O H

CH2

O OH HHC CH2

CH2

HO

H H

HHN C C

O

H2C CH2

CH2

HN C C

OH

HC CH2

HO

N C C N C C N C C

H O H2C CH2 O

CH2

OH HC CH2

HO

H H

H

CH2 CH2

glycine X - proline Y - hydroxyproline

Hodge and Petruska 1963

Collagen Fibril Axial D-Periodicity BBML - All R

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d

05

101520

0 02 04 06 08 1

Hei

ght (

nm)

Length (microns)

httpremfdartmoutheduimagesindexhtml

Randall Nature and Structure of Collagen 1953

TEM SEM

AFM

D-Spacing = 67 nm

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Collagen in Bone Fibril D-Spacing Many studies have shown observable D-spacing content that near theoretical value

Sasaki 2002 67 plusmn 2 nm Bozek 2005 665 plusmn 14 nm Ge 2007 666 plusmn 38 nm

Habelitz J Structural Bio 2002

A 2002 study in dentin got me interestedhellip

n = 322 wet n = 363 dry

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Collagen in Bone Fibril D-Spacing

bull Internal structure bull Wallace et al Bone 2010 46 1349-1354 bull Wallace et al J Stuct Bio 2011 173 146-152 bull Kemp et al J Stuct Bio 2012 180 428-438 bull Warden et al Endocrinology 2013 154(9)

3178-3187 bull Gallant et al Bone 2014 61 191-200 bull Bart et al Conn Tiss Res 2014 In Press

bull Enzymatic and non-

enzymatic cross-linking bull Hammond et al Bone 2014 60 26-32 bull Diaz Gonzalez et al J Biomech 2014 47(3)

681-686 bull Voytik-Harbin et al In Preparation 2014

D-periodic spacing important structural feature that captures aspects related to

Mouse Femur Mid-diaphysis BBML - All R

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2D Fast Fourier Transform Analysis

Harmonics

Fibril D-Spacing

Erickson B Wallace JM et al Biotechnology Journal 2013

Raw AFM Image 2D FFT Power Spectrum

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2D Fast Fourier Transform Analysis

Harmonics

Fibril D-Spacing

Raw AFM Image 2D FFT Power Spectrum

0

20000

40000

60000

80000

100000

120000

0 001 002 003 004 005 006

Am

plitu

de (

microV2 )

Position (1nm)

D-periodic spacing 0015 (1nm) ~ 67 nm

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d

D-Spacing Distribution

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Overall Mean 670 plusmn 00 nm

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ights Reserve

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D-Spacing Distribution

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Overall Mean 670 plusmn 00 nm

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

4 Samples Total of n = 255 fibrils Overall Mean 682 plusmn 12 nm

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Cumulative Distribution Function

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

This population 255 fibrils - Each fibril 1255 = 0392 - Data range 628-716 nm

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CDF vs Histogram 62 63 64 65 66 67 68 69 70 71 72 73 74

0

10

20

30

40

50

60

70

80

90

100

0

5

10

15

20

25

30

35

40

45

50

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

HistoCDF

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Changes in D-Spacing with Disease

0

5

10

15

20

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

0

5

10

15

20

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

Wallace et al Bone 2010 46 1349-1354

KS Test plt0001

Control 680 plusmn 26 nm

Estrogen Dep 659 plusmn 31 nm

t-test on mean p=0048

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Changes in D-Spacing with Disease

Wallace et al Bone 2010 46 1349-1354

0

10

20

30

40

50

60

70

80

90

100

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

KS Test plt0001

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Question Model and Tissue Morphology Publication

Tissue Specificity in Mice Male mouse 8 weeks Femur dentin tail tendon

No differences in bone vs dentin both ∆ vs tendon

Wallace et al Langmuir 2011 26(10) 7349-7354

Gender Effects Male and female mice 16 weeks Femur No differences NA

Estrogen Depletion OVX in sheep 5 years + 2 Radius Mean and population shift Wallace et al Bone

2010 46 1349-1354

Estrogen Depletion OVX in female 5 years + 2 Skin (dermis) Significant population shift Erickson et al Biotech

J 2013 8(1) 1174-126

Osteoporosis and Mechanical Loading

OVX in ulnar loading in rats Ulna

No loading effect population shift with OVX

Warden et al Endocrinology 2013 154(9) 3178-3187

Osteogenesis Imperfecta Brtl+ model male mice 2 months Femur

No mean ∆ widening of distribution

Wallace et al J Stuct Bio 2011 173 146-152

Osteogenesis Imperfecta Oimoim model male mice 12 weeks Femur Mean and population shift Bart et al Conn Tiss

Res 2014 In Press

Osteogenesis Imperfecta and Tissue Hydration

Brtl+ model male mice 6 months Tail Tendon Significant population shift Kemp et al J Stuct Bio

2012 180 428-438

Type 2 Diabetes Male ZDSD rats 30 weeks Tibia Significant population shift Hammond et al Bone

2014 60 26-32

Type 2 Diabetes Female ZDSD rats 32 weeks Tail Tendon Significant population shift

Diaz Gonzalez et al J Biomech 2014 47(3)

681-686

Raloxifene (in vivo and in vitro) Female dog treated with Raloxifene Femur Significant population shift Gallant et al Bone

2014 61 191-200 BBML - All R

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Issues to Consider Site Selection and Sampling

KS Test plt0001

0023 of bonersquos length

15 m

m =

150

00 micro

m

Wallace et al Langmuir 2011 26(10) 7349-7354

100 microm x 100 microm

35 microm x 35 microm

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Issues to Consider Site Selection and Sampling

60

62

64

66

68

70

72

74

1 (35)

2 (33)

3 (40)

4 (39)

5 (35)

6 (29)

7 (26)

8 (23)

9 (28)

WT (288)

D-P

erio

dic

Gap

Ove

rlap

Spac

ing

(nm

)

Axial Location (Proximal End = 1 Distal End = 9)

( of Fibrils)

91

23

45

87

6

Wallace et al Langmuir 2011 26(10) 7349-7354

288 fibrils for population n=1 for mean comparisons BBML - All R

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Issues to Consider Effects of Hydration

Wet Tendon Dry Tendon Kemp et al J StructBio 2012 180 (3) 428-438

Collagen and bone exist in a hydrated environment in vivo bull Hydration state can impact morphology and mechanics

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Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

Nonsignificant mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet (n=255) 682 plusmn 12 nm

WT Dry (n=257) 686 plusmn 08 nm

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

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Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

Brtl DryBrtl Wet

KS Test plt0001

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

WT Case 04 nm mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet 682 plusmn 12 nm

WT Dry 686 plusmn 08 nm

Brtl+ Wet 675 plusmn 14 nm

Brtl+ Dry 689 plusmn 10 nm

Brtl+ Case 14 nm increase with drying (plt0001) bull Entire population shifted upward (plt0001)

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

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Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT WetBrtl Wet

KS Test plt0001

Brtl+ distribution shifted downward vs WT when wet

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryBrtl Dry

KS Test plt0001

Brtl+ distribution shifted upward vs WT when dry

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

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Issues to Consider AFM calibration

bull Absolute distance measurements require accurate piezo calibration

bull AFM systems have large scan ranges (150 microm to lt 1 microm)

bull Scanner non-linearities can introduce substantial error over that range

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

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Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

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Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

100 nm 10 80 602 1198 100 nm 7 60 827 1439 100 nm 5 40 814 1427 100 nm 35 20 917 1724 100 nm 2 10 937 1774

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

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AFM calibration ndash Take Away Message

Calibration using a scan size and feature size comparable to features of interest is highly recommended

Calibration addresses absolute accuracy not precision (differential sensitivity between measurements)

bull Does not limit ability to differentiate between groups using the same AFM with the same calibration parameters

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AFM Applications in Bone Research Indentation Mechanics

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AFM-Based Indentation

In addition to high resolution imaging AFM can be used to extract nanoscale mechanical data

ApproachWithdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

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AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Can

tilev

er D

efle

ctio

n (V

olts

)

1 Ramp into a rigid sample ndash measure the slope

Deflection sensitivity x 119899119899119899119899119881119881

2 Calculate cantilever spring constant

3 Hookersquos law takes deflection (volts) and converts to units of force F = kx

Spring constant k 119873119873119899119899

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AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Forc

e (n

N)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

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AFM-Based Indentation Cantilever Calibration Fo

rce

(nN

)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

Tip-Sample Separation (nm)

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AFM-Based Indentation Parameters

Approach Withdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

Peak Force

Area Between Curves Energy Dissipation

Contact Mechanics Fit for Elastic Modulus (E)

Adhesion Force

Indentation Depth (δ)

BBML - All R

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Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

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Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

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Hertz Contact Equation

wwwwikipediacom

z

r

δ

R

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119877119877 ∙ 12057512057532

Es Sample modulus νs Poissonrsquos ratio R tip radius of curvature δ load point displacement

Relevant Hertz Assumptions bull Strains are small and within the elastic limit bull Indenter is infinitely stiff (only sample deforms) bull Indentation depth ltlt radius of curvature of indenter bull Surfaces are continuous non-conforming (must be flat) bull Load is normal to surface

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d

Proper Indentation Model is Essential

δ

r

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119903119903 ∙ 12057512057532

δ

α

119865119865 =2120587120587∙

1198641198641199041199041 minus 1205841205841199041199042

∙ tan120572120572 ∙ 1205751205752

Es Sample modulus νs Poissonrsquos ratio R tip radius α opening angle

Hertz Model Spherical Indenter

depth ltlt radius of curvature

Sneddon Model Conical Indenter

depth ge radius of curvature

BBML - All R

ights Reserve

d

Indentation in OI Tendon Study

x

z

yBrtl+ and WT mice 6 month old males

4 tails per group

2-3 fascicles per tail

25-30 fibrils per tail

4-5 locations per fibril

Indent force 20 nN wet (Sneddon) 50 nN dry (Hertz) BBML - A

ll Rights R

eserved

OI Tendon Differential Effects of Dehydration

Dry 1268 plusmn 718 MPa

Wet 305 plusmn 243 MPa

Dry 1737 plusmn 973 MPa

Wet 301 plusmn 202 MPa

Kemp et al J StructBio 2012 180 (3) 428-438

bull Significant stiffening of fibrils with dehydration

bull More pronounced modulus modulus as a function of disease

BBML - All R

ights Reserve

d

OI Tendon Differential Effects of Dehydration

Kemp et al J StructBio 2012 180 (3) 428-438

Diseased phenotype not present when wet phenotype observed when dried

0

10

20

30

40

50

60

0 20 40 60 80 100 120 140 160 180 200

Gro

up S

ampl

es (

)

Indent Modulus (MPa)

WT WetBrtl Wet

0

2

4

6

8

10

12

14

16

18

20

025

050

075

010

0012

5015

0017

5020

0022

5025

0027

5030

0032

5035

0037

5040

0042

5045

0047

5050

00

Gro

up S

ampl

es (

)Indent Modulus (MPa)

WT DryBrtl Dry

555 WT Indents 305 plusmn 243 MPa

614 Brtl+ Indents 301 plusmn 202 MPa

KS Test p=0129 t Test p=0464 464 WT Indents

1268 plusmn 718 MPa 479 Brtl+ Indents 1737 plusmn 973 MPa

KS Test plt0001 T Test plt0001

BBML - All R

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d

Indentation in Diabetic Tendon (Poster 0495)

x

z

y

Control and Diabetic Rat Tail Tendon

4-5 rats per group

2-3 fascicles per rat

~70 fibrilstail

4-5 locationsfibril

20 nN (Sneddon)

BBML - All R

ights Reserve

d

Multiscale Diabetes Study (Poster 0495)

0

5

10

15

20

25

300 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 gt25

Perc

enta

ge o

f Gro

up S

ampl

es

Elastic Modulus (MPa)

CD (1530 indents)ZDSD (913 indents)

CD (n=5) 381 plusmn 149 MPa

ZDSD (n=3) 706 plusmn 263 MPa

0

100

200

300

400

500

600

700

800

900

0 2 4 6 8 10

Mic

rosc

ale

Mod

ulus

(MPa

)

Nanoscale Modulus (MPa)

plt0001

r2=0531 p=0040

bull Increased indentation modulus in diabetic tendon

bull Strong relationship between nanoscale indent modulus and microscale tensile modulus

BBML - All R

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d

Indentation Limitations and Weaknesses

Proper probe selection for intended application bull Cantilever must be stiff relative to sample bull Sharp probes may damage sample easily dull

R or α must be determined for each probe bull Properties provided by manufacturer are unreliable bull Vary from probe to probe can dull during indent process

Spring constant must be determined for each probe

bull Value provided by manufacturer is inaccurate bull Thermal tuning OK for ldquosoftrdquo probes poor performance

for stiffer probes bull Sader method (geometry) added mass reference

cantilevers

BBML - All R

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d

Indentation Limitations and Weaknesses

Veeco Application Technical note Ben Ohler Practical Advice on the Determination of Cantilever Spring Constants

BBML - All R

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d

Indentation Limitations and Weaknesses

bull Sample processing

bull Sample hydration

bull Proper force range

bull Substrate effects for thin samples

bull Proper modulus model and range of data goodness of fit

Bearing in mind these assumptions and limitations AFM can provide true ldquonanoscalerdquo mechanical properties both in terms of forces and deformations

BBML - All R

ights Reserve

d

A Note on Peak Force QNM for Bruker AFMs bull Mechanical properties mapped pixel-by-pixel at same

resolution as the height image (PeakForce QNM)

Height Modulus BBML - All R

ights Reserve

d

A Note on Peak Force QNM

Correct probe stiffness must be used based on sample Pittenger ndash Veeco Product Bulletin

BBML - All R

ights Reserve

d

A Note on Peak Force QNM Extremely sensitive to calibration (details rarely given) Absolute radius and spring constant known

bull especially important from probe to probe bull radius uarr with indent depth ndash must determine R at depth

you expect to image bull Depth may vary as a function of sample (or at different

regions within the sample)

Relative reference material of ldquoknownrdquo modulus used to determine a ratio of 119896119896 119877119877 bull ldquoknownrdquo modulus may come from tension compression

and not indentation bull R is related to depth indentation depth on sample must

be same as on reference BBML - All R

ights Reserve

d

A Note on Peak Force QNM bull Modulus fit on the fly ndash no ability to monitor fit (or range) bull Local sample topography and sample tilt play a role in

measured parameters bull Assuming all modulus values in an image are correct how to

quantify

512 x 512 pixels = 262144 measurements

n=1 n=262144

n from selected ROIs

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ights Reserve

d

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ights Reserve

d

bull Imaging in air on mica ndash substrate effect

bull Same imaging force or indent depth for all

bull For each area were all modulus values pooled

Lamprou et al PLoS ONE 2013 BBML - All R

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d

AFM Applications in Bone Research Other Cool Mechanical Techniques

BBML - All R

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d

ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo Bonds within or between collagen fibrils in bone that rupture to dissipate energy as a toughening mechanism

Thompson et al Nature 2001

Fantner et al Nature Mat 2005

Hansma et al JMNI 2005

Fantner et al Nano Letters 2007 BBML - All R

ights Reserve

d

Other Mechanical Techniques

Jimenez-Palomar J Mech Beh Bio Mat 2012

van der Rijt Macro Bio 2004

Yang J Biomed Mater Res 2007

Yang Biophysical Journal 2008 BBML - All R

ights Reserve

d

Other Cool Techniques bull Piezoresponse Force Microscopy (PFM)

bull Electrochemical AFM (EC-AFM)

bull Force Modulation Microscopy (FMM)

bull Lateral Force Microscopy (LFM)

bull Magnetic Force Microscopy (MFM)

bull Conductive AFM (C-AFM)

bull Force Pulling

bull Cantilever Bending

bull Tensile testing of individual fibrils

bull 3 point bending of fibrils

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Summary Things to rememberhellip

bull Imaging mode imaging medium and probe selection

bull Morphology Measurements bull Sample preparation and hydration state bull Measuring fibril diameter bull Site selection and proper sampling bull Appropriate system calibration

bull Mechanical Measurements

bull Probe selection (stiffness and tip radius) bull Probe calibration (radius angle spring constant) bull Indentation force range and mechanical model bull Hydration in biological samples bull PeakForce QNM

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Acknowledgements BBML

Max Hammond Armando Diaz Gonzalez Zachary Bart Silvia Canelon Creasy Clauser

IU School of Medicine Dr David Burr Dr Matt Allen Dr Stuart Warden Dr Lilian Plotkin

Funding Sources NIHNIDCR 1F32DE018840-01 A1 IUPUI BME Departmental Startup Funds IUPUI Research Support Funds Grant IUPUI Biomechanics and Biomaterials Research Center

NIH NICHD Dr Joan Marini

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Bone Biology and Mechanics Lab (BBML) wwwiupuiedu~bbml Facebook BoneBiologyMechanicsLab

BBML - All R

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• Slide Number 1
• General Seminar Outline
• Bonersquos Hierarchical Structure
• Critical Gap in Understanding Collagen
• Methods For Analyzing Collagen Structure
• Methods For Imaging Collagen Structure
• Cryo-TEM
• Atomic Force Microscopy (AFM)
• AFM Image of Pentacene (C22H14)
• AFM Image of Hexabenzocoronene
• Basics of AFM Imaging The Probe
• Basics of AFM Imaging System Components
• van der Waals Potential Energy Curve
• Contact Mode ndash Monitor Deflection
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Peak Force Tapping Mode ndash Monitor Force
• AFM Applications in Bone Research Imaging
• AFM Imaging of Collagen
• Imaging of Osteoclast Activity
• Mineralized collagen deposition in vitro
• Imaging of Canilicular Structure
• AFM Imaging in Bone
• AFM Imaging in Bone
• Removing Mineral in Bone to Expose Collagen
• Removing Mineral in Bone to Expose Collagen
• Demineralization with EDTA
• Demineralization with EDTA
• Demineralization with EDTA
• Issues to Consider Effects of Acid
• Slide Number 32
• Slide Number 33
• Analysis of Collagen Structure in Bone
• Analysis of Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril D-Spacing
• Slide Number 39
• Collagen in Bone Fibril D-Spacing
• Collagen in Bone Fibril D-Spacing
• 2D Fast Fourier Transform Analysis
• 2D Fast Fourier Transform Analysis
• D-Spacing Distribution
• D-Spacing Distribution
• Cumulative Distribution Function
• CDF vs Histogram
• Changes in D-Spacing with Disease
• Changes in D-Spacing with Disease
• Slide Number 50
• Issues to Consider Site Selection and Sampling
• Issues to Consider Site Selection and Sampling
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• AFM calibration ndash Take Away Message
• AFM Applications in Bone ResearchIndentation Mechanics
• AFM-Based Indentation
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Parameters
• Hertz Contact Equation
• Hertz Contact Equation
• Hertz Contact Equation
• Proper Indentation Model is Essential
• Indentation in OI Tendon Study
• OI Tendon Differential Effects of Dehydration
• OI Tendon Differential Effects of Dehydration
• Indentation in Diabetic Tendon (Poster 0495)
• Multiscale Diabetes Study (Poster 0495)
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• A Note on Peak Force QNM for Bruker AFMs
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• Slide Number 83
• Slide Number 84
• AFM Applications in Bone ResearchOther Cool Mechanical Techniques
• ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo
• Other Mechanical Techniques
• Other Cool Techniques
• Summary Things to rememberhellip
• Acknowledgements
• Bone Biology and Mechanics Lab (BBML)

Demineralization with EDTA

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Issues to Consider Effects of Acid

Immature collagen crosslinks are very sensitive to acid bull Using acids to demineralize bone may inadvertently

destroy crosslinks distort structure

Fixation of tissue may be necessary prior to demineralization bull Fixation (glutaraldehyde or paraformadehyde) may

preserve structural integrity

bull Fixation effects on collagen structure are not fully known

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100 microm x 100 microm

50 microm x 50 microm

25 microm x 25 microm

10 microm x 10 microm

35 microm x 35 microm

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d

35 microm x 35 microm

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Analysis of Collagen Structure in Bone Spatial organization of newly formed bone following a surgically-induced injury (rat tibial hole)

Baranauskas J Vac Sci Tech 2001 Baranauskas App Surface Sci 2005

ldquohellipstatistical diameter of the fibers is 159 nm in good agreement with the literature values for type I collagen fibersrdquo

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Analysis of Collagen in Bone Fibril Diameter Fibril Diameter easiest to measure property first attempts were a bit crude better with increased resolution

Sasaki J Mat Sci Tech 2002 Thalhammer J Arch Sci 2001

Bozec Ultramicroscopy 2005

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Collagen in Bone Fibril Diameter

Caution must be taken when reporting and interpreting diameters measured from fibrils in bone bull Fibril packing - width may not be fully exposed

Wallace et al Langmuir 2010

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Collagen in Bone Fibril Diameter

Caution must be taken when reporting and interpreting diameters measured from fibrils in bone bull Fibril packing - width may not be fully exposed

Rigozzi ndash J Struct Bio - 2011

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Collagen in Bone Fibril D-Spacing

300 nm 15 nm

N C C N C C N C C N C C

H O H2C CH2 O H

CH2

O OH HHC CH2

CH2

HO

H H

HHN C C

O

H2C CH2

CH2

HN C C

OH

HC CH2

HO

N C C N C C N C C

H O H2C CH2 O

CH2

OH HC CH2

HO

H H

H

CH2 CH2

glycine X - proline Y - hydroxyproline

Hodge and Petruska 1963

Collagen Fibril Axial D-Periodicity BBML - All R

ights Reserve

d

05

101520

0 02 04 06 08 1

Hei

ght (

nm)

Length (microns)

httpremfdartmoutheduimagesindexhtml

Randall Nature and Structure of Collagen 1953

TEM SEM

AFM

D-Spacing = 67 nm

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Collagen in Bone Fibril D-Spacing Many studies have shown observable D-spacing content that near theoretical value

Sasaki 2002 67 plusmn 2 nm Bozek 2005 665 plusmn 14 nm Ge 2007 666 plusmn 38 nm

Habelitz J Structural Bio 2002

A 2002 study in dentin got me interestedhellip

n = 322 wet n = 363 dry

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Collagen in Bone Fibril D-Spacing

bull Internal structure bull Wallace et al Bone 2010 46 1349-1354 bull Wallace et al J Stuct Bio 2011 173 146-152 bull Kemp et al J Stuct Bio 2012 180 428-438 bull Warden et al Endocrinology 2013 154(9)

3178-3187 bull Gallant et al Bone 2014 61 191-200 bull Bart et al Conn Tiss Res 2014 In Press

bull Enzymatic and non-

enzymatic cross-linking bull Hammond et al Bone 2014 60 26-32 bull Diaz Gonzalez et al J Biomech 2014 47(3)

681-686 bull Voytik-Harbin et al In Preparation 2014

D-periodic spacing important structural feature that captures aspects related to

Mouse Femur Mid-diaphysis BBML - All R

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2D Fast Fourier Transform Analysis

Harmonics

Fibril D-Spacing

Erickson B Wallace JM et al Biotechnology Journal 2013

Raw AFM Image 2D FFT Power Spectrum

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2D Fast Fourier Transform Analysis

Harmonics

Fibril D-Spacing

Raw AFM Image 2D FFT Power Spectrum

0

20000

40000

60000

80000

100000

120000

0 001 002 003 004 005 006

Am

plitu

de (

microV2 )

Position (1nm)

D-periodic spacing 0015 (1nm) ~ 67 nm

BBML - All R

ights Reserve

d

D-Spacing Distribution

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Overall Mean 670 plusmn 00 nm

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ights Reserve

d

D-Spacing Distribution

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Overall Mean 670 plusmn 00 nm

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

4 Samples Total of n = 255 fibrils Overall Mean 682 plusmn 12 nm

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Cumulative Distribution Function

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

This population 255 fibrils - Each fibril 1255 = 0392 - Data range 628-716 nm

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CDF vs Histogram 62 63 64 65 66 67 68 69 70 71 72 73 74

0

10

20

30

40

50

60

70

80

90

100

0

5

10

15

20

25

30

35

40

45

50

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

HistoCDF

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d

Changes in D-Spacing with Disease

0

5

10

15

20

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

0

5

10

15

20

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

Wallace et al Bone 2010 46 1349-1354

KS Test plt0001

Control 680 plusmn 26 nm

Estrogen Dep 659 plusmn 31 nm

t-test on mean p=0048

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Changes in D-Spacing with Disease

Wallace et al Bone 2010 46 1349-1354

0

10

20

30

40

50

60

70

80

90

100

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

KS Test plt0001

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Question Model and Tissue Morphology Publication

Tissue Specificity in Mice Male mouse 8 weeks Femur dentin tail tendon

No differences in bone vs dentin both ∆ vs tendon

Wallace et al Langmuir 2011 26(10) 7349-7354

Gender Effects Male and female mice 16 weeks Femur No differences NA

Estrogen Depletion OVX in sheep 5 years + 2 Radius Mean and population shift Wallace et al Bone

2010 46 1349-1354

Estrogen Depletion OVX in female 5 years + 2 Skin (dermis) Significant population shift Erickson et al Biotech

J 2013 8(1) 1174-126

Osteoporosis and Mechanical Loading

OVX in ulnar loading in rats Ulna

No loading effect population shift with OVX

Warden et al Endocrinology 2013 154(9) 3178-3187

Osteogenesis Imperfecta Brtl+ model male mice 2 months Femur

No mean ∆ widening of distribution

Wallace et al J Stuct Bio 2011 173 146-152

Osteogenesis Imperfecta Oimoim model male mice 12 weeks Femur Mean and population shift Bart et al Conn Tiss

Res 2014 In Press

Osteogenesis Imperfecta and Tissue Hydration

Brtl+ model male mice 6 months Tail Tendon Significant population shift Kemp et al J Stuct Bio

2012 180 428-438

Type 2 Diabetes Male ZDSD rats 30 weeks Tibia Significant population shift Hammond et al Bone

2014 60 26-32

Type 2 Diabetes Female ZDSD rats 32 weeks Tail Tendon Significant population shift

Diaz Gonzalez et al J Biomech 2014 47(3)

681-686

Raloxifene (in vivo and in vitro) Female dog treated with Raloxifene Femur Significant population shift Gallant et al Bone

2014 61 191-200 BBML - All R

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Issues to Consider Site Selection and Sampling

KS Test plt0001

0023 of bonersquos length

15 m

m =

150

00 micro

m

Wallace et al Langmuir 2011 26(10) 7349-7354

100 microm x 100 microm

35 microm x 35 microm

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Issues to Consider Site Selection and Sampling

60

62

64

66

68

70

72

74

1 (35)

2 (33)

3 (40)

4 (39)

5 (35)

6 (29)

7 (26)

8 (23)

9 (28)

WT (288)

D-P

erio

dic

Gap

Ove

rlap

Spac

ing

(nm

)

Axial Location (Proximal End = 1 Distal End = 9)

( of Fibrils)

91

23

45

87

6

Wallace et al Langmuir 2011 26(10) 7349-7354

288 fibrils for population n=1 for mean comparisons BBML - All R

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Issues to Consider Effects of Hydration

Wet Tendon Dry Tendon Kemp et al J StructBio 2012 180 (3) 428-438

Collagen and bone exist in a hydrated environment in vivo bull Hydration state can impact morphology and mechanics

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Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

Nonsignificant mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet (n=255) 682 plusmn 12 nm

WT Dry (n=257) 686 plusmn 08 nm

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

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Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

Brtl DryBrtl Wet

KS Test plt0001

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

WT Case 04 nm mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet 682 plusmn 12 nm

WT Dry 686 plusmn 08 nm

Brtl+ Wet 675 plusmn 14 nm

Brtl+ Dry 689 plusmn 10 nm

Brtl+ Case 14 nm increase with drying (plt0001) bull Entire population shifted upward (plt0001)

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT WetBrtl Wet

KS Test plt0001

Brtl+ distribution shifted downward vs WT when wet

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryBrtl Dry

KS Test plt0001

Brtl+ distribution shifted upward vs WT when dry

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider AFM calibration

bull Absolute distance measurements require accurate piezo calibration

bull AFM systems have large scan ranges (150 microm to lt 1 microm)

bull Scanner non-linearities can introduce substantial error over that range

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

ights Reserve

d

Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

BBML - All R

ights Reserve

d

Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

100 nm 10 80 602 1198 100 nm 7 60 827 1439 100 nm 5 40 814 1427 100 nm 35 20 917 1724 100 nm 2 10 937 1774

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

ights Reserve

d

AFM calibration ndash Take Away Message

Calibration using a scan size and feature size comparable to features of interest is highly recommended

Calibration addresses absolute accuracy not precision (differential sensitivity between measurements)

bull Does not limit ability to differentiate between groups using the same AFM with the same calibration parameters

BBML - All R

ights Reserve

d

AFM Applications in Bone Research Indentation Mechanics

BBML - All R

ights Reserve

d

AFM-Based Indentation

In addition to high resolution imaging AFM can be used to extract nanoscale mechanical data

ApproachWithdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Can

tilev

er D

efle

ctio

n (V

olts

)

1 Ramp into a rigid sample ndash measure the slope

Deflection sensitivity x 119899119899119899119899119881119881

2 Calculate cantilever spring constant

3 Hookersquos law takes deflection (volts) and converts to units of force F = kx

Spring constant k 119873119873119899119899

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Forc

e (n

N)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration Fo

rce

(nN

)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

Tip-Sample Separation (nm)

BBML - All R

ights Reserve

d

AFM-Based Indentation Parameters

Approach Withdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

Peak Force

Area Between Curves Energy Dissipation

Contact Mechanics Fit for Elastic Modulus (E)

Adhesion Force

Indentation Depth (δ)

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

z

r

δ

R

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119877119877 ∙ 12057512057532

Es Sample modulus νs Poissonrsquos ratio R tip radius of curvature δ load point displacement

Relevant Hertz Assumptions bull Strains are small and within the elastic limit bull Indenter is infinitely stiff (only sample deforms) bull Indentation depth ltlt radius of curvature of indenter bull Surfaces are continuous non-conforming (must be flat) bull Load is normal to surface

BBML - All R

ights Reserve

d

Proper Indentation Model is Essential

δ

r

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119903119903 ∙ 12057512057532

δ

α

119865119865 =2120587120587∙

1198641198641199041199041 minus 1205841205841199041199042

∙ tan120572120572 ∙ 1205751205752

Es Sample modulus νs Poissonrsquos ratio R tip radius α opening angle

Hertz Model Spherical Indenter

depth ltlt radius of curvature

Sneddon Model Conical Indenter

depth ge radius of curvature

BBML - All R

ights Reserve

d

Indentation in OI Tendon Study

x

z

yBrtl+ and WT mice 6 month old males

4 tails per group

2-3 fascicles per tail

25-30 fibrils per tail

4-5 locations per fibril

Indent force 20 nN wet (Sneddon) 50 nN dry (Hertz) BBML - A

ll Rights R

eserved

OI Tendon Differential Effects of Dehydration

Dry 1268 plusmn 718 MPa

Wet 305 plusmn 243 MPa

Dry 1737 plusmn 973 MPa

Wet 301 plusmn 202 MPa

Kemp et al J StructBio 2012 180 (3) 428-438

bull Significant stiffening of fibrils with dehydration

bull More pronounced modulus modulus as a function of disease

BBML - All R

ights Reserve

d

OI Tendon Differential Effects of Dehydration

Kemp et al J StructBio 2012 180 (3) 428-438

Diseased phenotype not present when wet phenotype observed when dried

0

10

20

30

40

50

60

0 20 40 60 80 100 120 140 160 180 200

Gro

up S

ampl

es (

)

Indent Modulus (MPa)

WT WetBrtl Wet

0

2

4

6

8

10

12

14

16

18

20

025

050

075

010

0012

5015

0017

5020

0022

5025

0027

5030

0032

5035

0037

5040

0042

5045

0047

5050

00

Gro

up S

ampl

es (

)Indent Modulus (MPa)

WT DryBrtl Dry

555 WT Indents 305 plusmn 243 MPa

614 Brtl+ Indents 301 plusmn 202 MPa

KS Test p=0129 t Test p=0464 464 WT Indents

1268 plusmn 718 MPa 479 Brtl+ Indents 1737 plusmn 973 MPa

KS Test plt0001 T Test plt0001

BBML - All R

ights Reserve

d

Indentation in Diabetic Tendon (Poster 0495)

x

z

y

Control and Diabetic Rat Tail Tendon

4-5 rats per group

2-3 fascicles per rat

~70 fibrilstail

4-5 locationsfibril

20 nN (Sneddon)

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ights Reserve

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Multiscale Diabetes Study (Poster 0495)

0

5

10

15

20

25

300 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 gt25

Perc

enta

ge o

f Gro

up S

ampl

es

Elastic Modulus (MPa)

CD (1530 indents)ZDSD (913 indents)

CD (n=5) 381 plusmn 149 MPa

ZDSD (n=3) 706 plusmn 263 MPa

0

100

200

300

400

500

600

700

800

900

0 2 4 6 8 10

Mic

rosc

ale

Mod

ulus

(MPa

)

Nanoscale Modulus (MPa)

plt0001

r2=0531 p=0040

bull Increased indentation modulus in diabetic tendon

bull Strong relationship between nanoscale indent modulus and microscale tensile modulus

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Indentation Limitations and Weaknesses

Proper probe selection for intended application bull Cantilever must be stiff relative to sample bull Sharp probes may damage sample easily dull

R or α must be determined for each probe bull Properties provided by manufacturer are unreliable bull Vary from probe to probe can dull during indent process

Spring constant must be determined for each probe

bull Value provided by manufacturer is inaccurate bull Thermal tuning OK for ldquosoftrdquo probes poor performance

for stiffer probes bull Sader method (geometry) added mass reference

cantilevers

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ights Reserve

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Indentation Limitations and Weaknesses

Veeco Application Technical note Ben Ohler Practical Advice on the Determination of Cantilever Spring Constants

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Indentation Limitations and Weaknesses

bull Sample processing

bull Sample hydration

bull Proper force range

bull Substrate effects for thin samples

bull Proper modulus model and range of data goodness of fit

Bearing in mind these assumptions and limitations AFM can provide true ldquonanoscalerdquo mechanical properties both in terms of forces and deformations

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ights Reserve

d

A Note on Peak Force QNM for Bruker AFMs bull Mechanical properties mapped pixel-by-pixel at same

resolution as the height image (PeakForce QNM)

Height Modulus BBML - All R

ights Reserve

d

A Note on Peak Force QNM

Correct probe stiffness must be used based on sample Pittenger ndash Veeco Product Bulletin

BBML - All R

ights Reserve

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A Note on Peak Force QNM Extremely sensitive to calibration (details rarely given) Absolute radius and spring constant known

bull especially important from probe to probe bull radius uarr with indent depth ndash must determine R at depth

you expect to image bull Depth may vary as a function of sample (or at different

regions within the sample)

Relative reference material of ldquoknownrdquo modulus used to determine a ratio of 119896119896 119877119877 bull ldquoknownrdquo modulus may come from tension compression

and not indentation bull R is related to depth indentation depth on sample must

be same as on reference BBML - All R

ights Reserve

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A Note on Peak Force QNM bull Modulus fit on the fly ndash no ability to monitor fit (or range) bull Local sample topography and sample tilt play a role in

measured parameters bull Assuming all modulus values in an image are correct how to

quantify

512 x 512 pixels = 262144 measurements

n=1 n=262144

n from selected ROIs

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bull Imaging in air on mica ndash substrate effect

bull Same imaging force or indent depth for all

bull For each area were all modulus values pooled

Lamprou et al PLoS ONE 2013 BBML - All R

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d

AFM Applications in Bone Research Other Cool Mechanical Techniques

BBML - All R

ights Reserve

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ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo Bonds within or between collagen fibrils in bone that rupture to dissipate energy as a toughening mechanism

Thompson et al Nature 2001

Fantner et al Nature Mat 2005

Hansma et al JMNI 2005

Fantner et al Nano Letters 2007 BBML - All R

ights Reserve

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Other Mechanical Techniques

Jimenez-Palomar J Mech Beh Bio Mat 2012

van der Rijt Macro Bio 2004

Yang J Biomed Mater Res 2007

Yang Biophysical Journal 2008 BBML - All R

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Other Cool Techniques bull Piezoresponse Force Microscopy (PFM)

bull Electrochemical AFM (EC-AFM)

bull Force Modulation Microscopy (FMM)

bull Lateral Force Microscopy (LFM)

bull Magnetic Force Microscopy (MFM)

bull Conductive AFM (C-AFM)

bull Force Pulling

bull Cantilever Bending

bull Tensile testing of individual fibrils

bull 3 point bending of fibrils

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Summary Things to rememberhellip

bull Imaging mode imaging medium and probe selection

bull Morphology Measurements bull Sample preparation and hydration state bull Measuring fibril diameter bull Site selection and proper sampling bull Appropriate system calibration

bull Mechanical Measurements

bull Probe selection (stiffness and tip radius) bull Probe calibration (radius angle spring constant) bull Indentation force range and mechanical model bull Hydration in biological samples bull PeakForce QNM

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Acknowledgements BBML

Max Hammond Armando Diaz Gonzalez Zachary Bart Silvia Canelon Creasy Clauser

IU School of Medicine Dr David Burr Dr Matt Allen Dr Stuart Warden Dr Lilian Plotkin

Funding Sources NIHNIDCR 1F32DE018840-01 A1 IUPUI BME Departmental Startup Funds IUPUI Research Support Funds Grant IUPUI Biomechanics and Biomaterials Research Center

NIH NICHD Dr Joan Marini

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Bone Biology and Mechanics Lab (BBML) wwwiupuiedu~bbml Facebook BoneBiologyMechanicsLab

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• Slide Number 1
• General Seminar Outline
• Bonersquos Hierarchical Structure
• Critical Gap in Understanding Collagen
• Methods For Analyzing Collagen Structure
• Methods For Imaging Collagen Structure
• Cryo-TEM
• Atomic Force Microscopy (AFM)
• AFM Image of Pentacene (C22H14)
• AFM Image of Hexabenzocoronene
• Basics of AFM Imaging The Probe
• Basics of AFM Imaging System Components
• van der Waals Potential Energy Curve
• Contact Mode ndash Monitor Deflection
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Peak Force Tapping Mode ndash Monitor Force
• AFM Applications in Bone Research Imaging
• AFM Imaging of Collagen
• Imaging of Osteoclast Activity
• Mineralized collagen deposition in vitro
• Imaging of Canilicular Structure
• AFM Imaging in Bone
• AFM Imaging in Bone
• Removing Mineral in Bone to Expose Collagen
• Removing Mineral in Bone to Expose Collagen
• Demineralization with EDTA
• Demineralization with EDTA
• Demineralization with EDTA
• Issues to Consider Effects of Acid
• Slide Number 32
• Slide Number 33
• Analysis of Collagen Structure in Bone
• Analysis of Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril D-Spacing
• Slide Number 39
• Collagen in Bone Fibril D-Spacing
• Collagen in Bone Fibril D-Spacing
• 2D Fast Fourier Transform Analysis
• 2D Fast Fourier Transform Analysis
• D-Spacing Distribution
• D-Spacing Distribution
• Cumulative Distribution Function
• CDF vs Histogram
• Changes in D-Spacing with Disease
• Changes in D-Spacing with Disease
• Slide Number 50
• Issues to Consider Site Selection and Sampling
• Issues to Consider Site Selection and Sampling
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• AFM calibration ndash Take Away Message
• AFM Applications in Bone ResearchIndentation Mechanics
• AFM-Based Indentation
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Parameters
• Hertz Contact Equation
• Hertz Contact Equation
• Hertz Contact Equation
• Proper Indentation Model is Essential
• Indentation in OI Tendon Study
• OI Tendon Differential Effects of Dehydration
• OI Tendon Differential Effects of Dehydration
• Indentation in Diabetic Tendon (Poster 0495)
• Multiscale Diabetes Study (Poster 0495)
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• A Note on Peak Force QNM for Bruker AFMs
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• Slide Number 83
• Slide Number 84
• AFM Applications in Bone ResearchOther Cool Mechanical Techniques
• ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo
• Other Mechanical Techniques
• Other Cool Techniques
• Summary Things to rememberhellip
• Acknowledgements
• Bone Biology and Mechanics Lab (BBML)

Issues to Consider Effects of Acid

Immature collagen crosslinks are very sensitive to acid bull Using acids to demineralize bone may inadvertently

destroy crosslinks distort structure

Fixation of tissue may be necessary prior to demineralization bull Fixation (glutaraldehyde or paraformadehyde) may

preserve structural integrity

bull Fixation effects on collagen structure are not fully known

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100 microm x 100 microm

50 microm x 50 microm

25 microm x 25 microm

10 microm x 10 microm

35 microm x 35 microm

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ights Reserve

d

35 microm x 35 microm

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ights Reserve

d

Analysis of Collagen Structure in Bone Spatial organization of newly formed bone following a surgically-induced injury (rat tibial hole)

Baranauskas J Vac Sci Tech 2001 Baranauskas App Surface Sci 2005

ldquohellipstatistical diameter of the fibers is 159 nm in good agreement with the literature values for type I collagen fibersrdquo

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Analysis of Collagen in Bone Fibril Diameter Fibril Diameter easiest to measure property first attempts were a bit crude better with increased resolution

Sasaki J Mat Sci Tech 2002 Thalhammer J Arch Sci 2001

Bozec Ultramicroscopy 2005

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Collagen in Bone Fibril Diameter

Caution must be taken when reporting and interpreting diameters measured from fibrils in bone bull Fibril packing - width may not be fully exposed

Wallace et al Langmuir 2010

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Collagen in Bone Fibril Diameter

Caution must be taken when reporting and interpreting diameters measured from fibrils in bone bull Fibril packing - width may not be fully exposed

Rigozzi ndash J Struct Bio - 2011

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Collagen in Bone Fibril D-Spacing

300 nm 15 nm

N C C N C C N C C N C C

H O H2C CH2 O H

CH2

O OH HHC CH2

CH2

HO

H H

HHN C C

O

H2C CH2

CH2

HN C C

OH

HC CH2

HO

N C C N C C N C C

H O H2C CH2 O

CH2

OH HC CH2

HO

H H

H

CH2 CH2

glycine X - proline Y - hydroxyproline

Hodge and Petruska 1963

Collagen Fibril Axial D-Periodicity BBML - All R

ights Reserve

d

05

101520

0 02 04 06 08 1

Hei

ght (

nm)

Length (microns)

httpremfdartmoutheduimagesindexhtml

Randall Nature and Structure of Collagen 1953

TEM SEM

AFM

D-Spacing = 67 nm

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Collagen in Bone Fibril D-Spacing Many studies have shown observable D-spacing content that near theoretical value

Sasaki 2002 67 plusmn 2 nm Bozek 2005 665 plusmn 14 nm Ge 2007 666 plusmn 38 nm

Habelitz J Structural Bio 2002

A 2002 study in dentin got me interestedhellip

n = 322 wet n = 363 dry

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Collagen in Bone Fibril D-Spacing

bull Internal structure bull Wallace et al Bone 2010 46 1349-1354 bull Wallace et al J Stuct Bio 2011 173 146-152 bull Kemp et al J Stuct Bio 2012 180 428-438 bull Warden et al Endocrinology 2013 154(9)

3178-3187 bull Gallant et al Bone 2014 61 191-200 bull Bart et al Conn Tiss Res 2014 In Press

bull Enzymatic and non-

enzymatic cross-linking bull Hammond et al Bone 2014 60 26-32 bull Diaz Gonzalez et al J Biomech 2014 47(3)

681-686 bull Voytik-Harbin et al In Preparation 2014

D-periodic spacing important structural feature that captures aspects related to

Mouse Femur Mid-diaphysis BBML - All R

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2D Fast Fourier Transform Analysis

Harmonics

Fibril D-Spacing

Erickson B Wallace JM et al Biotechnology Journal 2013

Raw AFM Image 2D FFT Power Spectrum

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2D Fast Fourier Transform Analysis

Harmonics

Fibril D-Spacing

Raw AFM Image 2D FFT Power Spectrum

0

20000

40000

60000

80000

100000

120000

0 001 002 003 004 005 006

Am

plitu

de (

microV2 )

Position (1nm)

D-periodic spacing 0015 (1nm) ~ 67 nm

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d

D-Spacing Distribution

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Overall Mean 670 plusmn 00 nm

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ights Reserve

d

D-Spacing Distribution

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Overall Mean 670 plusmn 00 nm

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

4 Samples Total of n = 255 fibrils Overall Mean 682 plusmn 12 nm

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Cumulative Distribution Function

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

This population 255 fibrils - Each fibril 1255 = 0392 - Data range 628-716 nm

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CDF vs Histogram 62 63 64 65 66 67 68 69 70 71 72 73 74

0

10

20

30

40

50

60

70

80

90

100

0

5

10

15

20

25

30

35

40

45

50

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

HistoCDF

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d

Changes in D-Spacing with Disease

0

5

10

15

20

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

0

5

10

15

20

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

Wallace et al Bone 2010 46 1349-1354

KS Test plt0001

Control 680 plusmn 26 nm

Estrogen Dep 659 plusmn 31 nm

t-test on mean p=0048

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Changes in D-Spacing with Disease

Wallace et al Bone 2010 46 1349-1354

0

10

20

30

40

50

60

70

80

90

100

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

KS Test plt0001

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Question Model and Tissue Morphology Publication

Tissue Specificity in Mice Male mouse 8 weeks Femur dentin tail tendon

No differences in bone vs dentin both ∆ vs tendon

Wallace et al Langmuir 2011 26(10) 7349-7354

Gender Effects Male and female mice 16 weeks Femur No differences NA

Estrogen Depletion OVX in sheep 5 years + 2 Radius Mean and population shift Wallace et al Bone

2010 46 1349-1354

Estrogen Depletion OVX in female 5 years + 2 Skin (dermis) Significant population shift Erickson et al Biotech

J 2013 8(1) 1174-126

Osteoporosis and Mechanical Loading

OVX in ulnar loading in rats Ulna

No loading effect population shift with OVX

Warden et al Endocrinology 2013 154(9) 3178-3187

Osteogenesis Imperfecta Brtl+ model male mice 2 months Femur

No mean ∆ widening of distribution

Wallace et al J Stuct Bio 2011 173 146-152

Osteogenesis Imperfecta Oimoim model male mice 12 weeks Femur Mean and population shift Bart et al Conn Tiss

Res 2014 In Press

Osteogenesis Imperfecta and Tissue Hydration

Brtl+ model male mice 6 months Tail Tendon Significant population shift Kemp et al J Stuct Bio

2012 180 428-438

Type 2 Diabetes Male ZDSD rats 30 weeks Tibia Significant population shift Hammond et al Bone

2014 60 26-32

Type 2 Diabetes Female ZDSD rats 32 weeks Tail Tendon Significant population shift

Diaz Gonzalez et al J Biomech 2014 47(3)

681-686

Raloxifene (in vivo and in vitro) Female dog treated with Raloxifene Femur Significant population shift Gallant et al Bone

2014 61 191-200 BBML - All R

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Issues to Consider Site Selection and Sampling

KS Test plt0001

0023 of bonersquos length

15 m

m =

150

00 micro

m

Wallace et al Langmuir 2011 26(10) 7349-7354

100 microm x 100 microm

35 microm x 35 microm

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Issues to Consider Site Selection and Sampling

60

62

64

66

68

70

72

74

1 (35)

2 (33)

3 (40)

4 (39)

5 (35)

6 (29)

7 (26)

8 (23)

9 (28)

WT (288)

D-P

erio

dic

Gap

Ove

rlap

Spac

ing

(nm

)

Axial Location (Proximal End = 1 Distal End = 9)

( of Fibrils)

91

23

45

87

6

Wallace et al Langmuir 2011 26(10) 7349-7354

288 fibrils for population n=1 for mean comparisons BBML - All R

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Issues to Consider Effects of Hydration

Wet Tendon Dry Tendon Kemp et al J StructBio 2012 180 (3) 428-438

Collagen and bone exist in a hydrated environment in vivo bull Hydration state can impact morphology and mechanics

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Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

Nonsignificant mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet (n=255) 682 plusmn 12 nm

WT Dry (n=257) 686 plusmn 08 nm

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

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Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

Brtl DryBrtl Wet

KS Test plt0001

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

WT Case 04 nm mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet 682 plusmn 12 nm

WT Dry 686 plusmn 08 nm

Brtl+ Wet 675 plusmn 14 nm

Brtl+ Dry 689 plusmn 10 nm

Brtl+ Case 14 nm increase with drying (plt0001) bull Entire population shifted upward (plt0001)

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT WetBrtl Wet

KS Test plt0001

Brtl+ distribution shifted downward vs WT when wet

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryBrtl Dry

KS Test plt0001

Brtl+ distribution shifted upward vs WT when dry

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider AFM calibration

bull Absolute distance measurements require accurate piezo calibration

bull AFM systems have large scan ranges (150 microm to lt 1 microm)

bull Scanner non-linearities can introduce substantial error over that range

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

ights Reserve

d

Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

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Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

100 nm 10 80 602 1198 100 nm 7 60 827 1439 100 nm 5 40 814 1427 100 nm 35 20 917 1724 100 nm 2 10 937 1774

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

ights Reserve

d

AFM calibration ndash Take Away Message

Calibration using a scan size and feature size comparable to features of interest is highly recommended

Calibration addresses absolute accuracy not precision (differential sensitivity between measurements)

bull Does not limit ability to differentiate between groups using the same AFM with the same calibration parameters

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ights Reserve

d

AFM Applications in Bone Research Indentation Mechanics

BBML - All R

ights Reserve

d

AFM-Based Indentation

In addition to high resolution imaging AFM can be used to extract nanoscale mechanical data

ApproachWithdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Can

tilev

er D

efle

ctio

n (V

olts

)

1 Ramp into a rigid sample ndash measure the slope

Deflection sensitivity x 119899119899119899119899119881119881

2 Calculate cantilever spring constant

3 Hookersquos law takes deflection (volts) and converts to units of force F = kx

Spring constant k 119873119873119899119899

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Forc

e (n

N)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration Fo

rce

(nN

)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

Tip-Sample Separation (nm)

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d

AFM-Based Indentation Parameters

Approach Withdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

Peak Force

Area Between Curves Energy Dissipation

Contact Mechanics Fit for Elastic Modulus (E)

Adhesion Force

Indentation Depth (δ)

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

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d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

z

r

δ

R

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119877119877 ∙ 12057512057532

Es Sample modulus νs Poissonrsquos ratio R tip radius of curvature δ load point displacement

Relevant Hertz Assumptions bull Strains are small and within the elastic limit bull Indenter is infinitely stiff (only sample deforms) bull Indentation depth ltlt radius of curvature of indenter bull Surfaces are continuous non-conforming (must be flat) bull Load is normal to surface

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d

Proper Indentation Model is Essential

δ

r

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119903119903 ∙ 12057512057532

δ

α

119865119865 =2120587120587∙

1198641198641199041199041 minus 1205841205841199041199042

∙ tan120572120572 ∙ 1205751205752

Es Sample modulus νs Poissonrsquos ratio R tip radius α opening angle

Hertz Model Spherical Indenter

depth ltlt radius of curvature

Sneddon Model Conical Indenter

depth ge radius of curvature

BBML - All R

ights Reserve

d

Indentation in OI Tendon Study

x

z

yBrtl+ and WT mice 6 month old males

4 tails per group

2-3 fascicles per tail

25-30 fibrils per tail

4-5 locations per fibril

Indent force 20 nN wet (Sneddon) 50 nN dry (Hertz) BBML - A

ll Rights R

eserved

OI Tendon Differential Effects of Dehydration

Dry 1268 plusmn 718 MPa

Wet 305 plusmn 243 MPa

Dry 1737 plusmn 973 MPa

Wet 301 plusmn 202 MPa

Kemp et al J StructBio 2012 180 (3) 428-438

bull Significant stiffening of fibrils with dehydration

bull More pronounced modulus modulus as a function of disease

BBML - All R

ights Reserve

d

OI Tendon Differential Effects of Dehydration

Kemp et al J StructBio 2012 180 (3) 428-438

Diseased phenotype not present when wet phenotype observed when dried

0

10

20

30

40

50

60

0 20 40 60 80 100 120 140 160 180 200

Gro

up S

ampl

es (

)

Indent Modulus (MPa)

WT WetBrtl Wet

0

2

4

6

8

10

12

14

16

18

20

025

050

075

010

0012

5015

0017

5020

0022

5025

0027

5030

0032

5035

0037

5040

0042

5045

0047

5050

00

Gro

up S

ampl

es (

)Indent Modulus (MPa)

WT DryBrtl Dry

555 WT Indents 305 plusmn 243 MPa

614 Brtl+ Indents 301 plusmn 202 MPa

KS Test p=0129 t Test p=0464 464 WT Indents

1268 plusmn 718 MPa 479 Brtl+ Indents 1737 plusmn 973 MPa

KS Test plt0001 T Test plt0001

BBML - All R

ights Reserve

d

Indentation in Diabetic Tendon (Poster 0495)

x

z

y

Control and Diabetic Rat Tail Tendon

4-5 rats per group

2-3 fascicles per rat

~70 fibrilstail

4-5 locationsfibril

20 nN (Sneddon)

BBML - All R

ights Reserve

d

Multiscale Diabetes Study (Poster 0495)

0

5

10

15

20

25

300 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 gt25

Perc

enta

ge o

f Gro

up S

ampl

es

Elastic Modulus (MPa)

CD (1530 indents)ZDSD (913 indents)

CD (n=5) 381 plusmn 149 MPa

ZDSD (n=3) 706 plusmn 263 MPa

0

100

200

300

400

500

600

700

800

900

0 2 4 6 8 10

Mic

rosc

ale

Mod

ulus

(MPa

)

Nanoscale Modulus (MPa)

plt0001

r2=0531 p=0040

bull Increased indentation modulus in diabetic tendon

bull Strong relationship between nanoscale indent modulus and microscale tensile modulus

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

Proper probe selection for intended application bull Cantilever must be stiff relative to sample bull Sharp probes may damage sample easily dull

R or α must be determined for each probe bull Properties provided by manufacturer are unreliable bull Vary from probe to probe can dull during indent process

Spring constant must be determined for each probe

bull Value provided by manufacturer is inaccurate bull Thermal tuning OK for ldquosoftrdquo probes poor performance

for stiffer probes bull Sader method (geometry) added mass reference

cantilevers

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

Veeco Application Technical note Ben Ohler Practical Advice on the Determination of Cantilever Spring Constants

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

bull Sample processing

bull Sample hydration

bull Proper force range

bull Substrate effects for thin samples

bull Proper modulus model and range of data goodness of fit

Bearing in mind these assumptions and limitations AFM can provide true ldquonanoscalerdquo mechanical properties both in terms of forces and deformations

BBML - All R

ights Reserve

d

A Note on Peak Force QNM for Bruker AFMs bull Mechanical properties mapped pixel-by-pixel at same

resolution as the height image (PeakForce QNM)

Height Modulus BBML - All R

ights Reserve

d

A Note on Peak Force QNM

Correct probe stiffness must be used based on sample Pittenger ndash Veeco Product Bulletin

BBML - All R

ights Reserve

d

A Note on Peak Force QNM Extremely sensitive to calibration (details rarely given) Absolute radius and spring constant known

bull especially important from probe to probe bull radius uarr with indent depth ndash must determine R at depth

you expect to image bull Depth may vary as a function of sample (or at different

regions within the sample)

Relative reference material of ldquoknownrdquo modulus used to determine a ratio of 119896119896 119877119877 bull ldquoknownrdquo modulus may come from tension compression

and not indentation bull R is related to depth indentation depth on sample must

be same as on reference BBML - All R

ights Reserve

d

A Note on Peak Force QNM bull Modulus fit on the fly ndash no ability to monitor fit (or range) bull Local sample topography and sample tilt play a role in

measured parameters bull Assuming all modulus values in an image are correct how to

quantify

512 x 512 pixels = 262144 measurements

n=1 n=262144

n from selected ROIs

BBML - All R

ights Reserve

d

BBML - All R

ights Reserve

d

bull Imaging in air on mica ndash substrate effect

bull Same imaging force or indent depth for all

bull For each area were all modulus values pooled

Lamprou et al PLoS ONE 2013 BBML - All R

ights Reserve

d

AFM Applications in Bone Research Other Cool Mechanical Techniques

BBML - All R

ights Reserve

d

ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo Bonds within or between collagen fibrils in bone that rupture to dissipate energy as a toughening mechanism

Thompson et al Nature 2001

Fantner et al Nature Mat 2005

Hansma et al JMNI 2005

Fantner et al Nano Letters 2007 BBML - All R

ights Reserve

d

Other Mechanical Techniques

Jimenez-Palomar J Mech Beh Bio Mat 2012

van der Rijt Macro Bio 2004

Yang J Biomed Mater Res 2007

Yang Biophysical Journal 2008 BBML - All R

ights Reserve

d

Other Cool Techniques bull Piezoresponse Force Microscopy (PFM)

bull Electrochemical AFM (EC-AFM)

bull Force Modulation Microscopy (FMM)

bull Lateral Force Microscopy (LFM)

bull Magnetic Force Microscopy (MFM)

bull Conductive AFM (C-AFM)

bull Force Pulling

bull Cantilever Bending

bull Tensile testing of individual fibrils

bull 3 point bending of fibrils

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ights Reserve

d

Summary Things to rememberhellip

bull Imaging mode imaging medium and probe selection

bull Morphology Measurements bull Sample preparation and hydration state bull Measuring fibril diameter bull Site selection and proper sampling bull Appropriate system calibration

bull Mechanical Measurements

bull Probe selection (stiffness and tip radius) bull Probe calibration (radius angle spring constant) bull Indentation force range and mechanical model bull Hydration in biological samples bull PeakForce QNM

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ights Reserve

d

Acknowledgements BBML

Max Hammond Armando Diaz Gonzalez Zachary Bart Silvia Canelon Creasy Clauser

IU School of Medicine Dr David Burr Dr Matt Allen Dr Stuart Warden Dr Lilian Plotkin

Funding Sources NIHNIDCR 1F32DE018840-01 A1 IUPUI BME Departmental Startup Funds IUPUI Research Support Funds Grant IUPUI Biomechanics and Biomaterials Research Center

NIH NICHD Dr Joan Marini

BBML - All R

ights Reserve

d

Bone Biology and Mechanics Lab (BBML) wwwiupuiedu~bbml Facebook BoneBiologyMechanicsLab

BBML - All R

ights Reserve

d

• Slide Number 1
• General Seminar Outline
• Bonersquos Hierarchical Structure
• Critical Gap in Understanding Collagen
• Methods For Analyzing Collagen Structure
• Methods For Imaging Collagen Structure
• Cryo-TEM
• Atomic Force Microscopy (AFM)
• AFM Image of Pentacene (C22H14)
• AFM Image of Hexabenzocoronene
• Basics of AFM Imaging The Probe
• Basics of AFM Imaging System Components
• van der Waals Potential Energy Curve
• Contact Mode ndash Monitor Deflection
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Peak Force Tapping Mode ndash Monitor Force
• AFM Applications in Bone Research Imaging
• AFM Imaging of Collagen
• Imaging of Osteoclast Activity
• Mineralized collagen deposition in vitro
• Imaging of Canilicular Structure
• AFM Imaging in Bone
• AFM Imaging in Bone
• Removing Mineral in Bone to Expose Collagen
• Removing Mineral in Bone to Expose Collagen
• Demineralization with EDTA
• Demineralization with EDTA
• Demineralization with EDTA
• Issues to Consider Effects of Acid
• Slide Number 32
• Slide Number 33
• Analysis of Collagen Structure in Bone
• Analysis of Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril D-Spacing
• Slide Number 39
• Collagen in Bone Fibril D-Spacing
• Collagen in Bone Fibril D-Spacing
• 2D Fast Fourier Transform Analysis
• 2D Fast Fourier Transform Analysis
• D-Spacing Distribution
• D-Spacing Distribution
• Cumulative Distribution Function
• CDF vs Histogram
• Changes in D-Spacing with Disease
• Changes in D-Spacing with Disease
• Slide Number 50
• Issues to Consider Site Selection and Sampling
• Issues to Consider Site Selection and Sampling
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• AFM calibration ndash Take Away Message
• AFM Applications in Bone ResearchIndentation Mechanics
• AFM-Based Indentation
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Parameters
• Hertz Contact Equation
• Hertz Contact Equation
• Hertz Contact Equation
• Proper Indentation Model is Essential
• Indentation in OI Tendon Study
• OI Tendon Differential Effects of Dehydration
• OI Tendon Differential Effects of Dehydration
• Indentation in Diabetic Tendon (Poster 0495)
• Multiscale Diabetes Study (Poster 0495)
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• A Note on Peak Force QNM for Bruker AFMs
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• Slide Number 83
• Slide Number 84
• AFM Applications in Bone ResearchOther Cool Mechanical Techniques
• ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo
• Other Mechanical Techniques
• Other Cool Techniques
• Summary Things to rememberhellip
• Acknowledgements
• Bone Biology and Mechanics Lab (BBML)

100 microm x 100 microm

50 microm x 50 microm

25 microm x 25 microm

10 microm x 10 microm

35 microm x 35 microm

BBML - All R

ights Reserve

d

35 microm x 35 microm

BBML - All R

ights Reserve

d

Analysis of Collagen Structure in Bone Spatial organization of newly formed bone following a surgically-induced injury (rat tibial hole)

Baranauskas J Vac Sci Tech 2001 Baranauskas App Surface Sci 2005

ldquohellipstatistical diameter of the fibers is 159 nm in good agreement with the literature values for type I collagen fibersrdquo

BBML - All R

ights Reserve

d

Analysis of Collagen in Bone Fibril Diameter Fibril Diameter easiest to measure property first attempts were a bit crude better with increased resolution

Sasaki J Mat Sci Tech 2002 Thalhammer J Arch Sci 2001

Bozec Ultramicroscopy 2005

BBML - All R

ights Reserve

d

Collagen in Bone Fibril Diameter

Caution must be taken when reporting and interpreting diameters measured from fibrils in bone bull Fibril packing - width may not be fully exposed

Wallace et al Langmuir 2010

BBML - All R

ights Reserve

d

Collagen in Bone Fibril Diameter

Caution must be taken when reporting and interpreting diameters measured from fibrils in bone bull Fibril packing - width may not be fully exposed

Rigozzi ndash J Struct Bio - 2011

BBML - All R

ights Reserve

d

Collagen in Bone Fibril D-Spacing

300 nm 15 nm

N C C N C C N C C N C C

H O H2C CH2 O H

CH2

O OH HHC CH2

CH2

HO

H H

HHN C C

O

H2C CH2

CH2

HN C C

OH

HC CH2

HO

N C C N C C N C C

H O H2C CH2 O

CH2

OH HC CH2

HO

H H

H

CH2 CH2

glycine X - proline Y - hydroxyproline

Hodge and Petruska 1963

Collagen Fibril Axial D-Periodicity BBML - All R

ights Reserve

d

05

101520

0 02 04 06 08 1

Hei

ght (

nm)

Length (microns)

httpremfdartmoutheduimagesindexhtml

Randall Nature and Structure of Collagen 1953

TEM SEM

AFM

D-Spacing = 67 nm

BBML - All R

ights Reserve

d

Collagen in Bone Fibril D-Spacing Many studies have shown observable D-spacing content that near theoretical value

Sasaki 2002 67 plusmn 2 nm Bozek 2005 665 plusmn 14 nm Ge 2007 666 plusmn 38 nm

Habelitz J Structural Bio 2002

A 2002 study in dentin got me interestedhellip

n = 322 wet n = 363 dry

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ights Reserve

d

Collagen in Bone Fibril D-Spacing

bull Internal structure bull Wallace et al Bone 2010 46 1349-1354 bull Wallace et al J Stuct Bio 2011 173 146-152 bull Kemp et al J Stuct Bio 2012 180 428-438 bull Warden et al Endocrinology 2013 154(9)

3178-3187 bull Gallant et al Bone 2014 61 191-200 bull Bart et al Conn Tiss Res 2014 In Press

bull Enzymatic and non-

enzymatic cross-linking bull Hammond et al Bone 2014 60 26-32 bull Diaz Gonzalez et al J Biomech 2014 47(3)

681-686 bull Voytik-Harbin et al In Preparation 2014

D-periodic spacing important structural feature that captures aspects related to

Mouse Femur Mid-diaphysis BBML - All R

ights Reserve

d

2D Fast Fourier Transform Analysis

Harmonics

Fibril D-Spacing

Erickson B Wallace JM et al Biotechnology Journal 2013

Raw AFM Image 2D FFT Power Spectrum

BBML - All R

ights Reserve

d

2D Fast Fourier Transform Analysis

Harmonics

Fibril D-Spacing

Raw AFM Image 2D FFT Power Spectrum

0

20000

40000

60000

80000

100000

120000

0 001 002 003 004 005 006

Am

plitu

de (

microV2 )

Position (1nm)

D-periodic spacing 0015 (1nm) ~ 67 nm

BBML - All R

ights Reserve

d

D-Spacing Distribution

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Overall Mean 670 plusmn 00 nm

BBML - All R

ights Reserve

d

D-Spacing Distribution

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Overall Mean 670 plusmn 00 nm

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

4 Samples Total of n = 255 fibrils Overall Mean 682 plusmn 12 nm

BBML - All R

ights Reserve

d

Cumulative Distribution Function

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

This population 255 fibrils - Each fibril 1255 = 0392 - Data range 628-716 nm

BBML - All R

ights Reserve

d

CDF vs Histogram 62 63 64 65 66 67 68 69 70 71 72 73 74

0

10

20

30

40

50

60

70

80

90

100

0

5

10

15

20

25

30

35

40

45

50

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

HistoCDF

BBML - All R

ights Reserve

d

Changes in D-Spacing with Disease

0

5

10

15

20

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

0

5

10

15

20

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

Wallace et al Bone 2010 46 1349-1354

KS Test plt0001

Control 680 plusmn 26 nm

Estrogen Dep 659 plusmn 31 nm

t-test on mean p=0048

BBML - All R

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Changes in D-Spacing with Disease

Wallace et al Bone 2010 46 1349-1354

0

10

20

30

40

50

60

70

80

90

100

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

KS Test plt0001

BBML - All R

ights Reserve

d

Question Model and Tissue Morphology Publication

Tissue Specificity in Mice Male mouse 8 weeks Femur dentin tail tendon

No differences in bone vs dentin both ∆ vs tendon

Wallace et al Langmuir 2011 26(10) 7349-7354

Gender Effects Male and female mice 16 weeks Femur No differences NA

Estrogen Depletion OVX in sheep 5 years + 2 Radius Mean and population shift Wallace et al Bone

2010 46 1349-1354

Estrogen Depletion OVX in female 5 years + 2 Skin (dermis) Significant population shift Erickson et al Biotech

J 2013 8(1) 1174-126

Osteoporosis and Mechanical Loading

OVX in ulnar loading in rats Ulna

No loading effect population shift with OVX

Warden et al Endocrinology 2013 154(9) 3178-3187

Osteogenesis Imperfecta Brtl+ model male mice 2 months Femur

No mean ∆ widening of distribution

Wallace et al J Stuct Bio 2011 173 146-152

Osteogenesis Imperfecta Oimoim model male mice 12 weeks Femur Mean and population shift Bart et al Conn Tiss

Res 2014 In Press

Osteogenesis Imperfecta and Tissue Hydration

Brtl+ model male mice 6 months Tail Tendon Significant population shift Kemp et al J Stuct Bio

2012 180 428-438

Type 2 Diabetes Male ZDSD rats 30 weeks Tibia Significant population shift Hammond et al Bone

2014 60 26-32

Type 2 Diabetes Female ZDSD rats 32 weeks Tail Tendon Significant population shift

Diaz Gonzalez et al J Biomech 2014 47(3)

681-686

Raloxifene (in vivo and in vitro) Female dog treated with Raloxifene Femur Significant population shift Gallant et al Bone

2014 61 191-200 BBML - All R

ights Reserve

d

Issues to Consider Site Selection and Sampling

KS Test plt0001

0023 of bonersquos length

15 m

m =

150

00 micro

m

Wallace et al Langmuir 2011 26(10) 7349-7354

100 microm x 100 microm

35 microm x 35 microm

BBML - All R

ights Reserve

d

Issues to Consider Site Selection and Sampling

60

62

64

66

68

70

72

74

1 (35)

2 (33)

3 (40)

4 (39)

5 (35)

6 (29)

7 (26)

8 (23)

9 (28)

WT (288)

D-P

erio

dic

Gap

Ove

rlap

Spac

ing

(nm

)

Axial Location (Proximal End = 1 Distal End = 9)

( of Fibrils)

91

23

45

87

6

Wallace et al Langmuir 2011 26(10) 7349-7354

288 fibrils for population n=1 for mean comparisons BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

Wet Tendon Dry Tendon Kemp et al J StructBio 2012 180 (3) 428-438

Collagen and bone exist in a hydrated environment in vivo bull Hydration state can impact morphology and mechanics

BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

Nonsignificant mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet (n=255) 682 plusmn 12 nm

WT Dry (n=257) 686 plusmn 08 nm

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

Brtl DryBrtl Wet

KS Test plt0001

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

WT Case 04 nm mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet 682 plusmn 12 nm

WT Dry 686 plusmn 08 nm

Brtl+ Wet 675 plusmn 14 nm

Brtl+ Dry 689 plusmn 10 nm

Brtl+ Case 14 nm increase with drying (plt0001) bull Entire population shifted upward (plt0001)

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT WetBrtl Wet

KS Test plt0001

Brtl+ distribution shifted downward vs WT when wet

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryBrtl Dry

KS Test plt0001

Brtl+ distribution shifted upward vs WT when dry

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider AFM calibration

bull Absolute distance measurements require accurate piezo calibration

bull AFM systems have large scan ranges (150 microm to lt 1 microm)

bull Scanner non-linearities can introduce substantial error over that range

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

ights Reserve

d

Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

BBML - All R

ights Reserve

d

Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

100 nm 10 80 602 1198 100 nm 7 60 827 1439 100 nm 5 40 814 1427 100 nm 35 20 917 1724 100 nm 2 10 937 1774

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

ights Reserve

d

AFM calibration ndash Take Away Message

Calibration using a scan size and feature size comparable to features of interest is highly recommended

Calibration addresses absolute accuracy not precision (differential sensitivity between measurements)

bull Does not limit ability to differentiate between groups using the same AFM with the same calibration parameters

BBML - All R

ights Reserve

d

AFM Applications in Bone Research Indentation Mechanics

BBML - All R

ights Reserve

d

AFM-Based Indentation

In addition to high resolution imaging AFM can be used to extract nanoscale mechanical data

ApproachWithdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Can

tilev

er D

efle

ctio

n (V

olts

)

1 Ramp into a rigid sample ndash measure the slope

Deflection sensitivity x 119899119899119899119899119881119881

2 Calculate cantilever spring constant

3 Hookersquos law takes deflection (volts) and converts to units of force F = kx

Spring constant k 119873119873119899119899

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Forc

e (n

N)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration Fo

rce

(nN

)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

Tip-Sample Separation (nm)

BBML - All R

ights Reserve

d

AFM-Based Indentation Parameters

Approach Withdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

Peak Force

Area Between Curves Energy Dissipation

Contact Mechanics Fit for Elastic Modulus (E)

Adhesion Force

Indentation Depth (δ)

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

z

r

δ

R

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119877119877 ∙ 12057512057532

Es Sample modulus νs Poissonrsquos ratio R tip radius of curvature δ load point displacement

Relevant Hertz Assumptions bull Strains are small and within the elastic limit bull Indenter is infinitely stiff (only sample deforms) bull Indentation depth ltlt radius of curvature of indenter bull Surfaces are continuous non-conforming (must be flat) bull Load is normal to surface

BBML - All R

ights Reserve

d

Proper Indentation Model is Essential

δ

r

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119903119903 ∙ 12057512057532

δ

α

119865119865 =2120587120587∙

1198641198641199041199041 minus 1205841205841199041199042

∙ tan120572120572 ∙ 1205751205752

Es Sample modulus νs Poissonrsquos ratio R tip radius α opening angle

Hertz Model Spherical Indenter

depth ltlt radius of curvature

Sneddon Model Conical Indenter

depth ge radius of curvature

BBML - All R

ights Reserve

d

Indentation in OI Tendon Study

x

z

yBrtl+ and WT mice 6 month old males

4 tails per group

2-3 fascicles per tail

25-30 fibrils per tail

4-5 locations per fibril

Indent force 20 nN wet (Sneddon) 50 nN dry (Hertz) BBML - A

ll Rights R

eserved

OI Tendon Differential Effects of Dehydration

Dry 1268 plusmn 718 MPa

Wet 305 plusmn 243 MPa

Dry 1737 plusmn 973 MPa

Wet 301 plusmn 202 MPa

Kemp et al J StructBio 2012 180 (3) 428-438

bull Significant stiffening of fibrils with dehydration

bull More pronounced modulus modulus as a function of disease

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ights Reserve

d

OI Tendon Differential Effects of Dehydration

Kemp et al J StructBio 2012 180 (3) 428-438

Diseased phenotype not present when wet phenotype observed when dried

0

10

20

30

40

50

60

0 20 40 60 80 100 120 140 160 180 200

Gro

up S

ampl

es (

)

Indent Modulus (MPa)

WT WetBrtl Wet

0

2

4

6

8

10

12

14

16

18

20

025

050

075

010

0012

5015

0017

5020

0022

5025

0027

5030

0032

5035

0037

5040

0042

5045

0047

5050

00

Gro

up S

ampl

es (

)Indent Modulus (MPa)

WT DryBrtl Dry

555 WT Indents 305 plusmn 243 MPa

614 Brtl+ Indents 301 plusmn 202 MPa

KS Test p=0129 t Test p=0464 464 WT Indents

1268 plusmn 718 MPa 479 Brtl+ Indents 1737 plusmn 973 MPa

KS Test plt0001 T Test plt0001

BBML - All R

ights Reserve

d

Indentation in Diabetic Tendon (Poster 0495)

x

z

y

Control and Diabetic Rat Tail Tendon

4-5 rats per group

2-3 fascicles per rat

~70 fibrilstail

4-5 locationsfibril

20 nN (Sneddon)

BBML - All R

ights Reserve

d

Multiscale Diabetes Study (Poster 0495)

0

5

10

15

20

25

300 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 gt25

Perc

enta

ge o

f Gro

up S

ampl

es

Elastic Modulus (MPa)

CD (1530 indents)ZDSD (913 indents)

CD (n=5) 381 plusmn 149 MPa

ZDSD (n=3) 706 plusmn 263 MPa

0

100

200

300

400

500

600

700

800

900

0 2 4 6 8 10

Mic

rosc

ale

Mod

ulus

(MPa

)

Nanoscale Modulus (MPa)

plt0001

r2=0531 p=0040

bull Increased indentation modulus in diabetic tendon

bull Strong relationship between nanoscale indent modulus and microscale tensile modulus

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

Proper probe selection for intended application bull Cantilever must be stiff relative to sample bull Sharp probes may damage sample easily dull

R or α must be determined for each probe bull Properties provided by manufacturer are unreliable bull Vary from probe to probe can dull during indent process

Spring constant must be determined for each probe

bull Value provided by manufacturer is inaccurate bull Thermal tuning OK for ldquosoftrdquo probes poor performance

for stiffer probes bull Sader method (geometry) added mass reference

cantilevers

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

Veeco Application Technical note Ben Ohler Practical Advice on the Determination of Cantilever Spring Constants

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

bull Sample processing

bull Sample hydration

bull Proper force range

bull Substrate effects for thin samples

bull Proper modulus model and range of data goodness of fit

Bearing in mind these assumptions and limitations AFM can provide true ldquonanoscalerdquo mechanical properties both in terms of forces and deformations

BBML - All R

ights Reserve

d

A Note on Peak Force QNM for Bruker AFMs bull Mechanical properties mapped pixel-by-pixel at same

resolution as the height image (PeakForce QNM)

Height Modulus BBML - All R

ights Reserve

d

A Note on Peak Force QNM

Correct probe stiffness must be used based on sample Pittenger ndash Veeco Product Bulletin

BBML - All R

ights Reserve

d

A Note on Peak Force QNM Extremely sensitive to calibration (details rarely given) Absolute radius and spring constant known

bull especially important from probe to probe bull radius uarr with indent depth ndash must determine R at depth

you expect to image bull Depth may vary as a function of sample (or at different

regions within the sample)

Relative reference material of ldquoknownrdquo modulus used to determine a ratio of 119896119896 119877119877 bull ldquoknownrdquo modulus may come from tension compression

and not indentation bull R is related to depth indentation depth on sample must

be same as on reference BBML - All R

ights Reserve

d

A Note on Peak Force QNM bull Modulus fit on the fly ndash no ability to monitor fit (or range) bull Local sample topography and sample tilt play a role in

measured parameters bull Assuming all modulus values in an image are correct how to

quantify

512 x 512 pixels = 262144 measurements

n=1 n=262144

n from selected ROIs

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ights Reserve

d

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ights Reserve

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bull Imaging in air on mica ndash substrate effect

bull Same imaging force or indent depth for all

bull For each area were all modulus values pooled

Lamprou et al PLoS ONE 2013 BBML - All R

ights Reserve

d

AFM Applications in Bone Research Other Cool Mechanical Techniques

BBML - All R

ights Reserve

d

ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo Bonds within or between collagen fibrils in bone that rupture to dissipate energy as a toughening mechanism

Thompson et al Nature 2001

Fantner et al Nature Mat 2005

Hansma et al JMNI 2005

Fantner et al Nano Letters 2007 BBML - All R

ights Reserve

d

Other Mechanical Techniques

Jimenez-Palomar J Mech Beh Bio Mat 2012

van der Rijt Macro Bio 2004

Yang J Biomed Mater Res 2007

Yang Biophysical Journal 2008 BBML - All R

ights Reserve

d

Other Cool Techniques bull Piezoresponse Force Microscopy (PFM)

bull Electrochemical AFM (EC-AFM)

bull Force Modulation Microscopy (FMM)

bull Lateral Force Microscopy (LFM)

bull Magnetic Force Microscopy (MFM)

bull Conductive AFM (C-AFM)

bull Force Pulling

bull Cantilever Bending

bull Tensile testing of individual fibrils

bull 3 point bending of fibrils

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ights Reserve

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Summary Things to rememberhellip

bull Imaging mode imaging medium and probe selection

bull Morphology Measurements bull Sample preparation and hydration state bull Measuring fibril diameter bull Site selection and proper sampling bull Appropriate system calibration

bull Mechanical Measurements

bull Probe selection (stiffness and tip radius) bull Probe calibration (radius angle spring constant) bull Indentation force range and mechanical model bull Hydration in biological samples bull PeakForce QNM

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d

Acknowledgements BBML

Max Hammond Armando Diaz Gonzalez Zachary Bart Silvia Canelon Creasy Clauser

IU School of Medicine Dr David Burr Dr Matt Allen Dr Stuart Warden Dr Lilian Plotkin

Funding Sources NIHNIDCR 1F32DE018840-01 A1 IUPUI BME Departmental Startup Funds IUPUI Research Support Funds Grant IUPUI Biomechanics and Biomaterials Research Center

NIH NICHD Dr Joan Marini

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ights Reserve

d

Bone Biology and Mechanics Lab (BBML) wwwiupuiedu~bbml Facebook BoneBiologyMechanicsLab

BBML - All R

ights Reserve

d

• Slide Number 1
• General Seminar Outline
• Bonersquos Hierarchical Structure
• Critical Gap in Understanding Collagen
• Methods For Analyzing Collagen Structure
• Methods For Imaging Collagen Structure
• Cryo-TEM
• Atomic Force Microscopy (AFM)
• AFM Image of Pentacene (C22H14)
• AFM Image of Hexabenzocoronene
• Basics of AFM Imaging The Probe
• Basics of AFM Imaging System Components
• van der Waals Potential Energy Curve
• Contact Mode ndash Monitor Deflection
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Peak Force Tapping Mode ndash Monitor Force
• AFM Applications in Bone Research Imaging
• AFM Imaging of Collagen
• Imaging of Osteoclast Activity
• Mineralized collagen deposition in vitro
• Imaging of Canilicular Structure
• AFM Imaging in Bone
• AFM Imaging in Bone
• Removing Mineral in Bone to Expose Collagen
• Removing Mineral in Bone to Expose Collagen
• Demineralization with EDTA
• Demineralization with EDTA
• Demineralization with EDTA
• Issues to Consider Effects of Acid
• Slide Number 32
• Slide Number 33
• Analysis of Collagen Structure in Bone
• Analysis of Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril D-Spacing
• Slide Number 39
• Collagen in Bone Fibril D-Spacing
• Collagen in Bone Fibril D-Spacing
• 2D Fast Fourier Transform Analysis
• 2D Fast Fourier Transform Analysis
• D-Spacing Distribution
• D-Spacing Distribution
• Cumulative Distribution Function
• CDF vs Histogram
• Changes in D-Spacing with Disease
• Changes in D-Spacing with Disease
• Slide Number 50
• Issues to Consider Site Selection and Sampling
• Issues to Consider Site Selection and Sampling
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• AFM calibration ndash Take Away Message
• AFM Applications in Bone ResearchIndentation Mechanics
• AFM-Based Indentation
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Parameters
• Hertz Contact Equation
• Hertz Contact Equation
• Hertz Contact Equation
• Proper Indentation Model is Essential
• Indentation in OI Tendon Study
• OI Tendon Differential Effects of Dehydration
• OI Tendon Differential Effects of Dehydration
• Indentation in Diabetic Tendon (Poster 0495)
• Multiscale Diabetes Study (Poster 0495)
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• A Note on Peak Force QNM for Bruker AFMs
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• Slide Number 83
• Slide Number 84
• AFM Applications in Bone ResearchOther Cool Mechanical Techniques
• ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo
• Other Mechanical Techniques
• Other Cool Techniques
• Summary Things to rememberhellip
• Acknowledgements
• Bone Biology and Mechanics Lab (BBML)

35 microm x 35 microm

BBML - All R

ights Reserve

d

Analysis of Collagen Structure in Bone Spatial organization of newly formed bone following a surgically-induced injury (rat tibial hole)

Baranauskas J Vac Sci Tech 2001 Baranauskas App Surface Sci 2005

ldquohellipstatistical diameter of the fibers is 159 nm in good agreement with the literature values for type I collagen fibersrdquo

BBML - All R

ights Reserve

d

Analysis of Collagen in Bone Fibril Diameter Fibril Diameter easiest to measure property first attempts were a bit crude better with increased resolution

Sasaki J Mat Sci Tech 2002 Thalhammer J Arch Sci 2001

Bozec Ultramicroscopy 2005

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Collagen in Bone Fibril Diameter

Caution must be taken when reporting and interpreting diameters measured from fibrils in bone bull Fibril packing - width may not be fully exposed

Wallace et al Langmuir 2010

BBML - All R

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Collagen in Bone Fibril Diameter

Caution must be taken when reporting and interpreting diameters measured from fibrils in bone bull Fibril packing - width may not be fully exposed

Rigozzi ndash J Struct Bio - 2011

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ights Reserve

d

Collagen in Bone Fibril D-Spacing

300 nm 15 nm

N C C N C C N C C N C C

H O H2C CH2 O H

CH2

O OH HHC CH2

CH2

HO

H H

HHN C C

O

H2C CH2

CH2

HN C C

OH

HC CH2

HO

N C C N C C N C C

H O H2C CH2 O

CH2

OH HC CH2

HO

H H

H

CH2 CH2

glycine X - proline Y - hydroxyproline

Hodge and Petruska 1963

Collagen Fibril Axial D-Periodicity BBML - All R

ights Reserve

d

05

101520

0 02 04 06 08 1

Hei

ght (

nm)

Length (microns)

httpremfdartmoutheduimagesindexhtml

Randall Nature and Structure of Collagen 1953

TEM SEM

AFM

D-Spacing = 67 nm

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d

Collagen in Bone Fibril D-Spacing Many studies have shown observable D-spacing content that near theoretical value

Sasaki 2002 67 plusmn 2 nm Bozek 2005 665 plusmn 14 nm Ge 2007 666 plusmn 38 nm

Habelitz J Structural Bio 2002

A 2002 study in dentin got me interestedhellip

n = 322 wet n = 363 dry

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Collagen in Bone Fibril D-Spacing

bull Internal structure bull Wallace et al Bone 2010 46 1349-1354 bull Wallace et al J Stuct Bio 2011 173 146-152 bull Kemp et al J Stuct Bio 2012 180 428-438 bull Warden et al Endocrinology 2013 154(9)

3178-3187 bull Gallant et al Bone 2014 61 191-200 bull Bart et al Conn Tiss Res 2014 In Press

bull Enzymatic and non-

enzymatic cross-linking bull Hammond et al Bone 2014 60 26-32 bull Diaz Gonzalez et al J Biomech 2014 47(3)

681-686 bull Voytik-Harbin et al In Preparation 2014

D-periodic spacing important structural feature that captures aspects related to

Mouse Femur Mid-diaphysis BBML - All R

ights Reserve

d

2D Fast Fourier Transform Analysis

Harmonics

Fibril D-Spacing

Erickson B Wallace JM et al Biotechnology Journal 2013

Raw AFM Image 2D FFT Power Spectrum

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ights Reserve

d

2D Fast Fourier Transform Analysis

Harmonics

Fibril D-Spacing

Raw AFM Image 2D FFT Power Spectrum

0

20000

40000

60000

80000

100000

120000

0 001 002 003 004 005 006

Am

plitu

de (

microV2 )

Position (1nm)

D-periodic spacing 0015 (1nm) ~ 67 nm

BBML - All R

ights Reserve

d

D-Spacing Distribution

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Overall Mean 670 plusmn 00 nm

BBML - All R

ights Reserve

d

D-Spacing Distribution

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Overall Mean 670 plusmn 00 nm

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

4 Samples Total of n = 255 fibrils Overall Mean 682 plusmn 12 nm

BBML - All R

ights Reserve

d

Cumulative Distribution Function

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

This population 255 fibrils - Each fibril 1255 = 0392 - Data range 628-716 nm

BBML - All R

ights Reserve

d

CDF vs Histogram 62 63 64 65 66 67 68 69 70 71 72 73 74

0

10

20

30

40

50

60

70

80

90

100

0

5

10

15

20

25

30

35

40

45

50

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

HistoCDF

BBML - All R

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d

Changes in D-Spacing with Disease

0

5

10

15

20

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

0

5

10

15

20

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

Wallace et al Bone 2010 46 1349-1354

KS Test plt0001

Control 680 plusmn 26 nm

Estrogen Dep 659 plusmn 31 nm

t-test on mean p=0048

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Changes in D-Spacing with Disease

Wallace et al Bone 2010 46 1349-1354

0

10

20

30

40

50

60

70

80

90

100

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

KS Test plt0001

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ights Reserve

d

Question Model and Tissue Morphology Publication

Tissue Specificity in Mice Male mouse 8 weeks Femur dentin tail tendon

No differences in bone vs dentin both ∆ vs tendon

Wallace et al Langmuir 2011 26(10) 7349-7354

Gender Effects Male and female mice 16 weeks Femur No differences NA

Estrogen Depletion OVX in sheep 5 years + 2 Radius Mean and population shift Wallace et al Bone

2010 46 1349-1354

Estrogen Depletion OVX in female 5 years + 2 Skin (dermis) Significant population shift Erickson et al Biotech

J 2013 8(1) 1174-126

Osteoporosis and Mechanical Loading

OVX in ulnar loading in rats Ulna

No loading effect population shift with OVX

Warden et al Endocrinology 2013 154(9) 3178-3187

Osteogenesis Imperfecta Brtl+ model male mice 2 months Femur

No mean ∆ widening of distribution

Wallace et al J Stuct Bio 2011 173 146-152

Osteogenesis Imperfecta Oimoim model male mice 12 weeks Femur Mean and population shift Bart et al Conn Tiss

Res 2014 In Press

Osteogenesis Imperfecta and Tissue Hydration

Brtl+ model male mice 6 months Tail Tendon Significant population shift Kemp et al J Stuct Bio

2012 180 428-438

Type 2 Diabetes Male ZDSD rats 30 weeks Tibia Significant population shift Hammond et al Bone

2014 60 26-32

Type 2 Diabetes Female ZDSD rats 32 weeks Tail Tendon Significant population shift

Diaz Gonzalez et al J Biomech 2014 47(3)

681-686

Raloxifene (in vivo and in vitro) Female dog treated with Raloxifene Femur Significant population shift Gallant et al Bone

2014 61 191-200 BBML - All R

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d

Issues to Consider Site Selection and Sampling

KS Test plt0001

0023 of bonersquos length

15 m

m =

150

00 micro

m

Wallace et al Langmuir 2011 26(10) 7349-7354

100 microm x 100 microm

35 microm x 35 microm

BBML - All R

ights Reserve

d

Issues to Consider Site Selection and Sampling

60

62

64

66

68

70

72

74

1 (35)

2 (33)

3 (40)

4 (39)

5 (35)

6 (29)

7 (26)

8 (23)

9 (28)

WT (288)

D-P

erio

dic

Gap

Ove

rlap

Spac

ing

(nm

)

Axial Location (Proximal End = 1 Distal End = 9)

( of Fibrils)

91

23

45

87

6

Wallace et al Langmuir 2011 26(10) 7349-7354

288 fibrils for population n=1 for mean comparisons BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

Wet Tendon Dry Tendon Kemp et al J StructBio 2012 180 (3) 428-438

Collagen and bone exist in a hydrated environment in vivo bull Hydration state can impact morphology and mechanics

BBML - All R

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Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

Nonsignificant mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet (n=255) 682 plusmn 12 nm

WT Dry (n=257) 686 plusmn 08 nm

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

Brtl DryBrtl Wet

KS Test plt0001

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

WT Case 04 nm mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet 682 plusmn 12 nm

WT Dry 686 plusmn 08 nm

Brtl+ Wet 675 plusmn 14 nm

Brtl+ Dry 689 plusmn 10 nm

Brtl+ Case 14 nm increase with drying (plt0001) bull Entire population shifted upward (plt0001)

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT WetBrtl Wet

KS Test plt0001

Brtl+ distribution shifted downward vs WT when wet

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryBrtl Dry

KS Test plt0001

Brtl+ distribution shifted upward vs WT when dry

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider AFM calibration

bull Absolute distance measurements require accurate piezo calibration

bull AFM systems have large scan ranges (150 microm to lt 1 microm)

bull Scanner non-linearities can introduce substantial error over that range

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

ights Reserve

d

Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

BBML - All R

ights Reserve

d

Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

100 nm 10 80 602 1198 100 nm 7 60 827 1439 100 nm 5 40 814 1427 100 nm 35 20 917 1724 100 nm 2 10 937 1774

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

ights Reserve

d

AFM calibration ndash Take Away Message

Calibration using a scan size and feature size comparable to features of interest is highly recommended

Calibration addresses absolute accuracy not precision (differential sensitivity between measurements)

bull Does not limit ability to differentiate between groups using the same AFM with the same calibration parameters

BBML - All R

ights Reserve

d

AFM Applications in Bone Research Indentation Mechanics

BBML - All R

ights Reserve

d

AFM-Based Indentation

In addition to high resolution imaging AFM can be used to extract nanoscale mechanical data

ApproachWithdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Can

tilev

er D

efle

ctio

n (V

olts

)

1 Ramp into a rigid sample ndash measure the slope

Deflection sensitivity x 119899119899119899119899119881119881

2 Calculate cantilever spring constant

3 Hookersquos law takes deflection (volts) and converts to units of force F = kx

Spring constant k 119873119873119899119899

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Forc

e (n

N)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration Fo

rce

(nN

)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

Tip-Sample Separation (nm)

BBML - All R

ights Reserve

d

AFM-Based Indentation Parameters

Approach Withdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

Peak Force

Area Between Curves Energy Dissipation

Contact Mechanics Fit for Elastic Modulus (E)

Adhesion Force

Indentation Depth (δ)

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

z

r

δ

R

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119877119877 ∙ 12057512057532

Es Sample modulus νs Poissonrsquos ratio R tip radius of curvature δ load point displacement

Relevant Hertz Assumptions bull Strains are small and within the elastic limit bull Indenter is infinitely stiff (only sample deforms) bull Indentation depth ltlt radius of curvature of indenter bull Surfaces are continuous non-conforming (must be flat) bull Load is normal to surface

BBML - All R

ights Reserve

d

Proper Indentation Model is Essential

δ

r

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119903119903 ∙ 12057512057532

δ

α

119865119865 =2120587120587∙

1198641198641199041199041 minus 1205841205841199041199042

∙ tan120572120572 ∙ 1205751205752

Es Sample modulus νs Poissonrsquos ratio R tip radius α opening angle

Hertz Model Spherical Indenter

depth ltlt radius of curvature

Sneddon Model Conical Indenter

depth ge radius of curvature

BBML - All R

ights Reserve

d

Indentation in OI Tendon Study

x

z

yBrtl+ and WT mice 6 month old males

4 tails per group

2-3 fascicles per tail

25-30 fibrils per tail

4-5 locations per fibril

Indent force 20 nN wet (Sneddon) 50 nN dry (Hertz) BBML - A

ll Rights R

eserved

OI Tendon Differential Effects of Dehydration

Dry 1268 plusmn 718 MPa

Wet 305 plusmn 243 MPa

Dry 1737 plusmn 973 MPa

Wet 301 plusmn 202 MPa

Kemp et al J StructBio 2012 180 (3) 428-438

bull Significant stiffening of fibrils with dehydration

bull More pronounced modulus modulus as a function of disease

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ights Reserve

d

OI Tendon Differential Effects of Dehydration

Kemp et al J StructBio 2012 180 (3) 428-438

Diseased phenotype not present when wet phenotype observed when dried

0

10

20

30

40

50

60

0 20 40 60 80 100 120 140 160 180 200

Gro

up S

ampl

es (

)

Indent Modulus (MPa)

WT WetBrtl Wet

0

2

4

6

8

10

12

14

16

18

20

025

050

075

010

0012

5015

0017

5020

0022

5025

0027

5030

0032

5035

0037

5040

0042

5045

0047

5050

00

Gro

up S

ampl

es (

)Indent Modulus (MPa)

WT DryBrtl Dry

555 WT Indents 305 plusmn 243 MPa

614 Brtl+ Indents 301 plusmn 202 MPa

KS Test p=0129 t Test p=0464 464 WT Indents

1268 plusmn 718 MPa 479 Brtl+ Indents 1737 plusmn 973 MPa

KS Test plt0001 T Test plt0001

BBML - All R

ights Reserve

d

Indentation in Diabetic Tendon (Poster 0495)

x

z

y

Control and Diabetic Rat Tail Tendon

4-5 rats per group

2-3 fascicles per rat

~70 fibrilstail

4-5 locationsfibril

20 nN (Sneddon)

BBML - All R

ights Reserve

d

Multiscale Diabetes Study (Poster 0495)

0

5

10

15

20

25

300 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 gt25

Perc

enta

ge o

f Gro

up S

ampl

es

Elastic Modulus (MPa)

CD (1530 indents)ZDSD (913 indents)

CD (n=5) 381 plusmn 149 MPa

ZDSD (n=3) 706 plusmn 263 MPa

0

100

200

300

400

500

600

700

800

900

0 2 4 6 8 10

Mic

rosc

ale

Mod

ulus

(MPa

)

Nanoscale Modulus (MPa)

plt0001

r2=0531 p=0040

bull Increased indentation modulus in diabetic tendon

bull Strong relationship between nanoscale indent modulus and microscale tensile modulus

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ights Reserve

d

Indentation Limitations and Weaknesses

Proper probe selection for intended application bull Cantilever must be stiff relative to sample bull Sharp probes may damage sample easily dull

R or α must be determined for each probe bull Properties provided by manufacturer are unreliable bull Vary from probe to probe can dull during indent process

Spring constant must be determined for each probe

bull Value provided by manufacturer is inaccurate bull Thermal tuning OK for ldquosoftrdquo probes poor performance

for stiffer probes bull Sader method (geometry) added mass reference

cantilevers

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

Veeco Application Technical note Ben Ohler Practical Advice on the Determination of Cantilever Spring Constants

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

bull Sample processing

bull Sample hydration

bull Proper force range

bull Substrate effects for thin samples

bull Proper modulus model and range of data goodness of fit

Bearing in mind these assumptions and limitations AFM can provide true ldquonanoscalerdquo mechanical properties both in terms of forces and deformations

BBML - All R

ights Reserve

d

A Note on Peak Force QNM for Bruker AFMs bull Mechanical properties mapped pixel-by-pixel at same

resolution as the height image (PeakForce QNM)

Height Modulus BBML - All R

ights Reserve

d

A Note on Peak Force QNM

Correct probe stiffness must be used based on sample Pittenger ndash Veeco Product Bulletin

BBML - All R

ights Reserve

d

A Note on Peak Force QNM Extremely sensitive to calibration (details rarely given) Absolute radius and spring constant known

bull especially important from probe to probe bull radius uarr with indent depth ndash must determine R at depth

you expect to image bull Depth may vary as a function of sample (or at different

regions within the sample)

Relative reference material of ldquoknownrdquo modulus used to determine a ratio of 119896119896 119877119877 bull ldquoknownrdquo modulus may come from tension compression

and not indentation bull R is related to depth indentation depth on sample must

be same as on reference BBML - All R

ights Reserve

d

A Note on Peak Force QNM bull Modulus fit on the fly ndash no ability to monitor fit (or range) bull Local sample topography and sample tilt play a role in

measured parameters bull Assuming all modulus values in an image are correct how to

quantify

512 x 512 pixels = 262144 measurements

n=1 n=262144

n from selected ROIs

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ights Reserve

d

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ights Reserve

d

bull Imaging in air on mica ndash substrate effect

bull Same imaging force or indent depth for all

bull For each area were all modulus values pooled

Lamprou et al PLoS ONE 2013 BBML - All R

ights Reserve

d

AFM Applications in Bone Research Other Cool Mechanical Techniques

BBML - All R

ights Reserve

d

ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo Bonds within or between collagen fibrils in bone that rupture to dissipate energy as a toughening mechanism

Thompson et al Nature 2001

Fantner et al Nature Mat 2005

Hansma et al JMNI 2005

Fantner et al Nano Letters 2007 BBML - All R

ights Reserve

d

Other Mechanical Techniques

Jimenez-Palomar J Mech Beh Bio Mat 2012

van der Rijt Macro Bio 2004

Yang J Biomed Mater Res 2007

Yang Biophysical Journal 2008 BBML - All R

ights Reserve

d

Other Cool Techniques bull Piezoresponse Force Microscopy (PFM)

bull Electrochemical AFM (EC-AFM)

bull Force Modulation Microscopy (FMM)

bull Lateral Force Microscopy (LFM)

bull Magnetic Force Microscopy (MFM)

bull Conductive AFM (C-AFM)

bull Force Pulling

bull Cantilever Bending

bull Tensile testing of individual fibrils

bull 3 point bending of fibrils

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ights Reserve

d

Summary Things to rememberhellip

bull Imaging mode imaging medium and probe selection

bull Morphology Measurements bull Sample preparation and hydration state bull Measuring fibril diameter bull Site selection and proper sampling bull Appropriate system calibration

bull Mechanical Measurements

bull Probe selection (stiffness and tip radius) bull Probe calibration (radius angle spring constant) bull Indentation force range and mechanical model bull Hydration in biological samples bull PeakForce QNM

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d

Acknowledgements BBML

Max Hammond Armando Diaz Gonzalez Zachary Bart Silvia Canelon Creasy Clauser

IU School of Medicine Dr David Burr Dr Matt Allen Dr Stuart Warden Dr Lilian Plotkin

Funding Sources NIHNIDCR 1F32DE018840-01 A1 IUPUI BME Departmental Startup Funds IUPUI Research Support Funds Grant IUPUI Biomechanics and Biomaterials Research Center

NIH NICHD Dr Joan Marini

BBML - All R

ights Reserve

d

Bone Biology and Mechanics Lab (BBML) wwwiupuiedu~bbml Facebook BoneBiologyMechanicsLab

BBML - All R

ights Reserve

d

• Slide Number 1
• General Seminar Outline
• Bonersquos Hierarchical Structure
• Critical Gap in Understanding Collagen
• Methods For Analyzing Collagen Structure
• Methods For Imaging Collagen Structure
• Cryo-TEM
• Atomic Force Microscopy (AFM)
• AFM Image of Pentacene (C22H14)
• AFM Image of Hexabenzocoronene
• Basics of AFM Imaging The Probe
• Basics of AFM Imaging System Components
• van der Waals Potential Energy Curve
• Contact Mode ndash Monitor Deflection
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Peak Force Tapping Mode ndash Monitor Force
• AFM Applications in Bone Research Imaging
• AFM Imaging of Collagen
• Imaging of Osteoclast Activity
• Mineralized collagen deposition in vitro
• Imaging of Canilicular Structure
• AFM Imaging in Bone
• AFM Imaging in Bone
• Removing Mineral in Bone to Expose Collagen
• Removing Mineral in Bone to Expose Collagen
• Demineralization with EDTA
• Demineralization with EDTA
• Demineralization with EDTA
• Issues to Consider Effects of Acid
• Slide Number 32
• Slide Number 33
• Analysis of Collagen Structure in Bone
• Analysis of Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril D-Spacing
• Slide Number 39
• Collagen in Bone Fibril D-Spacing
• Collagen in Bone Fibril D-Spacing
• 2D Fast Fourier Transform Analysis
• 2D Fast Fourier Transform Analysis
• D-Spacing Distribution
• D-Spacing Distribution
• Cumulative Distribution Function
• CDF vs Histogram
• Changes in D-Spacing with Disease
• Changes in D-Spacing with Disease
• Slide Number 50
• Issues to Consider Site Selection and Sampling
• Issues to Consider Site Selection and Sampling
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• AFM calibration ndash Take Away Message
• AFM Applications in Bone ResearchIndentation Mechanics
• AFM-Based Indentation
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Parameters
• Hertz Contact Equation
• Hertz Contact Equation
• Hertz Contact Equation
• Proper Indentation Model is Essential
• Indentation in OI Tendon Study
• OI Tendon Differential Effects of Dehydration
• OI Tendon Differential Effects of Dehydration
• Indentation in Diabetic Tendon (Poster 0495)
• Multiscale Diabetes Study (Poster 0495)
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• A Note on Peak Force QNM for Bruker AFMs
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• Slide Number 83
• Slide Number 84
• AFM Applications in Bone ResearchOther Cool Mechanical Techniques
• ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo
• Other Mechanical Techniques
• Other Cool Techniques
• Summary Things to rememberhellip
• Acknowledgements
• Bone Biology and Mechanics Lab (BBML)

Analysis of Collagen Structure in Bone Spatial organization of newly formed bone following a surgically-induced injury (rat tibial hole)

Baranauskas J Vac Sci Tech 2001 Baranauskas App Surface Sci 2005

ldquohellipstatistical diameter of the fibers is 159 nm in good agreement with the literature values for type I collagen fibersrdquo

BBML - All R

ights Reserve

d

Analysis of Collagen in Bone Fibril Diameter Fibril Diameter easiest to measure property first attempts were a bit crude better with increased resolution

Sasaki J Mat Sci Tech 2002 Thalhammer J Arch Sci 2001

Bozec Ultramicroscopy 2005

BBML - All R

ights Reserve

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Collagen in Bone Fibril Diameter

Caution must be taken when reporting and interpreting diameters measured from fibrils in bone bull Fibril packing - width may not be fully exposed

Wallace et al Langmuir 2010

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ights Reserve

d

Collagen in Bone Fibril Diameter

Caution must be taken when reporting and interpreting diameters measured from fibrils in bone bull Fibril packing - width may not be fully exposed

Rigozzi ndash J Struct Bio - 2011

BBML - All R

ights Reserve

d

Collagen in Bone Fibril D-Spacing

300 nm 15 nm

N C C N C C N C C N C C

H O H2C CH2 O H

CH2

O OH HHC CH2

CH2

HO

H H

HHN C C

O

H2C CH2

CH2

HN C C

OH

HC CH2

HO

N C C N C C N C C

H O H2C CH2 O

CH2

OH HC CH2

HO

H H

H

CH2 CH2

glycine X - proline Y - hydroxyproline

Hodge and Petruska 1963

Collagen Fibril Axial D-Periodicity BBML - All R

ights Reserve

d

05

101520

0 02 04 06 08 1

Hei

ght (

nm)

Length (microns)

httpremfdartmoutheduimagesindexhtml

Randall Nature and Structure of Collagen 1953

TEM SEM

AFM

D-Spacing = 67 nm

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ights Reserve

d

Collagen in Bone Fibril D-Spacing Many studies have shown observable D-spacing content that near theoretical value

Sasaki 2002 67 plusmn 2 nm Bozek 2005 665 plusmn 14 nm Ge 2007 666 plusmn 38 nm

Habelitz J Structural Bio 2002

A 2002 study in dentin got me interestedhellip

n = 322 wet n = 363 dry

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ights Reserve

d

Collagen in Bone Fibril D-Spacing

bull Internal structure bull Wallace et al Bone 2010 46 1349-1354 bull Wallace et al J Stuct Bio 2011 173 146-152 bull Kemp et al J Stuct Bio 2012 180 428-438 bull Warden et al Endocrinology 2013 154(9)

3178-3187 bull Gallant et al Bone 2014 61 191-200 bull Bart et al Conn Tiss Res 2014 In Press

bull Enzymatic and non-

enzymatic cross-linking bull Hammond et al Bone 2014 60 26-32 bull Diaz Gonzalez et al J Biomech 2014 47(3)

681-686 bull Voytik-Harbin et al In Preparation 2014

D-periodic spacing important structural feature that captures aspects related to

Mouse Femur Mid-diaphysis BBML - All R

ights Reserve

d

2D Fast Fourier Transform Analysis

Harmonics

Fibril D-Spacing

Erickson B Wallace JM et al Biotechnology Journal 2013

Raw AFM Image 2D FFT Power Spectrum

BBML - All R

ights Reserve

d

2D Fast Fourier Transform Analysis

Harmonics

Fibril D-Spacing

Raw AFM Image 2D FFT Power Spectrum

0

20000

40000

60000

80000

100000

120000

0 001 002 003 004 005 006

Am

plitu

de (

microV2 )

Position (1nm)

D-periodic spacing 0015 (1nm) ~ 67 nm

BBML - All R

ights Reserve

d

D-Spacing Distribution

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Overall Mean 670 plusmn 00 nm

BBML - All R

ights Reserve

d

D-Spacing Distribution

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Overall Mean 670 plusmn 00 nm

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

4 Samples Total of n = 255 fibrils Overall Mean 682 plusmn 12 nm

BBML - All R

ights Reserve

d

Cumulative Distribution Function

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

This population 255 fibrils - Each fibril 1255 = 0392 - Data range 628-716 nm

BBML - All R

ights Reserve

d

CDF vs Histogram 62 63 64 65 66 67 68 69 70 71 72 73 74

0

10

20

30

40

50

60

70

80

90

100

0

5

10

15

20

25

30

35

40

45

50

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

HistoCDF

BBML - All R

ights Reserve

d

Changes in D-Spacing with Disease

0

5

10

15

20

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

0

5

10

15

20

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

Wallace et al Bone 2010 46 1349-1354

KS Test plt0001

Control 680 plusmn 26 nm

Estrogen Dep 659 plusmn 31 nm

t-test on mean p=0048

BBML - All R

ights Reserve

d

Changes in D-Spacing with Disease

Wallace et al Bone 2010 46 1349-1354

0

10

20

30

40

50

60

70

80

90

100

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

KS Test plt0001

BBML - All R

ights Reserve

d

Question Model and Tissue Morphology Publication

Tissue Specificity in Mice Male mouse 8 weeks Femur dentin tail tendon

No differences in bone vs dentin both ∆ vs tendon

Wallace et al Langmuir 2011 26(10) 7349-7354

Gender Effects Male and female mice 16 weeks Femur No differences NA

Estrogen Depletion OVX in sheep 5 years + 2 Radius Mean and population shift Wallace et al Bone

2010 46 1349-1354

Estrogen Depletion OVX in female 5 years + 2 Skin (dermis) Significant population shift Erickson et al Biotech

J 2013 8(1) 1174-126

Osteoporosis and Mechanical Loading

OVX in ulnar loading in rats Ulna

No loading effect population shift with OVX

Warden et al Endocrinology 2013 154(9) 3178-3187

Osteogenesis Imperfecta Brtl+ model male mice 2 months Femur

No mean ∆ widening of distribution

Wallace et al J Stuct Bio 2011 173 146-152

Osteogenesis Imperfecta Oimoim model male mice 12 weeks Femur Mean and population shift Bart et al Conn Tiss

Res 2014 In Press

Osteogenesis Imperfecta and Tissue Hydration

Brtl+ model male mice 6 months Tail Tendon Significant population shift Kemp et al J Stuct Bio

2012 180 428-438

Type 2 Diabetes Male ZDSD rats 30 weeks Tibia Significant population shift Hammond et al Bone

2014 60 26-32

Type 2 Diabetes Female ZDSD rats 32 weeks Tail Tendon Significant population shift

Diaz Gonzalez et al J Biomech 2014 47(3)

681-686

Raloxifene (in vivo and in vitro) Female dog treated with Raloxifene Femur Significant population shift Gallant et al Bone

2014 61 191-200 BBML - All R

ights Reserve

d

Issues to Consider Site Selection and Sampling

KS Test plt0001

0023 of bonersquos length

15 m

m =

150

00 micro

m

Wallace et al Langmuir 2011 26(10) 7349-7354

100 microm x 100 microm

35 microm x 35 microm

BBML - All R

ights Reserve

d

Issues to Consider Site Selection and Sampling

60

62

64

66

68

70

72

74

1 (35)

2 (33)

3 (40)

4 (39)

5 (35)

6 (29)

7 (26)

8 (23)

9 (28)

WT (288)

D-P

erio

dic

Gap

Ove

rlap

Spac

ing

(nm

)

Axial Location (Proximal End = 1 Distal End = 9)

( of Fibrils)

91

23

45

87

6

Wallace et al Langmuir 2011 26(10) 7349-7354

288 fibrils for population n=1 for mean comparisons BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

Wet Tendon Dry Tendon Kemp et al J StructBio 2012 180 (3) 428-438

Collagen and bone exist in a hydrated environment in vivo bull Hydration state can impact morphology and mechanics

BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

Nonsignificant mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet (n=255) 682 plusmn 12 nm

WT Dry (n=257) 686 plusmn 08 nm

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

Brtl DryBrtl Wet

KS Test plt0001

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

WT Case 04 nm mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet 682 plusmn 12 nm

WT Dry 686 plusmn 08 nm

Brtl+ Wet 675 plusmn 14 nm

Brtl+ Dry 689 plusmn 10 nm

Brtl+ Case 14 nm increase with drying (plt0001) bull Entire population shifted upward (plt0001)

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT WetBrtl Wet

KS Test plt0001

Brtl+ distribution shifted downward vs WT when wet

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryBrtl Dry

KS Test plt0001

Brtl+ distribution shifted upward vs WT when dry

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider AFM calibration

bull Absolute distance measurements require accurate piezo calibration

bull AFM systems have large scan ranges (150 microm to lt 1 microm)

bull Scanner non-linearities can introduce substantial error over that range

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

ights Reserve

d

Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

BBML - All R

ights Reserve

d

Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

100 nm 10 80 602 1198 100 nm 7 60 827 1439 100 nm 5 40 814 1427 100 nm 35 20 917 1724 100 nm 2 10 937 1774

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

ights Reserve

d

AFM calibration ndash Take Away Message

Calibration using a scan size and feature size comparable to features of interest is highly recommended

Calibration addresses absolute accuracy not precision (differential sensitivity between measurements)

bull Does not limit ability to differentiate between groups using the same AFM with the same calibration parameters

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ights Reserve

d

AFM Applications in Bone Research Indentation Mechanics

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ights Reserve

d

AFM-Based Indentation

In addition to high resolution imaging AFM can be used to extract nanoscale mechanical data

ApproachWithdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

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ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Can

tilev

er D

efle

ctio

n (V

olts

)

1 Ramp into a rigid sample ndash measure the slope

Deflection sensitivity x 119899119899119899119899119881119881

2 Calculate cantilever spring constant

3 Hookersquos law takes deflection (volts) and converts to units of force F = kx

Spring constant k 119873119873119899119899

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Forc

e (n

N)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration Fo

rce

(nN

)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

Tip-Sample Separation (nm)

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ights Reserve

d

AFM-Based Indentation Parameters

Approach Withdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

Peak Force

Area Between Curves Energy Dissipation

Contact Mechanics Fit for Elastic Modulus (E)

Adhesion Force

Indentation Depth (δ)

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ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

z

r

δ

R

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119877119877 ∙ 12057512057532

Es Sample modulus νs Poissonrsquos ratio R tip radius of curvature δ load point displacement

Relevant Hertz Assumptions bull Strains are small and within the elastic limit bull Indenter is infinitely stiff (only sample deforms) bull Indentation depth ltlt radius of curvature of indenter bull Surfaces are continuous non-conforming (must be flat) bull Load is normal to surface

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ights Reserve

d

Proper Indentation Model is Essential

δ

r

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119903119903 ∙ 12057512057532

δ

α

119865119865 =2120587120587∙

1198641198641199041199041 minus 1205841205841199041199042

∙ tan120572120572 ∙ 1205751205752

Es Sample modulus νs Poissonrsquos ratio R tip radius α opening angle

Hertz Model Spherical Indenter

depth ltlt radius of curvature

Sneddon Model Conical Indenter

depth ge radius of curvature

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ights Reserve

d

Indentation in OI Tendon Study

x

z

yBrtl+ and WT mice 6 month old males

4 tails per group

2-3 fascicles per tail

25-30 fibrils per tail

4-5 locations per fibril

Indent force 20 nN wet (Sneddon) 50 nN dry (Hertz) BBML - A

ll Rights R

eserved

OI Tendon Differential Effects of Dehydration

Dry 1268 plusmn 718 MPa

Wet 305 plusmn 243 MPa

Dry 1737 plusmn 973 MPa

Wet 301 plusmn 202 MPa

Kemp et al J StructBio 2012 180 (3) 428-438

bull Significant stiffening of fibrils with dehydration

bull More pronounced modulus modulus as a function of disease

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ights Reserve

d

OI Tendon Differential Effects of Dehydration

Kemp et al J StructBio 2012 180 (3) 428-438

Diseased phenotype not present when wet phenotype observed when dried

0

10

20

30

40

50

60

0 20 40 60 80 100 120 140 160 180 200

Gro

up S

ampl

es (

)

Indent Modulus (MPa)

WT WetBrtl Wet

0

2

4

6

8

10

12

14

16

18

20

025

050

075

010

0012

5015

0017

5020

0022

5025

0027

5030

0032

5035

0037

5040

0042

5045

0047

5050

00

Gro

up S

ampl

es (

)Indent Modulus (MPa)

WT DryBrtl Dry

555 WT Indents 305 plusmn 243 MPa

614 Brtl+ Indents 301 plusmn 202 MPa

KS Test p=0129 t Test p=0464 464 WT Indents

1268 plusmn 718 MPa 479 Brtl+ Indents 1737 plusmn 973 MPa

KS Test plt0001 T Test plt0001

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ights Reserve

d

Indentation in Diabetic Tendon (Poster 0495)

x

z

y

Control and Diabetic Rat Tail Tendon

4-5 rats per group

2-3 fascicles per rat

~70 fibrilstail

4-5 locationsfibril

20 nN (Sneddon)

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ights Reserve

d

Multiscale Diabetes Study (Poster 0495)

0

5

10

15

20

25

300 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 gt25

Perc

enta

ge o

f Gro

up S

ampl

es

Elastic Modulus (MPa)

CD (1530 indents)ZDSD (913 indents)

CD (n=5) 381 plusmn 149 MPa

ZDSD (n=3) 706 plusmn 263 MPa

0

100

200

300

400

500

600

700

800

900

0 2 4 6 8 10

Mic

rosc

ale

Mod

ulus

(MPa

)

Nanoscale Modulus (MPa)

plt0001

r2=0531 p=0040

bull Increased indentation modulus in diabetic tendon

bull Strong relationship between nanoscale indent modulus and microscale tensile modulus

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d

Indentation Limitations and Weaknesses

Proper probe selection for intended application bull Cantilever must be stiff relative to sample bull Sharp probes may damage sample easily dull

R or α must be determined for each probe bull Properties provided by manufacturer are unreliable bull Vary from probe to probe can dull during indent process

Spring constant must be determined for each probe

bull Value provided by manufacturer is inaccurate bull Thermal tuning OK for ldquosoftrdquo probes poor performance

for stiffer probes bull Sader method (geometry) added mass reference

cantilevers

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d

Indentation Limitations and Weaknesses

Veeco Application Technical note Ben Ohler Practical Advice on the Determination of Cantilever Spring Constants

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Indentation Limitations and Weaknesses

bull Sample processing

bull Sample hydration

bull Proper force range

bull Substrate effects for thin samples

bull Proper modulus model and range of data goodness of fit

Bearing in mind these assumptions and limitations AFM can provide true ldquonanoscalerdquo mechanical properties both in terms of forces and deformations

BBML - All R

ights Reserve

d

A Note on Peak Force QNM for Bruker AFMs bull Mechanical properties mapped pixel-by-pixel at same

resolution as the height image (PeakForce QNM)

Height Modulus BBML - All R

ights Reserve

d

A Note on Peak Force QNM

Correct probe stiffness must be used based on sample Pittenger ndash Veeco Product Bulletin

BBML - All R

ights Reserve

d

A Note on Peak Force QNM Extremely sensitive to calibration (details rarely given) Absolute radius and spring constant known

bull especially important from probe to probe bull radius uarr with indent depth ndash must determine R at depth

you expect to image bull Depth may vary as a function of sample (or at different

regions within the sample)

Relative reference material of ldquoknownrdquo modulus used to determine a ratio of 119896119896 119877119877 bull ldquoknownrdquo modulus may come from tension compression

and not indentation bull R is related to depth indentation depth on sample must

be same as on reference BBML - All R

ights Reserve

d

A Note on Peak Force QNM bull Modulus fit on the fly ndash no ability to monitor fit (or range) bull Local sample topography and sample tilt play a role in

measured parameters bull Assuming all modulus values in an image are correct how to

quantify

512 x 512 pixels = 262144 measurements

n=1 n=262144

n from selected ROIs

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d

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d

bull Imaging in air on mica ndash substrate effect

bull Same imaging force or indent depth for all

bull For each area were all modulus values pooled

Lamprou et al PLoS ONE 2013 BBML - All R

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d

AFM Applications in Bone Research Other Cool Mechanical Techniques

BBML - All R

ights Reserve

d

ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo Bonds within or between collagen fibrils in bone that rupture to dissipate energy as a toughening mechanism

Thompson et al Nature 2001

Fantner et al Nature Mat 2005

Hansma et al JMNI 2005

Fantner et al Nano Letters 2007 BBML - All R

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d

Other Mechanical Techniques

Jimenez-Palomar J Mech Beh Bio Mat 2012

van der Rijt Macro Bio 2004

Yang J Biomed Mater Res 2007

Yang Biophysical Journal 2008 BBML - All R

ights Reserve

d

Other Cool Techniques bull Piezoresponse Force Microscopy (PFM)

bull Electrochemical AFM (EC-AFM)

bull Force Modulation Microscopy (FMM)

bull Lateral Force Microscopy (LFM)

bull Magnetic Force Microscopy (MFM)

bull Conductive AFM (C-AFM)

bull Force Pulling

bull Cantilever Bending

bull Tensile testing of individual fibrils

bull 3 point bending of fibrils

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Summary Things to rememberhellip

bull Imaging mode imaging medium and probe selection

bull Morphology Measurements bull Sample preparation and hydration state bull Measuring fibril diameter bull Site selection and proper sampling bull Appropriate system calibration

bull Mechanical Measurements

bull Probe selection (stiffness and tip radius) bull Probe calibration (radius angle spring constant) bull Indentation force range and mechanical model bull Hydration in biological samples bull PeakForce QNM

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d

Acknowledgements BBML

Max Hammond Armando Diaz Gonzalez Zachary Bart Silvia Canelon Creasy Clauser

IU School of Medicine Dr David Burr Dr Matt Allen Dr Stuart Warden Dr Lilian Plotkin

Funding Sources NIHNIDCR 1F32DE018840-01 A1 IUPUI BME Departmental Startup Funds IUPUI Research Support Funds Grant IUPUI Biomechanics and Biomaterials Research Center

NIH NICHD Dr Joan Marini

BBML - All R

ights Reserve

d

Bone Biology and Mechanics Lab (BBML) wwwiupuiedu~bbml Facebook BoneBiologyMechanicsLab

BBML - All R

ights Reserve

d

• Slide Number 1
• General Seminar Outline
• Bonersquos Hierarchical Structure
• Critical Gap in Understanding Collagen
• Methods For Analyzing Collagen Structure
• Methods For Imaging Collagen Structure
• Cryo-TEM
• Atomic Force Microscopy (AFM)
• AFM Image of Pentacene (C22H14)
• AFM Image of Hexabenzocoronene
• Basics of AFM Imaging The Probe
• Basics of AFM Imaging System Components
• van der Waals Potential Energy Curve
• Contact Mode ndash Monitor Deflection
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Peak Force Tapping Mode ndash Monitor Force
• AFM Applications in Bone Research Imaging
• AFM Imaging of Collagen
• Imaging of Osteoclast Activity
• Mineralized collagen deposition in vitro
• Imaging of Canilicular Structure
• AFM Imaging in Bone
• AFM Imaging in Bone
• Removing Mineral in Bone to Expose Collagen
• Removing Mineral in Bone to Expose Collagen
• Demineralization with EDTA
• Demineralization with EDTA
• Demineralization with EDTA
• Issues to Consider Effects of Acid
• Slide Number 32
• Slide Number 33
• Analysis of Collagen Structure in Bone
• Analysis of Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril D-Spacing
• Slide Number 39
• Collagen in Bone Fibril D-Spacing
• Collagen in Bone Fibril D-Spacing
• 2D Fast Fourier Transform Analysis
• 2D Fast Fourier Transform Analysis
• D-Spacing Distribution
• D-Spacing Distribution
• Cumulative Distribution Function
• CDF vs Histogram
• Changes in D-Spacing with Disease
• Changes in D-Spacing with Disease
• Slide Number 50
• Issues to Consider Site Selection and Sampling
• Issues to Consider Site Selection and Sampling
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• AFM calibration ndash Take Away Message
• AFM Applications in Bone ResearchIndentation Mechanics
• AFM-Based Indentation
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Parameters
• Hertz Contact Equation
• Hertz Contact Equation
• Hertz Contact Equation
• Proper Indentation Model is Essential
• Indentation in OI Tendon Study
• OI Tendon Differential Effects of Dehydration
• OI Tendon Differential Effects of Dehydration
• Indentation in Diabetic Tendon (Poster 0495)
• Multiscale Diabetes Study (Poster 0495)
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• A Note on Peak Force QNM for Bruker AFMs
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• Slide Number 83
• Slide Number 84
• AFM Applications in Bone ResearchOther Cool Mechanical Techniques
• ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo
• Other Mechanical Techniques
• Other Cool Techniques
• Summary Things to rememberhellip
• Acknowledgements
• Bone Biology and Mechanics Lab (BBML)

Analysis of Collagen in Bone Fibril Diameter Fibril Diameter easiest to measure property first attempts were a bit crude better with increased resolution

Sasaki J Mat Sci Tech 2002 Thalhammer J Arch Sci 2001

Bozec Ultramicroscopy 2005

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Collagen in Bone Fibril Diameter

Caution must be taken when reporting and interpreting diameters measured from fibrils in bone bull Fibril packing - width may not be fully exposed

Wallace et al Langmuir 2010

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Collagen in Bone Fibril Diameter

Caution must be taken when reporting and interpreting diameters measured from fibrils in bone bull Fibril packing - width may not be fully exposed

Rigozzi ndash J Struct Bio - 2011

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d

Collagen in Bone Fibril D-Spacing

300 nm 15 nm

N C C N C C N C C N C C

H O H2C CH2 O H

CH2

O OH HHC CH2

CH2

HO

H H

HHN C C

O

H2C CH2

CH2

HN C C

OH

HC CH2

HO

N C C N C C N C C

H O H2C CH2 O

CH2

OH HC CH2

HO

H H

H

CH2 CH2

glycine X - proline Y - hydroxyproline

Hodge and Petruska 1963

Collagen Fibril Axial D-Periodicity BBML - All R

ights Reserve

d

05

101520

0 02 04 06 08 1

Hei

ght (

nm)

Length (microns)

httpremfdartmoutheduimagesindexhtml

Randall Nature and Structure of Collagen 1953

TEM SEM

AFM

D-Spacing = 67 nm

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d

Collagen in Bone Fibril D-Spacing Many studies have shown observable D-spacing content that near theoretical value

Sasaki 2002 67 plusmn 2 nm Bozek 2005 665 plusmn 14 nm Ge 2007 666 plusmn 38 nm

Habelitz J Structural Bio 2002

A 2002 study in dentin got me interestedhellip

n = 322 wet n = 363 dry

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Collagen in Bone Fibril D-Spacing

bull Internal structure bull Wallace et al Bone 2010 46 1349-1354 bull Wallace et al J Stuct Bio 2011 173 146-152 bull Kemp et al J Stuct Bio 2012 180 428-438 bull Warden et al Endocrinology 2013 154(9)

3178-3187 bull Gallant et al Bone 2014 61 191-200 bull Bart et al Conn Tiss Res 2014 In Press

bull Enzymatic and non-

enzymatic cross-linking bull Hammond et al Bone 2014 60 26-32 bull Diaz Gonzalez et al J Biomech 2014 47(3)

681-686 bull Voytik-Harbin et al In Preparation 2014

D-periodic spacing important structural feature that captures aspects related to

Mouse Femur Mid-diaphysis BBML - All R

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d

2D Fast Fourier Transform Analysis

Harmonics

Fibril D-Spacing

Erickson B Wallace JM et al Biotechnology Journal 2013

Raw AFM Image 2D FFT Power Spectrum

BBML - All R

ights Reserve

d

2D Fast Fourier Transform Analysis

Harmonics

Fibril D-Spacing

Raw AFM Image 2D FFT Power Spectrum

0

20000

40000

60000

80000

100000

120000

0 001 002 003 004 005 006

Am

plitu

de (

microV2 )

Position (1nm)

D-periodic spacing 0015 (1nm) ~ 67 nm

BBML - All R

ights Reserve

d

D-Spacing Distribution

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Overall Mean 670 plusmn 00 nm

BBML - All R

ights Reserve

d

D-Spacing Distribution

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Overall Mean 670 plusmn 00 nm

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

4 Samples Total of n = 255 fibrils Overall Mean 682 plusmn 12 nm

BBML - All R

ights Reserve

d

Cumulative Distribution Function

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

This population 255 fibrils - Each fibril 1255 = 0392 - Data range 628-716 nm

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ights Reserve

d

CDF vs Histogram 62 63 64 65 66 67 68 69 70 71 72 73 74

0

10

20

30

40

50

60

70

80

90

100

0

5

10

15

20

25

30

35

40

45

50

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

HistoCDF

BBML - All R

ights Reserve

d

Changes in D-Spacing with Disease

0

5

10

15

20

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

0

5

10

15

20

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

Wallace et al Bone 2010 46 1349-1354

KS Test plt0001

Control 680 plusmn 26 nm

Estrogen Dep 659 plusmn 31 nm

t-test on mean p=0048

BBML - All R

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Changes in D-Spacing with Disease

Wallace et al Bone 2010 46 1349-1354

0

10

20

30

40

50

60

70

80

90

100

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

KS Test plt0001

BBML - All R

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d

Question Model and Tissue Morphology Publication

Tissue Specificity in Mice Male mouse 8 weeks Femur dentin tail tendon

No differences in bone vs dentin both ∆ vs tendon

Wallace et al Langmuir 2011 26(10) 7349-7354

Gender Effects Male and female mice 16 weeks Femur No differences NA

Estrogen Depletion OVX in sheep 5 years + 2 Radius Mean and population shift Wallace et al Bone

2010 46 1349-1354

Estrogen Depletion OVX in female 5 years + 2 Skin (dermis) Significant population shift Erickson et al Biotech

J 2013 8(1) 1174-126

Osteoporosis and Mechanical Loading

OVX in ulnar loading in rats Ulna

No loading effect population shift with OVX

Warden et al Endocrinology 2013 154(9) 3178-3187

Osteogenesis Imperfecta Brtl+ model male mice 2 months Femur

No mean ∆ widening of distribution

Wallace et al J Stuct Bio 2011 173 146-152

Osteogenesis Imperfecta Oimoim model male mice 12 weeks Femur Mean and population shift Bart et al Conn Tiss

Res 2014 In Press

Osteogenesis Imperfecta and Tissue Hydration

Brtl+ model male mice 6 months Tail Tendon Significant population shift Kemp et al J Stuct Bio

2012 180 428-438

Type 2 Diabetes Male ZDSD rats 30 weeks Tibia Significant population shift Hammond et al Bone

2014 60 26-32

Type 2 Diabetes Female ZDSD rats 32 weeks Tail Tendon Significant population shift

Diaz Gonzalez et al J Biomech 2014 47(3)

681-686

Raloxifene (in vivo and in vitro) Female dog treated with Raloxifene Femur Significant population shift Gallant et al Bone

2014 61 191-200 BBML - All R

ights Reserve

d

Issues to Consider Site Selection and Sampling

KS Test plt0001

0023 of bonersquos length

15 m

m =

150

00 micro

m

Wallace et al Langmuir 2011 26(10) 7349-7354

100 microm x 100 microm

35 microm x 35 microm

BBML - All R

ights Reserve

d

Issues to Consider Site Selection and Sampling

60

62

64

66

68

70

72

74

1 (35)

2 (33)

3 (40)

4 (39)

5 (35)

6 (29)

7 (26)

8 (23)

9 (28)

WT (288)

D-P

erio

dic

Gap

Ove

rlap

Spac

ing

(nm

)

Axial Location (Proximal End = 1 Distal End = 9)

( of Fibrils)

91

23

45

87

6

Wallace et al Langmuir 2011 26(10) 7349-7354

288 fibrils for population n=1 for mean comparisons BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

Wet Tendon Dry Tendon Kemp et al J StructBio 2012 180 (3) 428-438

Collagen and bone exist in a hydrated environment in vivo bull Hydration state can impact morphology and mechanics

BBML - All R

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d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

Nonsignificant mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet (n=255) 682 plusmn 12 nm

WT Dry (n=257) 686 plusmn 08 nm

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

Brtl DryBrtl Wet

KS Test plt0001

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

WT Case 04 nm mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet 682 plusmn 12 nm

WT Dry 686 plusmn 08 nm

Brtl+ Wet 675 plusmn 14 nm

Brtl+ Dry 689 plusmn 10 nm

Brtl+ Case 14 nm increase with drying (plt0001) bull Entire population shifted upward (plt0001)

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT WetBrtl Wet

KS Test plt0001

Brtl+ distribution shifted downward vs WT when wet

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryBrtl Dry

KS Test plt0001

Brtl+ distribution shifted upward vs WT when dry

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider AFM calibration

bull Absolute distance measurements require accurate piezo calibration

bull AFM systems have large scan ranges (150 microm to lt 1 microm)

bull Scanner non-linearities can introduce substantial error over that range

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

ights Reserve

d

Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

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ights Reserve

d

Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

100 nm 10 80 602 1198 100 nm 7 60 827 1439 100 nm 5 40 814 1427 100 nm 35 20 917 1724 100 nm 2 10 937 1774

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

ights Reserve

d

AFM calibration ndash Take Away Message

Calibration using a scan size and feature size comparable to features of interest is highly recommended

Calibration addresses absolute accuracy not precision (differential sensitivity between measurements)

bull Does not limit ability to differentiate between groups using the same AFM with the same calibration parameters

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d

AFM Applications in Bone Research Indentation Mechanics

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ights Reserve

d

AFM-Based Indentation

In addition to high resolution imaging AFM can be used to extract nanoscale mechanical data

ApproachWithdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

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ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Can

tilev

er D

efle

ctio

n (V

olts

)

1 Ramp into a rigid sample ndash measure the slope

Deflection sensitivity x 119899119899119899119899119881119881

2 Calculate cantilever spring constant

3 Hookersquos law takes deflection (volts) and converts to units of force F = kx

Spring constant k 119873119873119899119899

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ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Forc

e (n

N)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration Fo

rce

(nN

)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

Tip-Sample Separation (nm)

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ights Reserve

d

AFM-Based Indentation Parameters

Approach Withdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

Peak Force

Area Between Curves Energy Dissipation

Contact Mechanics Fit for Elastic Modulus (E)

Adhesion Force

Indentation Depth (δ)

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ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

z

r

δ

R

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119877119877 ∙ 12057512057532

Es Sample modulus νs Poissonrsquos ratio R tip radius of curvature δ load point displacement

Relevant Hertz Assumptions bull Strains are small and within the elastic limit bull Indenter is infinitely stiff (only sample deforms) bull Indentation depth ltlt radius of curvature of indenter bull Surfaces are continuous non-conforming (must be flat) bull Load is normal to surface

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d

Proper Indentation Model is Essential

δ

r

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119903119903 ∙ 12057512057532

δ

α

119865119865 =2120587120587∙

1198641198641199041199041 minus 1205841205841199041199042

∙ tan120572120572 ∙ 1205751205752

Es Sample modulus νs Poissonrsquos ratio R tip radius α opening angle

Hertz Model Spherical Indenter

depth ltlt radius of curvature

Sneddon Model Conical Indenter

depth ge radius of curvature

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ights Reserve

d

Indentation in OI Tendon Study

x

z

yBrtl+ and WT mice 6 month old males

4 tails per group

2-3 fascicles per tail

25-30 fibrils per tail

4-5 locations per fibril

Indent force 20 nN wet (Sneddon) 50 nN dry (Hertz) BBML - A

ll Rights R

eserved

OI Tendon Differential Effects of Dehydration

Dry 1268 plusmn 718 MPa

Wet 305 plusmn 243 MPa

Dry 1737 plusmn 973 MPa

Wet 301 plusmn 202 MPa

Kemp et al J StructBio 2012 180 (3) 428-438

bull Significant stiffening of fibrils with dehydration

bull More pronounced modulus modulus as a function of disease

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d

OI Tendon Differential Effects of Dehydration

Kemp et al J StructBio 2012 180 (3) 428-438

Diseased phenotype not present when wet phenotype observed when dried

0

10

20

30

40

50

60

0 20 40 60 80 100 120 140 160 180 200

Gro

up S

ampl

es (

)

Indent Modulus (MPa)

WT WetBrtl Wet

0

2

4

6

8

10

12

14

16

18

20

025

050

075

010

0012

5015

0017

5020

0022

5025

0027

5030

0032

5035

0037

5040

0042

5045

0047

5050

00

Gro

up S

ampl

es (

)Indent Modulus (MPa)

WT DryBrtl Dry

555 WT Indents 305 plusmn 243 MPa

614 Brtl+ Indents 301 plusmn 202 MPa

KS Test p=0129 t Test p=0464 464 WT Indents

1268 plusmn 718 MPa 479 Brtl+ Indents 1737 plusmn 973 MPa

KS Test plt0001 T Test plt0001

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d

Indentation in Diabetic Tendon (Poster 0495)

x

z

y

Control and Diabetic Rat Tail Tendon

4-5 rats per group

2-3 fascicles per rat

~70 fibrilstail

4-5 locationsfibril

20 nN (Sneddon)

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d

Multiscale Diabetes Study (Poster 0495)

0

5

10

15

20

25

300 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 gt25

Perc

enta

ge o

f Gro

up S

ampl

es

Elastic Modulus (MPa)

CD (1530 indents)ZDSD (913 indents)

CD (n=5) 381 plusmn 149 MPa

ZDSD (n=3) 706 plusmn 263 MPa

0

100

200

300

400

500

600

700

800

900

0 2 4 6 8 10

Mic

rosc

ale

Mod

ulus

(MPa

)

Nanoscale Modulus (MPa)

plt0001

r2=0531 p=0040

bull Increased indentation modulus in diabetic tendon

bull Strong relationship between nanoscale indent modulus and microscale tensile modulus

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d

Indentation Limitations and Weaknesses

Proper probe selection for intended application bull Cantilever must be stiff relative to sample bull Sharp probes may damage sample easily dull

R or α must be determined for each probe bull Properties provided by manufacturer are unreliable bull Vary from probe to probe can dull during indent process

Spring constant must be determined for each probe

bull Value provided by manufacturer is inaccurate bull Thermal tuning OK for ldquosoftrdquo probes poor performance

for stiffer probes bull Sader method (geometry) added mass reference

cantilevers

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d

Indentation Limitations and Weaknesses

Veeco Application Technical note Ben Ohler Practical Advice on the Determination of Cantilever Spring Constants

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Indentation Limitations and Weaknesses

bull Sample processing

bull Sample hydration

bull Proper force range

bull Substrate effects for thin samples

bull Proper modulus model and range of data goodness of fit

Bearing in mind these assumptions and limitations AFM can provide true ldquonanoscalerdquo mechanical properties both in terms of forces and deformations

BBML - All R

ights Reserve

d

A Note on Peak Force QNM for Bruker AFMs bull Mechanical properties mapped pixel-by-pixel at same

resolution as the height image (PeakForce QNM)

Height Modulus BBML - All R

ights Reserve

d

A Note on Peak Force QNM

Correct probe stiffness must be used based on sample Pittenger ndash Veeco Product Bulletin

BBML - All R

ights Reserve

d

A Note on Peak Force QNM Extremely sensitive to calibration (details rarely given) Absolute radius and spring constant known

bull especially important from probe to probe bull radius uarr with indent depth ndash must determine R at depth

you expect to image bull Depth may vary as a function of sample (or at different

regions within the sample)

Relative reference material of ldquoknownrdquo modulus used to determine a ratio of 119896119896 119877119877 bull ldquoknownrdquo modulus may come from tension compression

and not indentation bull R is related to depth indentation depth on sample must

be same as on reference BBML - All R

ights Reserve

d

A Note on Peak Force QNM bull Modulus fit on the fly ndash no ability to monitor fit (or range) bull Local sample topography and sample tilt play a role in

measured parameters bull Assuming all modulus values in an image are correct how to

quantify

512 x 512 pixels = 262144 measurements

n=1 n=262144

n from selected ROIs

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d

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d

bull Imaging in air on mica ndash substrate effect

bull Same imaging force or indent depth for all

bull For each area were all modulus values pooled

Lamprou et al PLoS ONE 2013 BBML - All R

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d

AFM Applications in Bone Research Other Cool Mechanical Techniques

BBML - All R

ights Reserve

d

ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo Bonds within or between collagen fibrils in bone that rupture to dissipate energy as a toughening mechanism

Thompson et al Nature 2001

Fantner et al Nature Mat 2005

Hansma et al JMNI 2005

Fantner et al Nano Letters 2007 BBML - All R

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d

Other Mechanical Techniques

Jimenez-Palomar J Mech Beh Bio Mat 2012

van der Rijt Macro Bio 2004

Yang J Biomed Mater Res 2007

Yang Biophysical Journal 2008 BBML - All R

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d

Other Cool Techniques bull Piezoresponse Force Microscopy (PFM)

bull Electrochemical AFM (EC-AFM)

bull Force Modulation Microscopy (FMM)

bull Lateral Force Microscopy (LFM)

bull Magnetic Force Microscopy (MFM)

bull Conductive AFM (C-AFM)

bull Force Pulling

bull Cantilever Bending

bull Tensile testing of individual fibrils

bull 3 point bending of fibrils

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Summary Things to rememberhellip

bull Imaging mode imaging medium and probe selection

bull Morphology Measurements bull Sample preparation and hydration state bull Measuring fibril diameter bull Site selection and proper sampling bull Appropriate system calibration

bull Mechanical Measurements

bull Probe selection (stiffness and tip radius) bull Probe calibration (radius angle spring constant) bull Indentation force range and mechanical model bull Hydration in biological samples bull PeakForce QNM

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Acknowledgements BBML

Max Hammond Armando Diaz Gonzalez Zachary Bart Silvia Canelon Creasy Clauser

IU School of Medicine Dr David Burr Dr Matt Allen Dr Stuart Warden Dr Lilian Plotkin

Funding Sources NIHNIDCR 1F32DE018840-01 A1 IUPUI BME Departmental Startup Funds IUPUI Research Support Funds Grant IUPUI Biomechanics and Biomaterials Research Center

NIH NICHD Dr Joan Marini

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d

Bone Biology and Mechanics Lab (BBML) wwwiupuiedu~bbml Facebook BoneBiologyMechanicsLab

BBML - All R

ights Reserve

d

• Slide Number 1
• General Seminar Outline
• Bonersquos Hierarchical Structure
• Critical Gap in Understanding Collagen
• Methods For Analyzing Collagen Structure
• Methods For Imaging Collagen Structure
• Cryo-TEM
• Atomic Force Microscopy (AFM)
• AFM Image of Pentacene (C22H14)
• AFM Image of Hexabenzocoronene
• Basics of AFM Imaging The Probe
• Basics of AFM Imaging System Components
• van der Waals Potential Energy Curve
• Contact Mode ndash Monitor Deflection
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Peak Force Tapping Mode ndash Monitor Force
• AFM Applications in Bone Research Imaging
• AFM Imaging of Collagen
• Imaging of Osteoclast Activity
• Mineralized collagen deposition in vitro
• Imaging of Canilicular Structure
• AFM Imaging in Bone
• AFM Imaging in Bone
• Removing Mineral in Bone to Expose Collagen
• Removing Mineral in Bone to Expose Collagen
• Demineralization with EDTA
• Demineralization with EDTA
• Demineralization with EDTA
• Issues to Consider Effects of Acid
• Slide Number 32
• Slide Number 33
• Analysis of Collagen Structure in Bone
• Analysis of Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril D-Spacing
• Slide Number 39
• Collagen in Bone Fibril D-Spacing
• Collagen in Bone Fibril D-Spacing
• 2D Fast Fourier Transform Analysis
• 2D Fast Fourier Transform Analysis
• D-Spacing Distribution
• D-Spacing Distribution
• Cumulative Distribution Function
• CDF vs Histogram
• Changes in D-Spacing with Disease
• Changes in D-Spacing with Disease
• Slide Number 50
• Issues to Consider Site Selection and Sampling
• Issues to Consider Site Selection and Sampling
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• AFM calibration ndash Take Away Message
• AFM Applications in Bone ResearchIndentation Mechanics
• AFM-Based Indentation
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Parameters
• Hertz Contact Equation
• Hertz Contact Equation
• Hertz Contact Equation
• Proper Indentation Model is Essential
• Indentation in OI Tendon Study
• OI Tendon Differential Effects of Dehydration
• OI Tendon Differential Effects of Dehydration
• Indentation in Diabetic Tendon (Poster 0495)
• Multiscale Diabetes Study (Poster 0495)
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• A Note on Peak Force QNM for Bruker AFMs
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• Slide Number 83
• Slide Number 84
• AFM Applications in Bone ResearchOther Cool Mechanical Techniques
• ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo
• Other Mechanical Techniques
• Other Cool Techniques
• Summary Things to rememberhellip
• Acknowledgements
• Bone Biology and Mechanics Lab (BBML)

Collagen in Bone Fibril Diameter

Caution must be taken when reporting and interpreting diameters measured from fibrils in bone bull Fibril packing - width may not be fully exposed

Wallace et al Langmuir 2010

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Collagen in Bone Fibril Diameter

Caution must be taken when reporting and interpreting diameters measured from fibrils in bone bull Fibril packing - width may not be fully exposed

Rigozzi ndash J Struct Bio - 2011

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d

Collagen in Bone Fibril D-Spacing

300 nm 15 nm

N C C N C C N C C N C C

H O H2C CH2 O H

CH2

O OH HHC CH2

CH2

HO

H H

HHN C C

O

H2C CH2

CH2

HN C C

OH

HC CH2

HO

N C C N C C N C C

H O H2C CH2 O

CH2

OH HC CH2

HO

H H

H

CH2 CH2

glycine X - proline Y - hydroxyproline

Hodge and Petruska 1963

Collagen Fibril Axial D-Periodicity BBML - All R

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d

05

101520

0 02 04 06 08 1

Hei

ght (

nm)

Length (microns)

httpremfdartmoutheduimagesindexhtml

Randall Nature and Structure of Collagen 1953

TEM SEM

AFM

D-Spacing = 67 nm

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d

Collagen in Bone Fibril D-Spacing Many studies have shown observable D-spacing content that near theoretical value

Sasaki 2002 67 plusmn 2 nm Bozek 2005 665 plusmn 14 nm Ge 2007 666 plusmn 38 nm

Habelitz J Structural Bio 2002

A 2002 study in dentin got me interestedhellip

n = 322 wet n = 363 dry

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Collagen in Bone Fibril D-Spacing

bull Internal structure bull Wallace et al Bone 2010 46 1349-1354 bull Wallace et al J Stuct Bio 2011 173 146-152 bull Kemp et al J Stuct Bio 2012 180 428-438 bull Warden et al Endocrinology 2013 154(9)

3178-3187 bull Gallant et al Bone 2014 61 191-200 bull Bart et al Conn Tiss Res 2014 In Press

bull Enzymatic and non-

enzymatic cross-linking bull Hammond et al Bone 2014 60 26-32 bull Diaz Gonzalez et al J Biomech 2014 47(3)

681-686 bull Voytik-Harbin et al In Preparation 2014

D-periodic spacing important structural feature that captures aspects related to

Mouse Femur Mid-diaphysis BBML - All R

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d

2D Fast Fourier Transform Analysis

Harmonics

Fibril D-Spacing

Erickson B Wallace JM et al Biotechnology Journal 2013

Raw AFM Image 2D FFT Power Spectrum

BBML - All R

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d

2D Fast Fourier Transform Analysis

Harmonics

Fibril D-Spacing

Raw AFM Image 2D FFT Power Spectrum

0

20000

40000

60000

80000

100000

120000

0 001 002 003 004 005 006

Am

plitu

de (

microV2 )

Position (1nm)

D-periodic spacing 0015 (1nm) ~ 67 nm

BBML - All R

ights Reserve

d

D-Spacing Distribution

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Overall Mean 670 plusmn 00 nm

BBML - All R

ights Reserve

d

D-Spacing Distribution

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Overall Mean 670 plusmn 00 nm

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

4 Samples Total of n = 255 fibrils Overall Mean 682 plusmn 12 nm

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ights Reserve

d

Cumulative Distribution Function

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

This population 255 fibrils - Each fibril 1255 = 0392 - Data range 628-716 nm

BBML - All R

ights Reserve

d

CDF vs Histogram 62 63 64 65 66 67 68 69 70 71 72 73 74

0

10

20

30

40

50

60

70

80

90

100

0

5

10

15

20

25

30

35

40

45

50

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

HistoCDF

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ights Reserve

d

Changes in D-Spacing with Disease

0

5

10

15

20

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

0

5

10

15

20

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

Wallace et al Bone 2010 46 1349-1354

KS Test plt0001

Control 680 plusmn 26 nm

Estrogen Dep 659 plusmn 31 nm

t-test on mean p=0048

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Changes in D-Spacing with Disease

Wallace et al Bone 2010 46 1349-1354

0

10

20

30

40

50

60

70

80

90

100

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

KS Test plt0001

BBML - All R

ights Reserve

d

Question Model and Tissue Morphology Publication

Tissue Specificity in Mice Male mouse 8 weeks Femur dentin tail tendon

No differences in bone vs dentin both ∆ vs tendon

Wallace et al Langmuir 2011 26(10) 7349-7354

Gender Effects Male and female mice 16 weeks Femur No differences NA

Estrogen Depletion OVX in sheep 5 years + 2 Radius Mean and population shift Wallace et al Bone

2010 46 1349-1354

Estrogen Depletion OVX in female 5 years + 2 Skin (dermis) Significant population shift Erickson et al Biotech

J 2013 8(1) 1174-126

Osteoporosis and Mechanical Loading

OVX in ulnar loading in rats Ulna

No loading effect population shift with OVX

Warden et al Endocrinology 2013 154(9) 3178-3187

Osteogenesis Imperfecta Brtl+ model male mice 2 months Femur

No mean ∆ widening of distribution

Wallace et al J Stuct Bio 2011 173 146-152

Osteogenesis Imperfecta Oimoim model male mice 12 weeks Femur Mean and population shift Bart et al Conn Tiss

Res 2014 In Press

Osteogenesis Imperfecta and Tissue Hydration

Brtl+ model male mice 6 months Tail Tendon Significant population shift Kemp et al J Stuct Bio

2012 180 428-438

Type 2 Diabetes Male ZDSD rats 30 weeks Tibia Significant population shift Hammond et al Bone

2014 60 26-32

Type 2 Diabetes Female ZDSD rats 32 weeks Tail Tendon Significant population shift

Diaz Gonzalez et al J Biomech 2014 47(3)

681-686

Raloxifene (in vivo and in vitro) Female dog treated with Raloxifene Femur Significant population shift Gallant et al Bone

2014 61 191-200 BBML - All R

ights Reserve

d

Issues to Consider Site Selection and Sampling

KS Test plt0001

0023 of bonersquos length

15 m

m =

150

00 micro

m

Wallace et al Langmuir 2011 26(10) 7349-7354

100 microm x 100 microm

35 microm x 35 microm

BBML - All R

ights Reserve

d

Issues to Consider Site Selection and Sampling

60

62

64

66

68

70

72

74

1 (35)

2 (33)

3 (40)

4 (39)

5 (35)

6 (29)

7 (26)

8 (23)

9 (28)

WT (288)

D-P

erio

dic

Gap

Ove

rlap

Spac

ing

(nm

)

Axial Location (Proximal End = 1 Distal End = 9)

( of Fibrils)

91

23

45

87

6

Wallace et al Langmuir 2011 26(10) 7349-7354

288 fibrils for population n=1 for mean comparisons BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

Wet Tendon Dry Tendon Kemp et al J StructBio 2012 180 (3) 428-438

Collagen and bone exist in a hydrated environment in vivo bull Hydration state can impact morphology and mechanics

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ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

Nonsignificant mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet (n=255) 682 plusmn 12 nm

WT Dry (n=257) 686 plusmn 08 nm

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

Brtl DryBrtl Wet

KS Test plt0001

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

WT Case 04 nm mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet 682 plusmn 12 nm

WT Dry 686 plusmn 08 nm

Brtl+ Wet 675 plusmn 14 nm

Brtl+ Dry 689 plusmn 10 nm

Brtl+ Case 14 nm increase with drying (plt0001) bull Entire population shifted upward (plt0001)

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT WetBrtl Wet

KS Test plt0001

Brtl+ distribution shifted downward vs WT when wet

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryBrtl Dry

KS Test plt0001

Brtl+ distribution shifted upward vs WT when dry

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider AFM calibration

bull Absolute distance measurements require accurate piezo calibration

bull AFM systems have large scan ranges (150 microm to lt 1 microm)

bull Scanner non-linearities can introduce substantial error over that range

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

ights Reserve

d

Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

BBML - All R

ights Reserve

d

Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

100 nm 10 80 602 1198 100 nm 7 60 827 1439 100 nm 5 40 814 1427 100 nm 35 20 917 1724 100 nm 2 10 937 1774

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

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d

AFM calibration ndash Take Away Message

Calibration using a scan size and feature size comparable to features of interest is highly recommended

Calibration addresses absolute accuracy not precision (differential sensitivity between measurements)

bull Does not limit ability to differentiate between groups using the same AFM with the same calibration parameters

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d

AFM Applications in Bone Research Indentation Mechanics

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ights Reserve

d

AFM-Based Indentation

In addition to high resolution imaging AFM can be used to extract nanoscale mechanical data

ApproachWithdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Can

tilev

er D

efle

ctio

n (V

olts

)

1 Ramp into a rigid sample ndash measure the slope

Deflection sensitivity x 119899119899119899119899119881119881

2 Calculate cantilever spring constant

3 Hookersquos law takes deflection (volts) and converts to units of force F = kx

Spring constant k 119873119873119899119899

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Forc

e (n

N)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration Fo

rce

(nN

)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

Tip-Sample Separation (nm)

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ights Reserve

d

AFM-Based Indentation Parameters

Approach Withdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

Peak Force

Area Between Curves Energy Dissipation

Contact Mechanics Fit for Elastic Modulus (E)

Adhesion Force

Indentation Depth (δ)

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

z

r

δ

R

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119877119877 ∙ 12057512057532

Es Sample modulus νs Poissonrsquos ratio R tip radius of curvature δ load point displacement

Relevant Hertz Assumptions bull Strains are small and within the elastic limit bull Indenter is infinitely stiff (only sample deforms) bull Indentation depth ltlt radius of curvature of indenter bull Surfaces are continuous non-conforming (must be flat) bull Load is normal to surface

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ights Reserve

d

Proper Indentation Model is Essential

δ

r

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119903119903 ∙ 12057512057532

δ

α

119865119865 =2120587120587∙

1198641198641199041199041 minus 1205841205841199041199042

∙ tan120572120572 ∙ 1205751205752

Es Sample modulus νs Poissonrsquos ratio R tip radius α opening angle

Hertz Model Spherical Indenter

depth ltlt radius of curvature

Sneddon Model Conical Indenter

depth ge radius of curvature

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ights Reserve

d

Indentation in OI Tendon Study

x

z

yBrtl+ and WT mice 6 month old males

4 tails per group

2-3 fascicles per tail

25-30 fibrils per tail

4-5 locations per fibril

Indent force 20 nN wet (Sneddon) 50 nN dry (Hertz) BBML - A

ll Rights R

eserved

OI Tendon Differential Effects of Dehydration

Dry 1268 plusmn 718 MPa

Wet 305 plusmn 243 MPa

Dry 1737 plusmn 973 MPa

Wet 301 plusmn 202 MPa

Kemp et al J StructBio 2012 180 (3) 428-438

bull Significant stiffening of fibrils with dehydration

bull More pronounced modulus modulus as a function of disease

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ights Reserve

d

OI Tendon Differential Effects of Dehydration

Kemp et al J StructBio 2012 180 (3) 428-438

Diseased phenotype not present when wet phenotype observed when dried

0

10

20

30

40

50

60

0 20 40 60 80 100 120 140 160 180 200

Gro

up S

ampl

es (

)

Indent Modulus (MPa)

WT WetBrtl Wet

0

2

4

6

8

10

12

14

16

18

20

025

050

075

010

0012

5015

0017

5020

0022

5025

0027

5030

0032

5035

0037

5040

0042

5045

0047

5050

00

Gro

up S

ampl

es (

)Indent Modulus (MPa)

WT DryBrtl Dry

555 WT Indents 305 plusmn 243 MPa

614 Brtl+ Indents 301 plusmn 202 MPa

KS Test p=0129 t Test p=0464 464 WT Indents

1268 plusmn 718 MPa 479 Brtl+ Indents 1737 plusmn 973 MPa

KS Test plt0001 T Test plt0001

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ights Reserve

d

Indentation in Diabetic Tendon (Poster 0495)

x

z

y

Control and Diabetic Rat Tail Tendon

4-5 rats per group

2-3 fascicles per rat

~70 fibrilstail

4-5 locationsfibril

20 nN (Sneddon)

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ights Reserve

d

Multiscale Diabetes Study (Poster 0495)

0

5

10

15

20

25

300 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 gt25

Perc

enta

ge o

f Gro

up S

ampl

es

Elastic Modulus (MPa)

CD (1530 indents)ZDSD (913 indents)

CD (n=5) 381 plusmn 149 MPa

ZDSD (n=3) 706 plusmn 263 MPa

0

100

200

300

400

500

600

700

800

900

0 2 4 6 8 10

Mic

rosc

ale

Mod

ulus

(MPa

)

Nanoscale Modulus (MPa)

plt0001

r2=0531 p=0040

bull Increased indentation modulus in diabetic tendon

bull Strong relationship between nanoscale indent modulus and microscale tensile modulus

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ights Reserve

d

Indentation Limitations and Weaknesses

Proper probe selection for intended application bull Cantilever must be stiff relative to sample bull Sharp probes may damage sample easily dull

R or α must be determined for each probe bull Properties provided by manufacturer are unreliable bull Vary from probe to probe can dull during indent process

Spring constant must be determined for each probe

bull Value provided by manufacturer is inaccurate bull Thermal tuning OK for ldquosoftrdquo probes poor performance

for stiffer probes bull Sader method (geometry) added mass reference

cantilevers

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d

Indentation Limitations and Weaknesses

Veeco Application Technical note Ben Ohler Practical Advice on the Determination of Cantilever Spring Constants

BBML - All R

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d

Indentation Limitations and Weaknesses

bull Sample processing

bull Sample hydration

bull Proper force range

bull Substrate effects for thin samples

bull Proper modulus model and range of data goodness of fit

Bearing in mind these assumptions and limitations AFM can provide true ldquonanoscalerdquo mechanical properties both in terms of forces and deformations

BBML - All R

ights Reserve

d

A Note on Peak Force QNM for Bruker AFMs bull Mechanical properties mapped pixel-by-pixel at same

resolution as the height image (PeakForce QNM)

Height Modulus BBML - All R

ights Reserve

d

A Note on Peak Force QNM

Correct probe stiffness must be used based on sample Pittenger ndash Veeco Product Bulletin

BBML - All R

ights Reserve

d

A Note on Peak Force QNM Extremely sensitive to calibration (details rarely given) Absolute radius and spring constant known

bull especially important from probe to probe bull radius uarr with indent depth ndash must determine R at depth

you expect to image bull Depth may vary as a function of sample (or at different

regions within the sample)

Relative reference material of ldquoknownrdquo modulus used to determine a ratio of 119896119896 119877119877 bull ldquoknownrdquo modulus may come from tension compression

and not indentation bull R is related to depth indentation depth on sample must

be same as on reference BBML - All R

ights Reserve

d

A Note on Peak Force QNM bull Modulus fit on the fly ndash no ability to monitor fit (or range) bull Local sample topography and sample tilt play a role in

measured parameters bull Assuming all modulus values in an image are correct how to

quantify

512 x 512 pixels = 262144 measurements

n=1 n=262144

n from selected ROIs

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d

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d

bull Imaging in air on mica ndash substrate effect

bull Same imaging force or indent depth for all

bull For each area were all modulus values pooled

Lamprou et al PLoS ONE 2013 BBML - All R

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d

AFM Applications in Bone Research Other Cool Mechanical Techniques

BBML - All R

ights Reserve

d

ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo Bonds within or between collagen fibrils in bone that rupture to dissipate energy as a toughening mechanism

Thompson et al Nature 2001

Fantner et al Nature Mat 2005

Hansma et al JMNI 2005

Fantner et al Nano Letters 2007 BBML - All R

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d

Other Mechanical Techniques

Jimenez-Palomar J Mech Beh Bio Mat 2012

van der Rijt Macro Bio 2004

Yang J Biomed Mater Res 2007

Yang Biophysical Journal 2008 BBML - All R

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d

Other Cool Techniques bull Piezoresponse Force Microscopy (PFM)

bull Electrochemical AFM (EC-AFM)

bull Force Modulation Microscopy (FMM)

bull Lateral Force Microscopy (LFM)

bull Magnetic Force Microscopy (MFM)

bull Conductive AFM (C-AFM)

bull Force Pulling

bull Cantilever Bending

bull Tensile testing of individual fibrils

bull 3 point bending of fibrils

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Summary Things to rememberhellip

bull Imaging mode imaging medium and probe selection

bull Morphology Measurements bull Sample preparation and hydration state bull Measuring fibril diameter bull Site selection and proper sampling bull Appropriate system calibration

bull Mechanical Measurements

bull Probe selection (stiffness and tip radius) bull Probe calibration (radius angle spring constant) bull Indentation force range and mechanical model bull Hydration in biological samples bull PeakForce QNM

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d

Acknowledgements BBML

Max Hammond Armando Diaz Gonzalez Zachary Bart Silvia Canelon Creasy Clauser

IU School of Medicine Dr David Burr Dr Matt Allen Dr Stuart Warden Dr Lilian Plotkin

Funding Sources NIHNIDCR 1F32DE018840-01 A1 IUPUI BME Departmental Startup Funds IUPUI Research Support Funds Grant IUPUI Biomechanics and Biomaterials Research Center

NIH NICHD Dr Joan Marini

BBML - All R

ights Reserve

d

Bone Biology and Mechanics Lab (BBML) wwwiupuiedu~bbml Facebook BoneBiologyMechanicsLab

BBML - All R

ights Reserve

d

• Slide Number 1
• General Seminar Outline
• Bonersquos Hierarchical Structure
• Critical Gap in Understanding Collagen
• Methods For Analyzing Collagen Structure
• Methods For Imaging Collagen Structure
• Cryo-TEM
• Atomic Force Microscopy (AFM)
• AFM Image of Pentacene (C22H14)
• AFM Image of Hexabenzocoronene
• Basics of AFM Imaging The Probe
• Basics of AFM Imaging System Components
• van der Waals Potential Energy Curve
• Contact Mode ndash Monitor Deflection
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Peak Force Tapping Mode ndash Monitor Force
• AFM Applications in Bone Research Imaging
• AFM Imaging of Collagen
• Imaging of Osteoclast Activity
• Mineralized collagen deposition in vitro
• Imaging of Canilicular Structure
• AFM Imaging in Bone
• AFM Imaging in Bone
• Removing Mineral in Bone to Expose Collagen
• Removing Mineral in Bone to Expose Collagen
• Demineralization with EDTA
• Demineralization with EDTA
• Demineralization with EDTA
• Issues to Consider Effects of Acid
• Slide Number 32
• Slide Number 33
• Analysis of Collagen Structure in Bone
• Analysis of Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril D-Spacing
• Slide Number 39
• Collagen in Bone Fibril D-Spacing
• Collagen in Bone Fibril D-Spacing
• 2D Fast Fourier Transform Analysis
• 2D Fast Fourier Transform Analysis
• D-Spacing Distribution
• D-Spacing Distribution
• Cumulative Distribution Function
• CDF vs Histogram
• Changes in D-Spacing with Disease
• Changes in D-Spacing with Disease
• Slide Number 50
• Issues to Consider Site Selection and Sampling
• Issues to Consider Site Selection and Sampling
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• AFM calibration ndash Take Away Message
• AFM Applications in Bone ResearchIndentation Mechanics
• AFM-Based Indentation
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Parameters
• Hertz Contact Equation
• Hertz Contact Equation
• Hertz Contact Equation
• Proper Indentation Model is Essential
• Indentation in OI Tendon Study
• OI Tendon Differential Effects of Dehydration
• OI Tendon Differential Effects of Dehydration
• Indentation in Diabetic Tendon (Poster 0495)
• Multiscale Diabetes Study (Poster 0495)
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• A Note on Peak Force QNM for Bruker AFMs
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• Slide Number 83
• Slide Number 84
• AFM Applications in Bone ResearchOther Cool Mechanical Techniques
• ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo
• Other Mechanical Techniques
• Other Cool Techniques
• Summary Things to rememberhellip
• Acknowledgements
• Bone Biology and Mechanics Lab (BBML)

Collagen in Bone Fibril Diameter

Caution must be taken when reporting and interpreting diameters measured from fibrils in bone bull Fibril packing - width may not be fully exposed

Rigozzi ndash J Struct Bio - 2011

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d

Collagen in Bone Fibril D-Spacing

300 nm 15 nm

N C C N C C N C C N C C

H O H2C CH2 O H

CH2

O OH HHC CH2

CH2

HO

H H

HHN C C

O

H2C CH2

CH2

HN C C

OH

HC CH2

HO

N C C N C C N C C

H O H2C CH2 O

CH2

OH HC CH2

HO

H H

H

CH2 CH2

glycine X - proline Y - hydroxyproline

Hodge and Petruska 1963

Collagen Fibril Axial D-Periodicity BBML - All R

ights Reserve

d

05

101520

0 02 04 06 08 1

Hei

ght (

nm)

Length (microns)

httpremfdartmoutheduimagesindexhtml

Randall Nature and Structure of Collagen 1953

TEM SEM

AFM

D-Spacing = 67 nm

BBML - All R

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d

Collagen in Bone Fibril D-Spacing Many studies have shown observable D-spacing content that near theoretical value

Sasaki 2002 67 plusmn 2 nm Bozek 2005 665 plusmn 14 nm Ge 2007 666 plusmn 38 nm

Habelitz J Structural Bio 2002

A 2002 study in dentin got me interestedhellip

n = 322 wet n = 363 dry

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d

Collagen in Bone Fibril D-Spacing

bull Internal structure bull Wallace et al Bone 2010 46 1349-1354 bull Wallace et al J Stuct Bio 2011 173 146-152 bull Kemp et al J Stuct Bio 2012 180 428-438 bull Warden et al Endocrinology 2013 154(9)

3178-3187 bull Gallant et al Bone 2014 61 191-200 bull Bart et al Conn Tiss Res 2014 In Press

bull Enzymatic and non-

enzymatic cross-linking bull Hammond et al Bone 2014 60 26-32 bull Diaz Gonzalez et al J Biomech 2014 47(3)

681-686 bull Voytik-Harbin et al In Preparation 2014

D-periodic spacing important structural feature that captures aspects related to

Mouse Femur Mid-diaphysis BBML - All R

ights Reserve

d

2D Fast Fourier Transform Analysis

Harmonics

Fibril D-Spacing

Erickson B Wallace JM et al Biotechnology Journal 2013

Raw AFM Image 2D FFT Power Spectrum

BBML - All R

ights Reserve

d

2D Fast Fourier Transform Analysis

Harmonics

Fibril D-Spacing

Raw AFM Image 2D FFT Power Spectrum

0

20000

40000

60000

80000

100000

120000

0 001 002 003 004 005 006

Am

plitu

de (

microV2 )

Position (1nm)

D-periodic spacing 0015 (1nm) ~ 67 nm

BBML - All R

ights Reserve

d

D-Spacing Distribution

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Overall Mean 670 plusmn 00 nm

BBML - All R

ights Reserve

d

D-Spacing Distribution

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Overall Mean 670 plusmn 00 nm

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

4 Samples Total of n = 255 fibrils Overall Mean 682 plusmn 12 nm

BBML - All R

ights Reserve

d

Cumulative Distribution Function

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

This population 255 fibrils - Each fibril 1255 = 0392 - Data range 628-716 nm

BBML - All R

ights Reserve

d

CDF vs Histogram 62 63 64 65 66 67 68 69 70 71 72 73 74

0

10

20

30

40

50

60

70

80

90

100

0

5

10

15

20

25

30

35

40

45

50

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

HistoCDF

BBML - All R

ights Reserve

d

Changes in D-Spacing with Disease

0

5

10

15

20

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

0

5

10

15

20

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

Wallace et al Bone 2010 46 1349-1354

KS Test plt0001

Control 680 plusmn 26 nm

Estrogen Dep 659 plusmn 31 nm

t-test on mean p=0048

BBML - All R

ights Reserve

d

Changes in D-Spacing with Disease

Wallace et al Bone 2010 46 1349-1354

0

10

20

30

40

50

60

70

80

90

100

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

KS Test plt0001

BBML - All R

ights Reserve

d

Question Model and Tissue Morphology Publication

Tissue Specificity in Mice Male mouse 8 weeks Femur dentin tail tendon

No differences in bone vs dentin both ∆ vs tendon

Wallace et al Langmuir 2011 26(10) 7349-7354

Gender Effects Male and female mice 16 weeks Femur No differences NA

Estrogen Depletion OVX in sheep 5 years + 2 Radius Mean and population shift Wallace et al Bone

2010 46 1349-1354

Estrogen Depletion OVX in female 5 years + 2 Skin (dermis) Significant population shift Erickson et al Biotech

J 2013 8(1) 1174-126

Osteoporosis and Mechanical Loading

OVX in ulnar loading in rats Ulna

No loading effect population shift with OVX

Warden et al Endocrinology 2013 154(9) 3178-3187

Osteogenesis Imperfecta Brtl+ model male mice 2 months Femur

No mean ∆ widening of distribution

Wallace et al J Stuct Bio 2011 173 146-152

Osteogenesis Imperfecta Oimoim model male mice 12 weeks Femur Mean and population shift Bart et al Conn Tiss

Res 2014 In Press

Osteogenesis Imperfecta and Tissue Hydration

Brtl+ model male mice 6 months Tail Tendon Significant population shift Kemp et al J Stuct Bio

2012 180 428-438

Type 2 Diabetes Male ZDSD rats 30 weeks Tibia Significant population shift Hammond et al Bone

2014 60 26-32

Type 2 Diabetes Female ZDSD rats 32 weeks Tail Tendon Significant population shift

Diaz Gonzalez et al J Biomech 2014 47(3)

681-686

Raloxifene (in vivo and in vitro) Female dog treated with Raloxifene Femur Significant population shift Gallant et al Bone

2014 61 191-200 BBML - All R

ights Reserve

d

Issues to Consider Site Selection and Sampling

KS Test plt0001

0023 of bonersquos length

15 m

m =

150

00 micro

m

Wallace et al Langmuir 2011 26(10) 7349-7354

100 microm x 100 microm

35 microm x 35 microm

BBML - All R

ights Reserve

d

Issues to Consider Site Selection and Sampling

60

62

64

66

68

70

72

74

1 (35)

2 (33)

3 (40)

4 (39)

5 (35)

6 (29)

7 (26)

8 (23)

9 (28)

WT (288)

D-P

erio

dic

Gap

Ove

rlap

Spac

ing

(nm

)

Axial Location (Proximal End = 1 Distal End = 9)

( of Fibrils)

91

23

45

87

6

Wallace et al Langmuir 2011 26(10) 7349-7354

288 fibrils for population n=1 for mean comparisons BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

Wet Tendon Dry Tendon Kemp et al J StructBio 2012 180 (3) 428-438

Collagen and bone exist in a hydrated environment in vivo bull Hydration state can impact morphology and mechanics

BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

Nonsignificant mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet (n=255) 682 plusmn 12 nm

WT Dry (n=257) 686 plusmn 08 nm

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

Brtl DryBrtl Wet

KS Test plt0001

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

WT Case 04 nm mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet 682 plusmn 12 nm

WT Dry 686 plusmn 08 nm

Brtl+ Wet 675 plusmn 14 nm

Brtl+ Dry 689 plusmn 10 nm

Brtl+ Case 14 nm increase with drying (plt0001) bull Entire population shifted upward (plt0001)

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT WetBrtl Wet

KS Test plt0001

Brtl+ distribution shifted downward vs WT when wet

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryBrtl Dry

KS Test plt0001

Brtl+ distribution shifted upward vs WT when dry

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider AFM calibration

bull Absolute distance measurements require accurate piezo calibration

bull AFM systems have large scan ranges (150 microm to lt 1 microm)

bull Scanner non-linearities can introduce substantial error over that range

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

ights Reserve

d

Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

BBML - All R

ights Reserve

d

Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

100 nm 10 80 602 1198 100 nm 7 60 827 1439 100 nm 5 40 814 1427 100 nm 35 20 917 1724 100 nm 2 10 937 1774

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

ights Reserve

d

AFM calibration ndash Take Away Message

Calibration using a scan size and feature size comparable to features of interest is highly recommended

Calibration addresses absolute accuracy not precision (differential sensitivity between measurements)

bull Does not limit ability to differentiate between groups using the same AFM with the same calibration parameters

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ights Reserve

d

AFM Applications in Bone Research Indentation Mechanics

BBML - All R

ights Reserve

d

AFM-Based Indentation

In addition to high resolution imaging AFM can be used to extract nanoscale mechanical data

ApproachWithdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Can

tilev

er D

efle

ctio

n (V

olts

)

1 Ramp into a rigid sample ndash measure the slope

Deflection sensitivity x 119899119899119899119899119881119881

2 Calculate cantilever spring constant

3 Hookersquos law takes deflection (volts) and converts to units of force F = kx

Spring constant k 119873119873119899119899

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Forc

e (n

N)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration Fo

rce

(nN

)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

Tip-Sample Separation (nm)

BBML - All R

ights Reserve

d

AFM-Based Indentation Parameters

Approach Withdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

Peak Force

Area Between Curves Energy Dissipation

Contact Mechanics Fit for Elastic Modulus (E)

Adhesion Force

Indentation Depth (δ)

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

z

r

δ

R

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119877119877 ∙ 12057512057532

Es Sample modulus νs Poissonrsquos ratio R tip radius of curvature δ load point displacement

Relevant Hertz Assumptions bull Strains are small and within the elastic limit bull Indenter is infinitely stiff (only sample deforms) bull Indentation depth ltlt radius of curvature of indenter bull Surfaces are continuous non-conforming (must be flat) bull Load is normal to surface

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ights Reserve

d

Proper Indentation Model is Essential

δ

r

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119903119903 ∙ 12057512057532

δ

α

119865119865 =2120587120587∙

1198641198641199041199041 minus 1205841205841199041199042

∙ tan120572120572 ∙ 1205751205752

Es Sample modulus νs Poissonrsquos ratio R tip radius α opening angle

Hertz Model Spherical Indenter

depth ltlt radius of curvature

Sneddon Model Conical Indenter

depth ge radius of curvature

BBML - All R

ights Reserve

d

Indentation in OI Tendon Study

x

z

yBrtl+ and WT mice 6 month old males

4 tails per group

2-3 fascicles per tail

25-30 fibrils per tail

4-5 locations per fibril

Indent force 20 nN wet (Sneddon) 50 nN dry (Hertz) BBML - A

ll Rights R

eserved

OI Tendon Differential Effects of Dehydration

Dry 1268 plusmn 718 MPa

Wet 305 plusmn 243 MPa

Dry 1737 plusmn 973 MPa

Wet 301 plusmn 202 MPa

Kemp et al J StructBio 2012 180 (3) 428-438

bull Significant stiffening of fibrils with dehydration

bull More pronounced modulus modulus as a function of disease

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d

OI Tendon Differential Effects of Dehydration

Kemp et al J StructBio 2012 180 (3) 428-438

Diseased phenotype not present when wet phenotype observed when dried

0

10

20

30

40

50

60

0 20 40 60 80 100 120 140 160 180 200

Gro

up S

ampl

es (

)

Indent Modulus (MPa)

WT WetBrtl Wet

0

2

4

6

8

10

12

14

16

18

20

025

050

075

010

0012

5015

0017

5020

0022

5025

0027

5030

0032

5035

0037

5040

0042

5045

0047

5050

00

Gro

up S

ampl

es (

)Indent Modulus (MPa)

WT DryBrtl Dry

555 WT Indents 305 plusmn 243 MPa

614 Brtl+ Indents 301 plusmn 202 MPa

KS Test p=0129 t Test p=0464 464 WT Indents

1268 plusmn 718 MPa 479 Brtl+ Indents 1737 plusmn 973 MPa

KS Test plt0001 T Test plt0001

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ights Reserve

d

Indentation in Diabetic Tendon (Poster 0495)

x

z

y

Control and Diabetic Rat Tail Tendon

4-5 rats per group

2-3 fascicles per rat

~70 fibrilstail

4-5 locationsfibril

20 nN (Sneddon)

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d

Multiscale Diabetes Study (Poster 0495)

0

5

10

15

20

25

300 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 gt25

Perc

enta

ge o

f Gro

up S

ampl

es

Elastic Modulus (MPa)

CD (1530 indents)ZDSD (913 indents)

CD (n=5) 381 plusmn 149 MPa

ZDSD (n=3) 706 plusmn 263 MPa

0

100

200

300

400

500

600

700

800

900

0 2 4 6 8 10

Mic

rosc

ale

Mod

ulus

(MPa

)

Nanoscale Modulus (MPa)

plt0001

r2=0531 p=0040

bull Increased indentation modulus in diabetic tendon

bull Strong relationship between nanoscale indent modulus and microscale tensile modulus

BBML - All R

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d

Indentation Limitations and Weaknesses

Proper probe selection for intended application bull Cantilever must be stiff relative to sample bull Sharp probes may damage sample easily dull

R or α must be determined for each probe bull Properties provided by manufacturer are unreliable bull Vary from probe to probe can dull during indent process

Spring constant must be determined for each probe

bull Value provided by manufacturer is inaccurate bull Thermal tuning OK for ldquosoftrdquo probes poor performance

for stiffer probes bull Sader method (geometry) added mass reference

cantilevers

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d

Indentation Limitations and Weaknesses

Veeco Application Technical note Ben Ohler Practical Advice on the Determination of Cantilever Spring Constants

BBML - All R

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d

Indentation Limitations and Weaknesses

bull Sample processing

bull Sample hydration

bull Proper force range

bull Substrate effects for thin samples

bull Proper modulus model and range of data goodness of fit

Bearing in mind these assumptions and limitations AFM can provide true ldquonanoscalerdquo mechanical properties both in terms of forces and deformations

BBML - All R

ights Reserve

d

A Note on Peak Force QNM for Bruker AFMs bull Mechanical properties mapped pixel-by-pixel at same

resolution as the height image (PeakForce QNM)

Height Modulus BBML - All R

ights Reserve

d

A Note on Peak Force QNM

Correct probe stiffness must be used based on sample Pittenger ndash Veeco Product Bulletin

BBML - All R

ights Reserve

d

A Note on Peak Force QNM Extremely sensitive to calibration (details rarely given) Absolute radius and spring constant known

bull especially important from probe to probe bull radius uarr with indent depth ndash must determine R at depth

you expect to image bull Depth may vary as a function of sample (or at different

regions within the sample)

Relative reference material of ldquoknownrdquo modulus used to determine a ratio of 119896119896 119877119877 bull ldquoknownrdquo modulus may come from tension compression

and not indentation bull R is related to depth indentation depth on sample must

be same as on reference BBML - All R

ights Reserve

d

A Note on Peak Force QNM bull Modulus fit on the fly ndash no ability to monitor fit (or range) bull Local sample topography and sample tilt play a role in

measured parameters bull Assuming all modulus values in an image are correct how to

quantify

512 x 512 pixels = 262144 measurements

n=1 n=262144

n from selected ROIs

BBML - All R

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d

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d

bull Imaging in air on mica ndash substrate effect

bull Same imaging force or indent depth for all

bull For each area were all modulus values pooled

Lamprou et al PLoS ONE 2013 BBML - All R

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d

AFM Applications in Bone Research Other Cool Mechanical Techniques

BBML - All R

ights Reserve

d

ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo Bonds within or between collagen fibrils in bone that rupture to dissipate energy as a toughening mechanism

Thompson et al Nature 2001

Fantner et al Nature Mat 2005

Hansma et al JMNI 2005

Fantner et al Nano Letters 2007 BBML - All R

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d

Other Mechanical Techniques

Jimenez-Palomar J Mech Beh Bio Mat 2012

van der Rijt Macro Bio 2004

Yang J Biomed Mater Res 2007

Yang Biophysical Journal 2008 BBML - All R

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d

Other Cool Techniques bull Piezoresponse Force Microscopy (PFM)

bull Electrochemical AFM (EC-AFM)

bull Force Modulation Microscopy (FMM)

bull Lateral Force Microscopy (LFM)

bull Magnetic Force Microscopy (MFM)

bull Conductive AFM (C-AFM)

bull Force Pulling

bull Cantilever Bending

bull Tensile testing of individual fibrils

bull 3 point bending of fibrils

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d

Summary Things to rememberhellip

bull Imaging mode imaging medium and probe selection

bull Morphology Measurements bull Sample preparation and hydration state bull Measuring fibril diameter bull Site selection and proper sampling bull Appropriate system calibration

bull Mechanical Measurements

bull Probe selection (stiffness and tip radius) bull Probe calibration (radius angle spring constant) bull Indentation force range and mechanical model bull Hydration in biological samples bull PeakForce QNM

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d

Acknowledgements BBML

Max Hammond Armando Diaz Gonzalez Zachary Bart Silvia Canelon Creasy Clauser

IU School of Medicine Dr David Burr Dr Matt Allen Dr Stuart Warden Dr Lilian Plotkin

Funding Sources NIHNIDCR 1F32DE018840-01 A1 IUPUI BME Departmental Startup Funds IUPUI Research Support Funds Grant IUPUI Biomechanics and Biomaterials Research Center

NIH NICHD Dr Joan Marini

BBML - All R

ights Reserve

d

Bone Biology and Mechanics Lab (BBML) wwwiupuiedu~bbml Facebook BoneBiologyMechanicsLab

BBML - All R

ights Reserve

d

• Slide Number 1
• General Seminar Outline
• Bonersquos Hierarchical Structure
• Critical Gap in Understanding Collagen
• Methods For Analyzing Collagen Structure
• Methods For Imaging Collagen Structure
• Cryo-TEM
• Atomic Force Microscopy (AFM)
• AFM Image of Pentacene (C22H14)
• AFM Image of Hexabenzocoronene
• Basics of AFM Imaging The Probe
• Basics of AFM Imaging System Components
• van der Waals Potential Energy Curve
• Contact Mode ndash Monitor Deflection
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Peak Force Tapping Mode ndash Monitor Force
• AFM Applications in Bone Research Imaging
• AFM Imaging of Collagen
• Imaging of Osteoclast Activity
• Mineralized collagen deposition in vitro
• Imaging of Canilicular Structure
• AFM Imaging in Bone
• AFM Imaging in Bone
• Removing Mineral in Bone to Expose Collagen
• Removing Mineral in Bone to Expose Collagen
• Demineralization with EDTA
• Demineralization with EDTA
• Demineralization with EDTA
• Issues to Consider Effects of Acid
• Slide Number 32
• Slide Number 33
• Analysis of Collagen Structure in Bone
• Analysis of Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril D-Spacing
• Slide Number 39
• Collagen in Bone Fibril D-Spacing
• Collagen in Bone Fibril D-Spacing
• 2D Fast Fourier Transform Analysis
• 2D Fast Fourier Transform Analysis
• D-Spacing Distribution
• D-Spacing Distribution
• Cumulative Distribution Function
• CDF vs Histogram
• Changes in D-Spacing with Disease
• Changes in D-Spacing with Disease
• Slide Number 50
• Issues to Consider Site Selection and Sampling
• Issues to Consider Site Selection and Sampling
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• AFM calibration ndash Take Away Message
• AFM Applications in Bone ResearchIndentation Mechanics
• AFM-Based Indentation
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Parameters
• Hertz Contact Equation
• Hertz Contact Equation
• Hertz Contact Equation
• Proper Indentation Model is Essential
• Indentation in OI Tendon Study
• OI Tendon Differential Effects of Dehydration
• OI Tendon Differential Effects of Dehydration
• Indentation in Diabetic Tendon (Poster 0495)
• Multiscale Diabetes Study (Poster 0495)
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• A Note on Peak Force QNM for Bruker AFMs
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• Slide Number 83
• Slide Number 84
• AFM Applications in Bone ResearchOther Cool Mechanical Techniques
• ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo
• Other Mechanical Techniques
• Other Cool Techniques
• Summary Things to rememberhellip
• Acknowledgements
• Bone Biology and Mechanics Lab (BBML)

Collagen in Bone Fibril D-Spacing

300 nm 15 nm

N C C N C C N C C N C C

H O H2C CH2 O H

CH2

O OH HHC CH2

CH2

HO

H H

HHN C C

O

H2C CH2

CH2

HN C C

OH

HC CH2

HO

N C C N C C N C C

H O H2C CH2 O

CH2

OH HC CH2

HO

H H

H

CH2 CH2

glycine X - proline Y - hydroxyproline

Hodge and Petruska 1963

Collagen Fibril Axial D-Periodicity BBML - All R

ights Reserve

d

05

101520

0 02 04 06 08 1

Hei

ght (

nm)

Length (microns)

httpremfdartmoutheduimagesindexhtml

Randall Nature and Structure of Collagen 1953

TEM SEM

AFM

D-Spacing = 67 nm

BBML - All R

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d

Collagen in Bone Fibril D-Spacing Many studies have shown observable D-spacing content that near theoretical value

Sasaki 2002 67 plusmn 2 nm Bozek 2005 665 plusmn 14 nm Ge 2007 666 plusmn 38 nm

Habelitz J Structural Bio 2002

A 2002 study in dentin got me interestedhellip

n = 322 wet n = 363 dry

BBML - All R

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Collagen in Bone Fibril D-Spacing

bull Internal structure bull Wallace et al Bone 2010 46 1349-1354 bull Wallace et al J Stuct Bio 2011 173 146-152 bull Kemp et al J Stuct Bio 2012 180 428-438 bull Warden et al Endocrinology 2013 154(9)

3178-3187 bull Gallant et al Bone 2014 61 191-200 bull Bart et al Conn Tiss Res 2014 In Press

bull Enzymatic and non-

enzymatic cross-linking bull Hammond et al Bone 2014 60 26-32 bull Diaz Gonzalez et al J Biomech 2014 47(3)

681-686 bull Voytik-Harbin et al In Preparation 2014

D-periodic spacing important structural feature that captures aspects related to

Mouse Femur Mid-diaphysis BBML - All R

ights Reserve

d

2D Fast Fourier Transform Analysis

Harmonics

Fibril D-Spacing

Erickson B Wallace JM et al Biotechnology Journal 2013

Raw AFM Image 2D FFT Power Spectrum

BBML - All R

ights Reserve

d

2D Fast Fourier Transform Analysis

Harmonics

Fibril D-Spacing

Raw AFM Image 2D FFT Power Spectrum

0

20000

40000

60000

80000

100000

120000

0 001 002 003 004 005 006

Am

plitu

de (

microV2 )

Position (1nm)

D-periodic spacing 0015 (1nm) ~ 67 nm

BBML - All R

ights Reserve

d

D-Spacing Distribution

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Overall Mean 670 plusmn 00 nm

BBML - All R

ights Reserve

d

D-Spacing Distribution

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Overall Mean 670 plusmn 00 nm

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

4 Samples Total of n = 255 fibrils Overall Mean 682 plusmn 12 nm

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ights Reserve

d

Cumulative Distribution Function

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

This population 255 fibrils - Each fibril 1255 = 0392 - Data range 628-716 nm

BBML - All R

ights Reserve

d

CDF vs Histogram 62 63 64 65 66 67 68 69 70 71 72 73 74

0

10

20

30

40

50

60

70

80

90

100

0

5

10

15

20

25

30

35

40

45

50

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

HistoCDF

BBML - All R

ights Reserve

d

Changes in D-Spacing with Disease

0

5

10

15

20

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

0

5

10

15

20

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

Wallace et al Bone 2010 46 1349-1354

KS Test plt0001

Control 680 plusmn 26 nm

Estrogen Dep 659 plusmn 31 nm

t-test on mean p=0048

BBML - All R

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d

Changes in D-Spacing with Disease

Wallace et al Bone 2010 46 1349-1354

0

10

20

30

40

50

60

70

80

90

100

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

KS Test plt0001

BBML - All R

ights Reserve

d

Question Model and Tissue Morphology Publication

Tissue Specificity in Mice Male mouse 8 weeks Femur dentin tail tendon

No differences in bone vs dentin both ∆ vs tendon

Wallace et al Langmuir 2011 26(10) 7349-7354

Gender Effects Male and female mice 16 weeks Femur No differences NA

Estrogen Depletion OVX in sheep 5 years + 2 Radius Mean and population shift Wallace et al Bone

2010 46 1349-1354

Estrogen Depletion OVX in female 5 years + 2 Skin (dermis) Significant population shift Erickson et al Biotech

J 2013 8(1) 1174-126

Osteoporosis and Mechanical Loading

OVX in ulnar loading in rats Ulna

No loading effect population shift with OVX

Warden et al Endocrinology 2013 154(9) 3178-3187

Osteogenesis Imperfecta Brtl+ model male mice 2 months Femur

No mean ∆ widening of distribution

Wallace et al J Stuct Bio 2011 173 146-152

Osteogenesis Imperfecta Oimoim model male mice 12 weeks Femur Mean and population shift Bart et al Conn Tiss

Res 2014 In Press

Osteogenesis Imperfecta and Tissue Hydration

Brtl+ model male mice 6 months Tail Tendon Significant population shift Kemp et al J Stuct Bio

2012 180 428-438

Type 2 Diabetes Male ZDSD rats 30 weeks Tibia Significant population shift Hammond et al Bone

2014 60 26-32

Type 2 Diabetes Female ZDSD rats 32 weeks Tail Tendon Significant population shift

Diaz Gonzalez et al J Biomech 2014 47(3)

681-686

Raloxifene (in vivo and in vitro) Female dog treated with Raloxifene Femur Significant population shift Gallant et al Bone

2014 61 191-200 BBML - All R

ights Reserve

d

Issues to Consider Site Selection and Sampling

KS Test plt0001

0023 of bonersquos length

15 m

m =

150

00 micro

m

Wallace et al Langmuir 2011 26(10) 7349-7354

100 microm x 100 microm

35 microm x 35 microm

BBML - All R

ights Reserve

d

Issues to Consider Site Selection and Sampling

60

62

64

66

68

70

72

74

1 (35)

2 (33)

3 (40)

4 (39)

5 (35)

6 (29)

7 (26)

8 (23)

9 (28)

WT (288)

D-P

erio

dic

Gap

Ove

rlap

Spac

ing

(nm

)

Axial Location (Proximal End = 1 Distal End = 9)

( of Fibrils)

91

23

45

87

6

Wallace et al Langmuir 2011 26(10) 7349-7354

288 fibrils for population n=1 for mean comparisons BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

Wet Tendon Dry Tendon Kemp et al J StructBio 2012 180 (3) 428-438

Collagen and bone exist in a hydrated environment in vivo bull Hydration state can impact morphology and mechanics

BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

Nonsignificant mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet (n=255) 682 plusmn 12 nm

WT Dry (n=257) 686 plusmn 08 nm

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

Brtl DryBrtl Wet

KS Test plt0001

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

WT Case 04 nm mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet 682 plusmn 12 nm

WT Dry 686 plusmn 08 nm

Brtl+ Wet 675 plusmn 14 nm

Brtl+ Dry 689 plusmn 10 nm

Brtl+ Case 14 nm increase with drying (plt0001) bull Entire population shifted upward (plt0001)

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT WetBrtl Wet

KS Test plt0001

Brtl+ distribution shifted downward vs WT when wet

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryBrtl Dry

KS Test plt0001

Brtl+ distribution shifted upward vs WT when dry

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider AFM calibration

bull Absolute distance measurements require accurate piezo calibration

bull AFM systems have large scan ranges (150 microm to lt 1 microm)

bull Scanner non-linearities can introduce substantial error over that range

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

ights Reserve

d

Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

BBML - All R

ights Reserve

d

Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

100 nm 10 80 602 1198 100 nm 7 60 827 1439 100 nm 5 40 814 1427 100 nm 35 20 917 1724 100 nm 2 10 937 1774

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

ights Reserve

d

AFM calibration ndash Take Away Message

Calibration using a scan size and feature size comparable to features of interest is highly recommended

Calibration addresses absolute accuracy not precision (differential sensitivity between measurements)

bull Does not limit ability to differentiate between groups using the same AFM with the same calibration parameters

BBML - All R

ights Reserve

d

AFM Applications in Bone Research Indentation Mechanics

BBML - All R

ights Reserve

d

AFM-Based Indentation

In addition to high resolution imaging AFM can be used to extract nanoscale mechanical data

ApproachWithdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Can

tilev

er D

efle

ctio

n (V

olts

)

1 Ramp into a rigid sample ndash measure the slope

Deflection sensitivity x 119899119899119899119899119881119881

2 Calculate cantilever spring constant

3 Hookersquos law takes deflection (volts) and converts to units of force F = kx

Spring constant k 119873119873119899119899

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Forc

e (n

N)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration Fo

rce

(nN

)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

Tip-Sample Separation (nm)

BBML - All R

ights Reserve

d

AFM-Based Indentation Parameters

Approach Withdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

Peak Force

Area Between Curves Energy Dissipation

Contact Mechanics Fit for Elastic Modulus (E)

Adhesion Force

Indentation Depth (δ)

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

z

r

δ

R

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119877119877 ∙ 12057512057532

Es Sample modulus νs Poissonrsquos ratio R tip radius of curvature δ load point displacement

Relevant Hertz Assumptions bull Strains are small and within the elastic limit bull Indenter is infinitely stiff (only sample deforms) bull Indentation depth ltlt radius of curvature of indenter bull Surfaces are continuous non-conforming (must be flat) bull Load is normal to surface

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ights Reserve

d

Proper Indentation Model is Essential

δ

r

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119903119903 ∙ 12057512057532

δ

α

119865119865 =2120587120587∙

1198641198641199041199041 minus 1205841205841199041199042

∙ tan120572120572 ∙ 1205751205752

Es Sample modulus νs Poissonrsquos ratio R tip radius α opening angle

Hertz Model Spherical Indenter

depth ltlt radius of curvature

Sneddon Model Conical Indenter

depth ge radius of curvature

BBML - All R

ights Reserve

d

Indentation in OI Tendon Study

x

z

yBrtl+ and WT mice 6 month old males

4 tails per group

2-3 fascicles per tail

25-30 fibrils per tail

4-5 locations per fibril

Indent force 20 nN wet (Sneddon) 50 nN dry (Hertz) BBML - A

ll Rights R

eserved

OI Tendon Differential Effects of Dehydration

Dry 1268 plusmn 718 MPa

Wet 305 plusmn 243 MPa

Dry 1737 plusmn 973 MPa

Wet 301 plusmn 202 MPa

Kemp et al J StructBio 2012 180 (3) 428-438

bull Significant stiffening of fibrils with dehydration

bull More pronounced modulus modulus as a function of disease

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ights Reserve

d

OI Tendon Differential Effects of Dehydration

Kemp et al J StructBio 2012 180 (3) 428-438

Diseased phenotype not present when wet phenotype observed when dried

0

10

20

30

40

50

60

0 20 40 60 80 100 120 140 160 180 200

Gro

up S

ampl

es (

)

Indent Modulus (MPa)

WT WetBrtl Wet

0

2

4

6

8

10

12

14

16

18

20

025

050

075

010

0012

5015

0017

5020

0022

5025

0027

5030

0032

5035

0037

5040

0042

5045

0047

5050

00

Gro

up S

ampl

es (

)Indent Modulus (MPa)

WT DryBrtl Dry

555 WT Indents 305 plusmn 243 MPa

614 Brtl+ Indents 301 plusmn 202 MPa

KS Test p=0129 t Test p=0464 464 WT Indents

1268 plusmn 718 MPa 479 Brtl+ Indents 1737 plusmn 973 MPa

KS Test plt0001 T Test plt0001

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ights Reserve

d

Indentation in Diabetic Tendon (Poster 0495)

x

z

y

Control and Diabetic Rat Tail Tendon

4-5 rats per group

2-3 fascicles per rat

~70 fibrilstail

4-5 locationsfibril

20 nN (Sneddon)

BBML - All R

ights Reserve

d

Multiscale Diabetes Study (Poster 0495)

0

5

10

15

20

25

300 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 gt25

Perc

enta

ge o

f Gro

up S

ampl

es

Elastic Modulus (MPa)

CD (1530 indents)ZDSD (913 indents)

CD (n=5) 381 plusmn 149 MPa

ZDSD (n=3) 706 plusmn 263 MPa

0

100

200

300

400

500

600

700

800

900

0 2 4 6 8 10

Mic

rosc

ale

Mod

ulus

(MPa

)

Nanoscale Modulus (MPa)

plt0001

r2=0531 p=0040

bull Increased indentation modulus in diabetic tendon

bull Strong relationship between nanoscale indent modulus and microscale tensile modulus

BBML - All R

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d

Indentation Limitations and Weaknesses

Proper probe selection for intended application bull Cantilever must be stiff relative to sample bull Sharp probes may damage sample easily dull

R or α must be determined for each probe bull Properties provided by manufacturer are unreliable bull Vary from probe to probe can dull during indent process

Spring constant must be determined for each probe

bull Value provided by manufacturer is inaccurate bull Thermal tuning OK for ldquosoftrdquo probes poor performance

for stiffer probes bull Sader method (geometry) added mass reference

cantilevers

BBML - All R

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d

Indentation Limitations and Weaknesses

Veeco Application Technical note Ben Ohler Practical Advice on the Determination of Cantilever Spring Constants

BBML - All R

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d

Indentation Limitations and Weaknesses

bull Sample processing

bull Sample hydration

bull Proper force range

bull Substrate effects for thin samples

bull Proper modulus model and range of data goodness of fit

Bearing in mind these assumptions and limitations AFM can provide true ldquonanoscalerdquo mechanical properties both in terms of forces and deformations

BBML - All R

ights Reserve

d

A Note on Peak Force QNM for Bruker AFMs bull Mechanical properties mapped pixel-by-pixel at same

resolution as the height image (PeakForce QNM)

Height Modulus BBML - All R

ights Reserve

d

A Note on Peak Force QNM

Correct probe stiffness must be used based on sample Pittenger ndash Veeco Product Bulletin

BBML - All R

ights Reserve

d

A Note on Peak Force QNM Extremely sensitive to calibration (details rarely given) Absolute radius and spring constant known

bull especially important from probe to probe bull radius uarr with indent depth ndash must determine R at depth

you expect to image bull Depth may vary as a function of sample (or at different

regions within the sample)

Relative reference material of ldquoknownrdquo modulus used to determine a ratio of 119896119896 119877119877 bull ldquoknownrdquo modulus may come from tension compression

and not indentation bull R is related to depth indentation depth on sample must

be same as on reference BBML - All R

ights Reserve

d

A Note on Peak Force QNM bull Modulus fit on the fly ndash no ability to monitor fit (or range) bull Local sample topography and sample tilt play a role in

measured parameters bull Assuming all modulus values in an image are correct how to

quantify

512 x 512 pixels = 262144 measurements

n=1 n=262144

n from selected ROIs

BBML - All R

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d

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d

bull Imaging in air on mica ndash substrate effect

bull Same imaging force or indent depth for all

bull For each area were all modulus values pooled

Lamprou et al PLoS ONE 2013 BBML - All R

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d

AFM Applications in Bone Research Other Cool Mechanical Techniques

BBML - All R

ights Reserve

d

ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo Bonds within or between collagen fibrils in bone that rupture to dissipate energy as a toughening mechanism

Thompson et al Nature 2001

Fantner et al Nature Mat 2005

Hansma et al JMNI 2005

Fantner et al Nano Letters 2007 BBML - All R

ights Reserve

d

Other Mechanical Techniques

Jimenez-Palomar J Mech Beh Bio Mat 2012

van der Rijt Macro Bio 2004

Yang J Biomed Mater Res 2007

Yang Biophysical Journal 2008 BBML - All R

ights Reserve

d

Other Cool Techniques bull Piezoresponse Force Microscopy (PFM)

bull Electrochemical AFM (EC-AFM)

bull Force Modulation Microscopy (FMM)

bull Lateral Force Microscopy (LFM)

bull Magnetic Force Microscopy (MFM)

bull Conductive AFM (C-AFM)

bull Force Pulling

bull Cantilever Bending

bull Tensile testing of individual fibrils

bull 3 point bending of fibrils

BBML - All R

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d

Summary Things to rememberhellip

bull Imaging mode imaging medium and probe selection

bull Morphology Measurements bull Sample preparation and hydration state bull Measuring fibril diameter bull Site selection and proper sampling bull Appropriate system calibration

bull Mechanical Measurements

bull Probe selection (stiffness and tip radius) bull Probe calibration (radius angle spring constant) bull Indentation force range and mechanical model bull Hydration in biological samples bull PeakForce QNM

BBML - All R

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d

Acknowledgements BBML

Max Hammond Armando Diaz Gonzalez Zachary Bart Silvia Canelon Creasy Clauser

IU School of Medicine Dr David Burr Dr Matt Allen Dr Stuart Warden Dr Lilian Plotkin

Funding Sources NIHNIDCR 1F32DE018840-01 A1 IUPUI BME Departmental Startup Funds IUPUI Research Support Funds Grant IUPUI Biomechanics and Biomaterials Research Center

NIH NICHD Dr Joan Marini

BBML - All R

ights Reserve

d

Bone Biology and Mechanics Lab (BBML) wwwiupuiedu~bbml Facebook BoneBiologyMechanicsLab

BBML - All R

ights Reserve

d

• Slide Number 1
• General Seminar Outline
• Bonersquos Hierarchical Structure
• Critical Gap in Understanding Collagen
• Methods For Analyzing Collagen Structure
• Methods For Imaging Collagen Structure
• Cryo-TEM
• Atomic Force Microscopy (AFM)
• AFM Image of Pentacene (C22H14)
• AFM Image of Hexabenzocoronene
• Basics of AFM Imaging The Probe
• Basics of AFM Imaging System Components
• van der Waals Potential Energy Curve
• Contact Mode ndash Monitor Deflection
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Peak Force Tapping Mode ndash Monitor Force
• AFM Applications in Bone Research Imaging
• AFM Imaging of Collagen
• Imaging of Osteoclast Activity
• Mineralized collagen deposition in vitro
• Imaging of Canilicular Structure
• AFM Imaging in Bone
• AFM Imaging in Bone
• Removing Mineral in Bone to Expose Collagen
• Removing Mineral in Bone to Expose Collagen
• Demineralization with EDTA
• Demineralization with EDTA
• Demineralization with EDTA
• Issues to Consider Effects of Acid
• Slide Number 32
• Slide Number 33
• Analysis of Collagen Structure in Bone
• Analysis of Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril D-Spacing
• Slide Number 39
• Collagen in Bone Fibril D-Spacing
• Collagen in Bone Fibril D-Spacing
• 2D Fast Fourier Transform Analysis
• 2D Fast Fourier Transform Analysis
• D-Spacing Distribution
• D-Spacing Distribution
• Cumulative Distribution Function
• CDF vs Histogram
• Changes in D-Spacing with Disease
• Changes in D-Spacing with Disease
• Slide Number 50
• Issues to Consider Site Selection and Sampling
• Issues to Consider Site Selection and Sampling
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• AFM calibration ndash Take Away Message
• AFM Applications in Bone ResearchIndentation Mechanics
• AFM-Based Indentation
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Parameters
• Hertz Contact Equation
• Hertz Contact Equation
• Hertz Contact Equation
• Proper Indentation Model is Essential
• Indentation in OI Tendon Study
• OI Tendon Differential Effects of Dehydration
• OI Tendon Differential Effects of Dehydration
• Indentation in Diabetic Tendon (Poster 0495)
• Multiscale Diabetes Study (Poster 0495)
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• A Note on Peak Force QNM for Bruker AFMs
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• Slide Number 83
• Slide Number 84
• AFM Applications in Bone ResearchOther Cool Mechanical Techniques
• ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo
• Other Mechanical Techniques
• Other Cool Techniques
• Summary Things to rememberhellip
• Acknowledgements
• Bone Biology and Mechanics Lab (BBML)

05

101520

0 02 04 06 08 1

Hei

ght (

nm)

Length (microns)

httpremfdartmoutheduimagesindexhtml

Randall Nature and Structure of Collagen 1953

TEM SEM

AFM

D-Spacing = 67 nm

BBML - All R

ights Reserve

d

Collagen in Bone Fibril D-Spacing Many studies have shown observable D-spacing content that near theoretical value

Sasaki 2002 67 plusmn 2 nm Bozek 2005 665 plusmn 14 nm Ge 2007 666 plusmn 38 nm

Habelitz J Structural Bio 2002

A 2002 study in dentin got me interestedhellip

n = 322 wet n = 363 dry

BBML - All R

ights Reserve

d

Collagen in Bone Fibril D-Spacing

bull Internal structure bull Wallace et al Bone 2010 46 1349-1354 bull Wallace et al J Stuct Bio 2011 173 146-152 bull Kemp et al J Stuct Bio 2012 180 428-438 bull Warden et al Endocrinology 2013 154(9)

3178-3187 bull Gallant et al Bone 2014 61 191-200 bull Bart et al Conn Tiss Res 2014 In Press

bull Enzymatic and non-

enzymatic cross-linking bull Hammond et al Bone 2014 60 26-32 bull Diaz Gonzalez et al J Biomech 2014 47(3)

681-686 bull Voytik-Harbin et al In Preparation 2014

D-periodic spacing important structural feature that captures aspects related to

Mouse Femur Mid-diaphysis BBML - All R

ights Reserve

d

2D Fast Fourier Transform Analysis

Harmonics

Fibril D-Spacing

Erickson B Wallace JM et al Biotechnology Journal 2013

Raw AFM Image 2D FFT Power Spectrum

BBML - All R

ights Reserve

d

2D Fast Fourier Transform Analysis

Harmonics

Fibril D-Spacing

Raw AFM Image 2D FFT Power Spectrum

0

20000

40000

60000

80000

100000

120000

0 001 002 003 004 005 006

Am

plitu

de (

microV2 )

Position (1nm)

D-periodic spacing 0015 (1nm) ~ 67 nm

BBML - All R

ights Reserve

d

D-Spacing Distribution

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Overall Mean 670 plusmn 00 nm

BBML - All R

ights Reserve

d

D-Spacing Distribution

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Overall Mean 670 plusmn 00 nm

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

4 Samples Total of n = 255 fibrils Overall Mean 682 plusmn 12 nm

BBML - All R

ights Reserve

d

Cumulative Distribution Function

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

This population 255 fibrils - Each fibril 1255 = 0392 - Data range 628-716 nm

BBML - All R

ights Reserve

d

CDF vs Histogram 62 63 64 65 66 67 68 69 70 71 72 73 74

0

10

20

30

40

50

60

70

80

90

100

0

5

10

15

20

25

30

35

40

45

50

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

HistoCDF

BBML - All R

ights Reserve

d

Changes in D-Spacing with Disease

0

5

10

15

20

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

0

5

10

15

20

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

Wallace et al Bone 2010 46 1349-1354

KS Test plt0001

Control 680 plusmn 26 nm

Estrogen Dep 659 plusmn 31 nm

t-test on mean p=0048

BBML - All R

ights Reserve

d

Changes in D-Spacing with Disease

Wallace et al Bone 2010 46 1349-1354

0

10

20

30

40

50

60

70

80

90

100

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

KS Test plt0001

BBML - All R

ights Reserve

d

Question Model and Tissue Morphology Publication

Tissue Specificity in Mice Male mouse 8 weeks Femur dentin tail tendon

No differences in bone vs dentin both ∆ vs tendon

Wallace et al Langmuir 2011 26(10) 7349-7354

Gender Effects Male and female mice 16 weeks Femur No differences NA

Estrogen Depletion OVX in sheep 5 years + 2 Radius Mean and population shift Wallace et al Bone

2010 46 1349-1354

Estrogen Depletion OVX in female 5 years + 2 Skin (dermis) Significant population shift Erickson et al Biotech

J 2013 8(1) 1174-126

Osteoporosis and Mechanical Loading

OVX in ulnar loading in rats Ulna

No loading effect population shift with OVX

Warden et al Endocrinology 2013 154(9) 3178-3187

Osteogenesis Imperfecta Brtl+ model male mice 2 months Femur

No mean ∆ widening of distribution

Wallace et al J Stuct Bio 2011 173 146-152

Osteogenesis Imperfecta Oimoim model male mice 12 weeks Femur Mean and population shift Bart et al Conn Tiss

Res 2014 In Press

Osteogenesis Imperfecta and Tissue Hydration

Brtl+ model male mice 6 months Tail Tendon Significant population shift Kemp et al J Stuct Bio

2012 180 428-438

Type 2 Diabetes Male ZDSD rats 30 weeks Tibia Significant population shift Hammond et al Bone

2014 60 26-32

Type 2 Diabetes Female ZDSD rats 32 weeks Tail Tendon Significant population shift

Diaz Gonzalez et al J Biomech 2014 47(3)

681-686

Raloxifene (in vivo and in vitro) Female dog treated with Raloxifene Femur Significant population shift Gallant et al Bone

2014 61 191-200 BBML - All R

ights Reserve

d

Issues to Consider Site Selection and Sampling

KS Test plt0001

0023 of bonersquos length

15 m

m =

150

00 micro

m

Wallace et al Langmuir 2011 26(10) 7349-7354

100 microm x 100 microm

35 microm x 35 microm

BBML - All R

ights Reserve

d

Issues to Consider Site Selection and Sampling

60

62

64

66

68

70

72

74

1 (35)

2 (33)

3 (40)

4 (39)

5 (35)

6 (29)

7 (26)

8 (23)

9 (28)

WT (288)

D-P

erio

dic

Gap

Ove

rlap

Spac

ing

(nm

)

Axial Location (Proximal End = 1 Distal End = 9)

( of Fibrils)

91

23

45

87

6

Wallace et al Langmuir 2011 26(10) 7349-7354

288 fibrils for population n=1 for mean comparisons BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

Wet Tendon Dry Tendon Kemp et al J StructBio 2012 180 (3) 428-438

Collagen and bone exist in a hydrated environment in vivo bull Hydration state can impact morphology and mechanics

BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

Nonsignificant mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet (n=255) 682 plusmn 12 nm

WT Dry (n=257) 686 plusmn 08 nm

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

Brtl DryBrtl Wet

KS Test plt0001

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

WT Case 04 nm mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet 682 plusmn 12 nm

WT Dry 686 plusmn 08 nm

Brtl+ Wet 675 plusmn 14 nm

Brtl+ Dry 689 plusmn 10 nm

Brtl+ Case 14 nm increase with drying (plt0001) bull Entire population shifted upward (plt0001)

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT WetBrtl Wet

KS Test plt0001

Brtl+ distribution shifted downward vs WT when wet

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryBrtl Dry

KS Test plt0001

Brtl+ distribution shifted upward vs WT when dry

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider AFM calibration

bull Absolute distance measurements require accurate piezo calibration

bull AFM systems have large scan ranges (150 microm to lt 1 microm)

bull Scanner non-linearities can introduce substantial error over that range

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

ights Reserve

d

Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

BBML - All R

ights Reserve

d

Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

100 nm 10 80 602 1198 100 nm 7 60 827 1439 100 nm 5 40 814 1427 100 nm 35 20 917 1724 100 nm 2 10 937 1774

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

ights Reserve

d

AFM calibration ndash Take Away Message

Calibration using a scan size and feature size comparable to features of interest is highly recommended

Calibration addresses absolute accuracy not precision (differential sensitivity between measurements)

bull Does not limit ability to differentiate between groups using the same AFM with the same calibration parameters

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ights Reserve

d

AFM Applications in Bone Research Indentation Mechanics

BBML - All R

ights Reserve

d

AFM-Based Indentation

In addition to high resolution imaging AFM can be used to extract nanoscale mechanical data

ApproachWithdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Can

tilev

er D

efle

ctio

n (V

olts

)

1 Ramp into a rigid sample ndash measure the slope

Deflection sensitivity x 119899119899119899119899119881119881

2 Calculate cantilever spring constant

3 Hookersquos law takes deflection (volts) and converts to units of force F = kx

Spring constant k 119873119873119899119899

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Forc

e (n

N)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration Fo

rce

(nN

)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

Tip-Sample Separation (nm)

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ights Reserve

d

AFM-Based Indentation Parameters

Approach Withdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

Peak Force

Area Between Curves Energy Dissipation

Contact Mechanics Fit for Elastic Modulus (E)

Adhesion Force

Indentation Depth (δ)

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

z

r

δ

R

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119877119877 ∙ 12057512057532

Es Sample modulus νs Poissonrsquos ratio R tip radius of curvature δ load point displacement

Relevant Hertz Assumptions bull Strains are small and within the elastic limit bull Indenter is infinitely stiff (only sample deforms) bull Indentation depth ltlt radius of curvature of indenter bull Surfaces are continuous non-conforming (must be flat) bull Load is normal to surface

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ights Reserve

d

Proper Indentation Model is Essential

δ

r

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119903119903 ∙ 12057512057532

δ

α

119865119865 =2120587120587∙

1198641198641199041199041 minus 1205841205841199041199042

∙ tan120572120572 ∙ 1205751205752

Es Sample modulus νs Poissonrsquos ratio R tip radius α opening angle

Hertz Model Spherical Indenter

depth ltlt radius of curvature

Sneddon Model Conical Indenter

depth ge radius of curvature

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ights Reserve

d

Indentation in OI Tendon Study

x

z

yBrtl+ and WT mice 6 month old males

4 tails per group

2-3 fascicles per tail

25-30 fibrils per tail

4-5 locations per fibril

Indent force 20 nN wet (Sneddon) 50 nN dry (Hertz) BBML - A

ll Rights R

eserved

OI Tendon Differential Effects of Dehydration

Dry 1268 plusmn 718 MPa

Wet 305 plusmn 243 MPa

Dry 1737 plusmn 973 MPa

Wet 301 plusmn 202 MPa

Kemp et al J StructBio 2012 180 (3) 428-438

bull Significant stiffening of fibrils with dehydration

bull More pronounced modulus modulus as a function of disease

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ights Reserve

d

OI Tendon Differential Effects of Dehydration

Kemp et al J StructBio 2012 180 (3) 428-438

Diseased phenotype not present when wet phenotype observed when dried

0

10

20

30

40

50

60

0 20 40 60 80 100 120 140 160 180 200

Gro

up S

ampl

es (

)

Indent Modulus (MPa)

WT WetBrtl Wet

0

2

4

6

8

10

12

14

16

18

20

025

050

075

010

0012

5015

0017

5020

0022

5025

0027

5030

0032

5035

0037

5040

0042

5045

0047

5050

00

Gro

up S

ampl

es (

)Indent Modulus (MPa)

WT DryBrtl Dry

555 WT Indents 305 plusmn 243 MPa

614 Brtl+ Indents 301 plusmn 202 MPa

KS Test p=0129 t Test p=0464 464 WT Indents

1268 plusmn 718 MPa 479 Brtl+ Indents 1737 plusmn 973 MPa

KS Test plt0001 T Test plt0001

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ights Reserve

d

Indentation in Diabetic Tendon (Poster 0495)

x

z

y

Control and Diabetic Rat Tail Tendon

4-5 rats per group

2-3 fascicles per rat

~70 fibrilstail

4-5 locationsfibril

20 nN (Sneddon)

BBML - All R

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d

Multiscale Diabetes Study (Poster 0495)

0

5

10

15

20

25

300 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 gt25

Perc

enta

ge o

f Gro

up S

ampl

es

Elastic Modulus (MPa)

CD (1530 indents)ZDSD (913 indents)

CD (n=5) 381 plusmn 149 MPa

ZDSD (n=3) 706 plusmn 263 MPa

0

100

200

300

400

500

600

700

800

900

0 2 4 6 8 10

Mic

rosc

ale

Mod

ulus

(MPa

)

Nanoscale Modulus (MPa)

plt0001

r2=0531 p=0040

bull Increased indentation modulus in diabetic tendon

bull Strong relationship between nanoscale indent modulus and microscale tensile modulus

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d

Indentation Limitations and Weaknesses

Proper probe selection for intended application bull Cantilever must be stiff relative to sample bull Sharp probes may damage sample easily dull

R or α must be determined for each probe bull Properties provided by manufacturer are unreliable bull Vary from probe to probe can dull during indent process

Spring constant must be determined for each probe

bull Value provided by manufacturer is inaccurate bull Thermal tuning OK for ldquosoftrdquo probes poor performance

for stiffer probes bull Sader method (geometry) added mass reference

cantilevers

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d

Indentation Limitations and Weaknesses

Veeco Application Technical note Ben Ohler Practical Advice on the Determination of Cantilever Spring Constants

BBML - All R

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d

Indentation Limitations and Weaknesses

bull Sample processing

bull Sample hydration

bull Proper force range

bull Substrate effects for thin samples

bull Proper modulus model and range of data goodness of fit

Bearing in mind these assumptions and limitations AFM can provide true ldquonanoscalerdquo mechanical properties both in terms of forces and deformations

BBML - All R

ights Reserve

d

A Note on Peak Force QNM for Bruker AFMs bull Mechanical properties mapped pixel-by-pixel at same

resolution as the height image (PeakForce QNM)

Height Modulus BBML - All R

ights Reserve

d

A Note on Peak Force QNM

Correct probe stiffness must be used based on sample Pittenger ndash Veeco Product Bulletin

BBML - All R

ights Reserve

d

A Note on Peak Force QNM Extremely sensitive to calibration (details rarely given) Absolute radius and spring constant known

bull especially important from probe to probe bull radius uarr with indent depth ndash must determine R at depth

you expect to image bull Depth may vary as a function of sample (or at different

regions within the sample)

Relative reference material of ldquoknownrdquo modulus used to determine a ratio of 119896119896 119877119877 bull ldquoknownrdquo modulus may come from tension compression

and not indentation bull R is related to depth indentation depth on sample must

be same as on reference BBML - All R

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d

A Note on Peak Force QNM bull Modulus fit on the fly ndash no ability to monitor fit (or range) bull Local sample topography and sample tilt play a role in

measured parameters bull Assuming all modulus values in an image are correct how to

quantify

512 x 512 pixels = 262144 measurements

n=1 n=262144

n from selected ROIs

BBML - All R

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d

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d

bull Imaging in air on mica ndash substrate effect

bull Same imaging force or indent depth for all

bull For each area were all modulus values pooled

Lamprou et al PLoS ONE 2013 BBML - All R

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d

AFM Applications in Bone Research Other Cool Mechanical Techniques

BBML - All R

ights Reserve

d

ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo Bonds within or between collagen fibrils in bone that rupture to dissipate energy as a toughening mechanism

Thompson et al Nature 2001

Fantner et al Nature Mat 2005

Hansma et al JMNI 2005

Fantner et al Nano Letters 2007 BBML - All R

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d

Other Mechanical Techniques

Jimenez-Palomar J Mech Beh Bio Mat 2012

van der Rijt Macro Bio 2004

Yang J Biomed Mater Res 2007

Yang Biophysical Journal 2008 BBML - All R

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d

Other Cool Techniques bull Piezoresponse Force Microscopy (PFM)

bull Electrochemical AFM (EC-AFM)

bull Force Modulation Microscopy (FMM)

bull Lateral Force Microscopy (LFM)

bull Magnetic Force Microscopy (MFM)

bull Conductive AFM (C-AFM)

bull Force Pulling

bull Cantilever Bending

bull Tensile testing of individual fibrils

bull 3 point bending of fibrils

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d

Summary Things to rememberhellip

bull Imaging mode imaging medium and probe selection

bull Morphology Measurements bull Sample preparation and hydration state bull Measuring fibril diameter bull Site selection and proper sampling bull Appropriate system calibration

bull Mechanical Measurements

bull Probe selection (stiffness and tip radius) bull Probe calibration (radius angle spring constant) bull Indentation force range and mechanical model bull Hydration in biological samples bull PeakForce QNM

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d

Acknowledgements BBML

Max Hammond Armando Diaz Gonzalez Zachary Bart Silvia Canelon Creasy Clauser

IU School of Medicine Dr David Burr Dr Matt Allen Dr Stuart Warden Dr Lilian Plotkin

Funding Sources NIHNIDCR 1F32DE018840-01 A1 IUPUI BME Departmental Startup Funds IUPUI Research Support Funds Grant IUPUI Biomechanics and Biomaterials Research Center

NIH NICHD Dr Joan Marini

BBML - All R

ights Reserve

d

Bone Biology and Mechanics Lab (BBML) wwwiupuiedu~bbml Facebook BoneBiologyMechanicsLab

BBML - All R

ights Reserve

d

• Slide Number 1
• General Seminar Outline
• Bonersquos Hierarchical Structure
• Critical Gap in Understanding Collagen
• Methods For Analyzing Collagen Structure
• Methods For Imaging Collagen Structure
• Cryo-TEM
• Atomic Force Microscopy (AFM)
• AFM Image of Pentacene (C22H14)
• AFM Image of Hexabenzocoronene
• Basics of AFM Imaging The Probe
• Basics of AFM Imaging System Components
• van der Waals Potential Energy Curve
• Contact Mode ndash Monitor Deflection
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Peak Force Tapping Mode ndash Monitor Force
• AFM Applications in Bone Research Imaging
• AFM Imaging of Collagen
• Imaging of Osteoclast Activity
• Mineralized collagen deposition in vitro
• Imaging of Canilicular Structure
• AFM Imaging in Bone
• AFM Imaging in Bone
• Removing Mineral in Bone to Expose Collagen
• Removing Mineral in Bone to Expose Collagen
• Demineralization with EDTA
• Demineralization with EDTA
• Demineralization with EDTA
• Issues to Consider Effects of Acid
• Slide Number 32
• Slide Number 33
• Analysis of Collagen Structure in Bone
• Analysis of Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril D-Spacing
• Slide Number 39
• Collagen in Bone Fibril D-Spacing
• Collagen in Bone Fibril D-Spacing
• 2D Fast Fourier Transform Analysis
• 2D Fast Fourier Transform Analysis
• D-Spacing Distribution
• D-Spacing Distribution
• Cumulative Distribution Function
• CDF vs Histogram
• Changes in D-Spacing with Disease
• Changes in D-Spacing with Disease
• Slide Number 50
• Issues to Consider Site Selection and Sampling
• Issues to Consider Site Selection and Sampling
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• AFM calibration ndash Take Away Message
• AFM Applications in Bone ResearchIndentation Mechanics
• AFM-Based Indentation
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Parameters
• Hertz Contact Equation
• Hertz Contact Equation
• Hertz Contact Equation
• Proper Indentation Model is Essential
• Indentation in OI Tendon Study
• OI Tendon Differential Effects of Dehydration
• OI Tendon Differential Effects of Dehydration
• Indentation in Diabetic Tendon (Poster 0495)
• Multiscale Diabetes Study (Poster 0495)
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• A Note on Peak Force QNM for Bruker AFMs
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• Slide Number 83
• Slide Number 84
• AFM Applications in Bone ResearchOther Cool Mechanical Techniques
• ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo
• Other Mechanical Techniques
• Other Cool Techniques
• Summary Things to rememberhellip
• Acknowledgements
• Bone Biology and Mechanics Lab (BBML)

Collagen in Bone Fibril D-Spacing Many studies have shown observable D-spacing content that near theoretical value

Sasaki 2002 67 plusmn 2 nm Bozek 2005 665 plusmn 14 nm Ge 2007 666 plusmn 38 nm

Habelitz J Structural Bio 2002

A 2002 study in dentin got me interestedhellip

n = 322 wet n = 363 dry

BBML - All R

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Collagen in Bone Fibril D-Spacing

bull Internal structure bull Wallace et al Bone 2010 46 1349-1354 bull Wallace et al J Stuct Bio 2011 173 146-152 bull Kemp et al J Stuct Bio 2012 180 428-438 bull Warden et al Endocrinology 2013 154(9)

3178-3187 bull Gallant et al Bone 2014 61 191-200 bull Bart et al Conn Tiss Res 2014 In Press

bull Enzymatic and non-

enzymatic cross-linking bull Hammond et al Bone 2014 60 26-32 bull Diaz Gonzalez et al J Biomech 2014 47(3)

681-686 bull Voytik-Harbin et al In Preparation 2014

D-periodic spacing important structural feature that captures aspects related to

Mouse Femur Mid-diaphysis BBML - All R

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d

2D Fast Fourier Transform Analysis

Harmonics

Fibril D-Spacing

Erickson B Wallace JM et al Biotechnology Journal 2013

Raw AFM Image 2D FFT Power Spectrum

BBML - All R

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d

2D Fast Fourier Transform Analysis

Harmonics

Fibril D-Spacing

Raw AFM Image 2D FFT Power Spectrum

0

20000

40000

60000

80000

100000

120000

0 001 002 003 004 005 006

Am

plitu

de (

microV2 )

Position (1nm)

D-periodic spacing 0015 (1nm) ~ 67 nm

BBML - All R

ights Reserve

d

D-Spacing Distribution

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Overall Mean 670 plusmn 00 nm

BBML - All R

ights Reserve

d

D-Spacing Distribution

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Overall Mean 670 plusmn 00 nm

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

4 Samples Total of n = 255 fibrils Overall Mean 682 plusmn 12 nm

BBML - All R

ights Reserve

d

Cumulative Distribution Function

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

This population 255 fibrils - Each fibril 1255 = 0392 - Data range 628-716 nm

BBML - All R

ights Reserve

d

CDF vs Histogram 62 63 64 65 66 67 68 69 70 71 72 73 74

0

10

20

30

40

50

60

70

80

90

100

0

5

10

15

20

25

30

35

40

45

50

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

HistoCDF

BBML - All R

ights Reserve

d

Changes in D-Spacing with Disease

0

5

10

15

20

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

0

5

10

15

20

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

Wallace et al Bone 2010 46 1349-1354

KS Test plt0001

Control 680 plusmn 26 nm

Estrogen Dep 659 plusmn 31 nm

t-test on mean p=0048

BBML - All R

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d

Changes in D-Spacing with Disease

Wallace et al Bone 2010 46 1349-1354

0

10

20

30

40

50

60

70

80

90

100

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

KS Test plt0001

BBML - All R

ights Reserve

d

Question Model and Tissue Morphology Publication

Tissue Specificity in Mice Male mouse 8 weeks Femur dentin tail tendon

No differences in bone vs dentin both ∆ vs tendon

Wallace et al Langmuir 2011 26(10) 7349-7354

Gender Effects Male and female mice 16 weeks Femur No differences NA

Estrogen Depletion OVX in sheep 5 years + 2 Radius Mean and population shift Wallace et al Bone

2010 46 1349-1354

Estrogen Depletion OVX in female 5 years + 2 Skin (dermis) Significant population shift Erickson et al Biotech

J 2013 8(1) 1174-126

Osteoporosis and Mechanical Loading

OVX in ulnar loading in rats Ulna

No loading effect population shift with OVX

Warden et al Endocrinology 2013 154(9) 3178-3187

Osteogenesis Imperfecta Brtl+ model male mice 2 months Femur

No mean ∆ widening of distribution

Wallace et al J Stuct Bio 2011 173 146-152

Osteogenesis Imperfecta Oimoim model male mice 12 weeks Femur Mean and population shift Bart et al Conn Tiss

Res 2014 In Press

Osteogenesis Imperfecta and Tissue Hydration

Brtl+ model male mice 6 months Tail Tendon Significant population shift Kemp et al J Stuct Bio

2012 180 428-438

Type 2 Diabetes Male ZDSD rats 30 weeks Tibia Significant population shift Hammond et al Bone

2014 60 26-32

Type 2 Diabetes Female ZDSD rats 32 weeks Tail Tendon Significant population shift

Diaz Gonzalez et al J Biomech 2014 47(3)

681-686

Raloxifene (in vivo and in vitro) Female dog treated with Raloxifene Femur Significant population shift Gallant et al Bone

2014 61 191-200 BBML - All R

ights Reserve

d

Issues to Consider Site Selection and Sampling

KS Test plt0001

0023 of bonersquos length

15 m

m =

150

00 micro

m

Wallace et al Langmuir 2011 26(10) 7349-7354

100 microm x 100 microm

35 microm x 35 microm

BBML - All R

ights Reserve

d

Issues to Consider Site Selection and Sampling

60

62

64

66

68

70

72

74

1 (35)

2 (33)

3 (40)

4 (39)

5 (35)

6 (29)

7 (26)

8 (23)

9 (28)

WT (288)

D-P

erio

dic

Gap

Ove

rlap

Spac

ing

(nm

)

Axial Location (Proximal End = 1 Distal End = 9)

( of Fibrils)

91

23

45

87

6

Wallace et al Langmuir 2011 26(10) 7349-7354

288 fibrils for population n=1 for mean comparisons BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

Wet Tendon Dry Tendon Kemp et al J StructBio 2012 180 (3) 428-438

Collagen and bone exist in a hydrated environment in vivo bull Hydration state can impact morphology and mechanics

BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

Nonsignificant mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet (n=255) 682 plusmn 12 nm

WT Dry (n=257) 686 plusmn 08 nm

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

Brtl DryBrtl Wet

KS Test plt0001

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

WT Case 04 nm mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet 682 plusmn 12 nm

WT Dry 686 plusmn 08 nm

Brtl+ Wet 675 plusmn 14 nm

Brtl+ Dry 689 plusmn 10 nm

Brtl+ Case 14 nm increase with drying (plt0001) bull Entire population shifted upward (plt0001)

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT WetBrtl Wet

KS Test plt0001

Brtl+ distribution shifted downward vs WT when wet

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryBrtl Dry

KS Test plt0001

Brtl+ distribution shifted upward vs WT when dry

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider AFM calibration

bull Absolute distance measurements require accurate piezo calibration

bull AFM systems have large scan ranges (150 microm to lt 1 microm)

bull Scanner non-linearities can introduce substantial error over that range

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

ights Reserve

d

Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

BBML - All R

ights Reserve

d

Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

100 nm 10 80 602 1198 100 nm 7 60 827 1439 100 nm 5 40 814 1427 100 nm 35 20 917 1724 100 nm 2 10 937 1774

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

ights Reserve

d

AFM calibration ndash Take Away Message

Calibration using a scan size and feature size comparable to features of interest is highly recommended

Calibration addresses absolute accuracy not precision (differential sensitivity between measurements)

bull Does not limit ability to differentiate between groups using the same AFM with the same calibration parameters

BBML - All R

ights Reserve

d

AFM Applications in Bone Research Indentation Mechanics

BBML - All R

ights Reserve

d

AFM-Based Indentation

In addition to high resolution imaging AFM can be used to extract nanoscale mechanical data

ApproachWithdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Can

tilev

er D

efle

ctio

n (V

olts

)

1 Ramp into a rigid sample ndash measure the slope

Deflection sensitivity x 119899119899119899119899119881119881

2 Calculate cantilever spring constant

3 Hookersquos law takes deflection (volts) and converts to units of force F = kx

Spring constant k 119873119873119899119899

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Forc

e (n

N)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration Fo

rce

(nN

)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

Tip-Sample Separation (nm)

BBML - All R

ights Reserve

d

AFM-Based Indentation Parameters

Approach Withdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

Peak Force

Area Between Curves Energy Dissipation

Contact Mechanics Fit for Elastic Modulus (E)

Adhesion Force

Indentation Depth (δ)

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

z

r

δ

R

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119877119877 ∙ 12057512057532

Es Sample modulus νs Poissonrsquos ratio R tip radius of curvature δ load point displacement

Relevant Hertz Assumptions bull Strains are small and within the elastic limit bull Indenter is infinitely stiff (only sample deforms) bull Indentation depth ltlt radius of curvature of indenter bull Surfaces are continuous non-conforming (must be flat) bull Load is normal to surface

BBML - All R

ights Reserve

d

Proper Indentation Model is Essential

δ

r

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119903119903 ∙ 12057512057532

δ

α

119865119865 =2120587120587∙

1198641198641199041199041 minus 1205841205841199041199042

∙ tan120572120572 ∙ 1205751205752

Es Sample modulus νs Poissonrsquos ratio R tip radius α opening angle

Hertz Model Spherical Indenter

depth ltlt radius of curvature

Sneddon Model Conical Indenter

depth ge radius of curvature

BBML - All R

ights Reserve

d

Indentation in OI Tendon Study

x

z

yBrtl+ and WT mice 6 month old males

4 tails per group

2-3 fascicles per tail

25-30 fibrils per tail

4-5 locations per fibril

Indent force 20 nN wet (Sneddon) 50 nN dry (Hertz) BBML - A

ll Rights R

eserved

OI Tendon Differential Effects of Dehydration

Dry 1268 plusmn 718 MPa

Wet 305 plusmn 243 MPa

Dry 1737 plusmn 973 MPa

Wet 301 plusmn 202 MPa

Kemp et al J StructBio 2012 180 (3) 428-438

bull Significant stiffening of fibrils with dehydration

bull More pronounced modulus modulus as a function of disease

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ights Reserve

d

OI Tendon Differential Effects of Dehydration

Kemp et al J StructBio 2012 180 (3) 428-438

Diseased phenotype not present when wet phenotype observed when dried

0

10

20

30

40

50

60

0 20 40 60 80 100 120 140 160 180 200

Gro

up S

ampl

es (

)

Indent Modulus (MPa)

WT WetBrtl Wet

0

2

4

6

8

10

12

14

16

18

20

025

050

075

010

0012

5015

0017

5020

0022

5025

0027

5030

0032

5035

0037

5040

0042

5045

0047

5050

00

Gro

up S

ampl

es (

)Indent Modulus (MPa)

WT DryBrtl Dry

555 WT Indents 305 plusmn 243 MPa

614 Brtl+ Indents 301 plusmn 202 MPa

KS Test p=0129 t Test p=0464 464 WT Indents

1268 plusmn 718 MPa 479 Brtl+ Indents 1737 plusmn 973 MPa

KS Test plt0001 T Test plt0001

BBML - All R

ights Reserve

d

Indentation in Diabetic Tendon (Poster 0495)

x

z

y

Control and Diabetic Rat Tail Tendon

4-5 rats per group

2-3 fascicles per rat

~70 fibrilstail

4-5 locationsfibril

20 nN (Sneddon)

BBML - All R

ights Reserve

d

Multiscale Diabetes Study (Poster 0495)

0

5

10

15

20

25

300 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 gt25

Perc

enta

ge o

f Gro

up S

ampl

es

Elastic Modulus (MPa)

CD (1530 indents)ZDSD (913 indents)

CD (n=5) 381 plusmn 149 MPa

ZDSD (n=3) 706 plusmn 263 MPa

0

100

200

300

400

500

600

700

800

900

0 2 4 6 8 10

Mic

rosc

ale

Mod

ulus

(MPa

)

Nanoscale Modulus (MPa)

plt0001

r2=0531 p=0040

bull Increased indentation modulus in diabetic tendon

bull Strong relationship between nanoscale indent modulus and microscale tensile modulus

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

Proper probe selection for intended application bull Cantilever must be stiff relative to sample bull Sharp probes may damage sample easily dull

R or α must be determined for each probe bull Properties provided by manufacturer are unreliable bull Vary from probe to probe can dull during indent process

Spring constant must be determined for each probe

bull Value provided by manufacturer is inaccurate bull Thermal tuning OK for ldquosoftrdquo probes poor performance

for stiffer probes bull Sader method (geometry) added mass reference

cantilevers

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

Veeco Application Technical note Ben Ohler Practical Advice on the Determination of Cantilever Spring Constants

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

bull Sample processing

bull Sample hydration

bull Proper force range

bull Substrate effects for thin samples

bull Proper modulus model and range of data goodness of fit

Bearing in mind these assumptions and limitations AFM can provide true ldquonanoscalerdquo mechanical properties both in terms of forces and deformations

BBML - All R

ights Reserve

d

A Note on Peak Force QNM for Bruker AFMs bull Mechanical properties mapped pixel-by-pixel at same

resolution as the height image (PeakForce QNM)

Height Modulus BBML - All R

ights Reserve

d

A Note on Peak Force QNM

Correct probe stiffness must be used based on sample Pittenger ndash Veeco Product Bulletin

BBML - All R

ights Reserve

d

A Note on Peak Force QNM Extremely sensitive to calibration (details rarely given) Absolute radius and spring constant known

bull especially important from probe to probe bull radius uarr with indent depth ndash must determine R at depth

you expect to image bull Depth may vary as a function of sample (or at different

regions within the sample)

Relative reference material of ldquoknownrdquo modulus used to determine a ratio of 119896119896 119877119877 bull ldquoknownrdquo modulus may come from tension compression

and not indentation bull R is related to depth indentation depth on sample must

be same as on reference BBML - All R

ights Reserve

d

A Note on Peak Force QNM bull Modulus fit on the fly ndash no ability to monitor fit (or range) bull Local sample topography and sample tilt play a role in

measured parameters bull Assuming all modulus values in an image are correct how to

quantify

512 x 512 pixels = 262144 measurements

n=1 n=262144

n from selected ROIs

BBML - All R

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d

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d

bull Imaging in air on mica ndash substrate effect

bull Same imaging force or indent depth for all

bull For each area were all modulus values pooled

Lamprou et al PLoS ONE 2013 BBML - All R

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d

AFM Applications in Bone Research Other Cool Mechanical Techniques

BBML - All R

ights Reserve

d

ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo Bonds within or between collagen fibrils in bone that rupture to dissipate energy as a toughening mechanism

Thompson et al Nature 2001

Fantner et al Nature Mat 2005

Hansma et al JMNI 2005

Fantner et al Nano Letters 2007 BBML - All R

ights Reserve

d

Other Mechanical Techniques

Jimenez-Palomar J Mech Beh Bio Mat 2012

van der Rijt Macro Bio 2004

Yang J Biomed Mater Res 2007

Yang Biophysical Journal 2008 BBML - All R

ights Reserve

d

Other Cool Techniques bull Piezoresponse Force Microscopy (PFM)

bull Electrochemical AFM (EC-AFM)

bull Force Modulation Microscopy (FMM)

bull Lateral Force Microscopy (LFM)

bull Magnetic Force Microscopy (MFM)

bull Conductive AFM (C-AFM)

bull Force Pulling

bull Cantilever Bending

bull Tensile testing of individual fibrils

bull 3 point bending of fibrils

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ights Reserve

d

Summary Things to rememberhellip

bull Imaging mode imaging medium and probe selection

bull Morphology Measurements bull Sample preparation and hydration state bull Measuring fibril diameter bull Site selection and proper sampling bull Appropriate system calibration

bull Mechanical Measurements

bull Probe selection (stiffness and tip radius) bull Probe calibration (radius angle spring constant) bull Indentation force range and mechanical model bull Hydration in biological samples bull PeakForce QNM

BBML - All R

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d

Acknowledgements BBML

Max Hammond Armando Diaz Gonzalez Zachary Bart Silvia Canelon Creasy Clauser

IU School of Medicine Dr David Burr Dr Matt Allen Dr Stuart Warden Dr Lilian Plotkin

Funding Sources NIHNIDCR 1F32DE018840-01 A1 IUPUI BME Departmental Startup Funds IUPUI Research Support Funds Grant IUPUI Biomechanics and Biomaterials Research Center

NIH NICHD Dr Joan Marini

BBML - All R

ights Reserve

d

Bone Biology and Mechanics Lab (BBML) wwwiupuiedu~bbml Facebook BoneBiologyMechanicsLab

BBML - All R

ights Reserve

d

• Slide Number 1
• General Seminar Outline
• Bonersquos Hierarchical Structure
• Critical Gap in Understanding Collagen
• Methods For Analyzing Collagen Structure
• Methods For Imaging Collagen Structure
• Cryo-TEM
• Atomic Force Microscopy (AFM)
• AFM Image of Pentacene (C22H14)
• AFM Image of Hexabenzocoronene
• Basics of AFM Imaging The Probe
• Basics of AFM Imaging System Components
• van der Waals Potential Energy Curve
• Contact Mode ndash Monitor Deflection
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Peak Force Tapping Mode ndash Monitor Force
• AFM Applications in Bone Research Imaging
• AFM Imaging of Collagen
• Imaging of Osteoclast Activity
• Mineralized collagen deposition in vitro
• Imaging of Canilicular Structure
• AFM Imaging in Bone
• AFM Imaging in Bone
• Removing Mineral in Bone to Expose Collagen
• Removing Mineral in Bone to Expose Collagen
• Demineralization with EDTA
• Demineralization with EDTA
• Demineralization with EDTA
• Issues to Consider Effects of Acid
• Slide Number 32
• Slide Number 33
• Analysis of Collagen Structure in Bone
• Analysis of Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril D-Spacing
• Slide Number 39
• Collagen in Bone Fibril D-Spacing
• Collagen in Bone Fibril D-Spacing
• 2D Fast Fourier Transform Analysis
• 2D Fast Fourier Transform Analysis
• D-Spacing Distribution
• D-Spacing Distribution
• Cumulative Distribution Function
• CDF vs Histogram
• Changes in D-Spacing with Disease
• Changes in D-Spacing with Disease
• Slide Number 50
• Issues to Consider Site Selection and Sampling
• Issues to Consider Site Selection and Sampling
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• AFM calibration ndash Take Away Message
• AFM Applications in Bone ResearchIndentation Mechanics
• AFM-Based Indentation
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Parameters
• Hertz Contact Equation
• Hertz Contact Equation
• Hertz Contact Equation
• Proper Indentation Model is Essential
• Indentation in OI Tendon Study
• OI Tendon Differential Effects of Dehydration
• OI Tendon Differential Effects of Dehydration
• Indentation in Diabetic Tendon (Poster 0495)
• Multiscale Diabetes Study (Poster 0495)
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• A Note on Peak Force QNM for Bruker AFMs
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• Slide Number 83
• Slide Number 84
• AFM Applications in Bone ResearchOther Cool Mechanical Techniques
• ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo
• Other Mechanical Techniques
• Other Cool Techniques
• Summary Things to rememberhellip
• Acknowledgements
• Bone Biology and Mechanics Lab (BBML)

Collagen in Bone Fibril D-Spacing

bull Internal structure bull Wallace et al Bone 2010 46 1349-1354 bull Wallace et al J Stuct Bio 2011 173 146-152 bull Kemp et al J Stuct Bio 2012 180 428-438 bull Warden et al Endocrinology 2013 154(9)

3178-3187 bull Gallant et al Bone 2014 61 191-200 bull Bart et al Conn Tiss Res 2014 In Press

bull Enzymatic and non-

enzymatic cross-linking bull Hammond et al Bone 2014 60 26-32 bull Diaz Gonzalez et al J Biomech 2014 47(3)

681-686 bull Voytik-Harbin et al In Preparation 2014

D-periodic spacing important structural feature that captures aspects related to

Mouse Femur Mid-diaphysis BBML - All R

ights Reserve

d

2D Fast Fourier Transform Analysis

Harmonics

Fibril D-Spacing

Erickson B Wallace JM et al Biotechnology Journal 2013

Raw AFM Image 2D FFT Power Spectrum

BBML - All R

ights Reserve

d

2D Fast Fourier Transform Analysis

Harmonics

Fibril D-Spacing

Raw AFM Image 2D FFT Power Spectrum

0

20000

40000

60000

80000

100000

120000

0 001 002 003 004 005 006

Am

plitu

de (

microV2 )

Position (1nm)

D-periodic spacing 0015 (1nm) ~ 67 nm

BBML - All R

ights Reserve

d

D-Spacing Distribution

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Overall Mean 670 plusmn 00 nm

BBML - All R

ights Reserve

d

D-Spacing Distribution

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Overall Mean 670 plusmn 00 nm

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

4 Samples Total of n = 255 fibrils Overall Mean 682 plusmn 12 nm

BBML - All R

ights Reserve

d

Cumulative Distribution Function

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

This population 255 fibrils - Each fibril 1255 = 0392 - Data range 628-716 nm

BBML - All R

ights Reserve

d

CDF vs Histogram 62 63 64 65 66 67 68 69 70 71 72 73 74

0

10

20

30

40

50

60

70

80

90

100

0

5

10

15

20

25

30

35

40

45

50

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

HistoCDF

BBML - All R

ights Reserve

d

Changes in D-Spacing with Disease

0

5

10

15

20

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

0

5

10

15

20

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

Wallace et al Bone 2010 46 1349-1354

KS Test plt0001

Control 680 plusmn 26 nm

Estrogen Dep 659 plusmn 31 nm

t-test on mean p=0048

BBML - All R

ights Reserve

d

Changes in D-Spacing with Disease

Wallace et al Bone 2010 46 1349-1354

0

10

20

30

40

50

60

70

80

90

100

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

KS Test plt0001

BBML - All R

ights Reserve

d

Question Model and Tissue Morphology Publication

Tissue Specificity in Mice Male mouse 8 weeks Femur dentin tail tendon

No differences in bone vs dentin both ∆ vs tendon

Wallace et al Langmuir 2011 26(10) 7349-7354

Gender Effects Male and female mice 16 weeks Femur No differences NA

Estrogen Depletion OVX in sheep 5 years + 2 Radius Mean and population shift Wallace et al Bone

2010 46 1349-1354

Estrogen Depletion OVX in female 5 years + 2 Skin (dermis) Significant population shift Erickson et al Biotech

J 2013 8(1) 1174-126

Osteoporosis and Mechanical Loading

OVX in ulnar loading in rats Ulna

No loading effect population shift with OVX

Warden et al Endocrinology 2013 154(9) 3178-3187

Osteogenesis Imperfecta Brtl+ model male mice 2 months Femur

No mean ∆ widening of distribution

Wallace et al J Stuct Bio 2011 173 146-152

Osteogenesis Imperfecta Oimoim model male mice 12 weeks Femur Mean and population shift Bart et al Conn Tiss

Res 2014 In Press

Osteogenesis Imperfecta and Tissue Hydration

Brtl+ model male mice 6 months Tail Tendon Significant population shift Kemp et al J Stuct Bio

2012 180 428-438

Type 2 Diabetes Male ZDSD rats 30 weeks Tibia Significant population shift Hammond et al Bone

2014 60 26-32

Type 2 Diabetes Female ZDSD rats 32 weeks Tail Tendon Significant population shift

Diaz Gonzalez et al J Biomech 2014 47(3)

681-686

Raloxifene (in vivo and in vitro) Female dog treated with Raloxifene Femur Significant population shift Gallant et al Bone

2014 61 191-200 BBML - All R

ights Reserve

d

Issues to Consider Site Selection and Sampling

KS Test plt0001

0023 of bonersquos length

15 m

m =

150

00 micro

m

Wallace et al Langmuir 2011 26(10) 7349-7354

100 microm x 100 microm

35 microm x 35 microm

BBML - All R

ights Reserve

d

Issues to Consider Site Selection and Sampling

60

62

64

66

68

70

72

74

1 (35)

2 (33)

3 (40)

4 (39)

5 (35)

6 (29)

7 (26)

8 (23)

9 (28)

WT (288)

D-P

erio

dic

Gap

Ove

rlap

Spac

ing

(nm

)

Axial Location (Proximal End = 1 Distal End = 9)

( of Fibrils)

91

23

45

87

6

Wallace et al Langmuir 2011 26(10) 7349-7354

288 fibrils for population n=1 for mean comparisons BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

Wet Tendon Dry Tendon Kemp et al J StructBio 2012 180 (3) 428-438

Collagen and bone exist in a hydrated environment in vivo bull Hydration state can impact morphology and mechanics

BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

Nonsignificant mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet (n=255) 682 plusmn 12 nm

WT Dry (n=257) 686 plusmn 08 nm

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

Brtl DryBrtl Wet

KS Test plt0001

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

WT Case 04 nm mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet 682 plusmn 12 nm

WT Dry 686 plusmn 08 nm

Brtl+ Wet 675 plusmn 14 nm

Brtl+ Dry 689 plusmn 10 nm

Brtl+ Case 14 nm increase with drying (plt0001) bull Entire population shifted upward (plt0001)

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT WetBrtl Wet

KS Test plt0001

Brtl+ distribution shifted downward vs WT when wet

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryBrtl Dry

KS Test plt0001

Brtl+ distribution shifted upward vs WT when dry

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider AFM calibration

bull Absolute distance measurements require accurate piezo calibration

bull AFM systems have large scan ranges (150 microm to lt 1 microm)

bull Scanner non-linearities can introduce substantial error over that range

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

ights Reserve

d

Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

BBML - All R

ights Reserve

d

Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

100 nm 10 80 602 1198 100 nm 7 60 827 1439 100 nm 5 40 814 1427 100 nm 35 20 917 1724 100 nm 2 10 937 1774

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

ights Reserve

d

AFM calibration ndash Take Away Message

Calibration using a scan size and feature size comparable to features of interest is highly recommended

Calibration addresses absolute accuracy not precision (differential sensitivity between measurements)

bull Does not limit ability to differentiate between groups using the same AFM with the same calibration parameters

BBML - All R

ights Reserve

d

AFM Applications in Bone Research Indentation Mechanics

BBML - All R

ights Reserve

d

AFM-Based Indentation

In addition to high resolution imaging AFM can be used to extract nanoscale mechanical data

ApproachWithdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Can

tilev

er D

efle

ctio

n (V

olts

)

1 Ramp into a rigid sample ndash measure the slope

Deflection sensitivity x 119899119899119899119899119881119881

2 Calculate cantilever spring constant

3 Hookersquos law takes deflection (volts) and converts to units of force F = kx

Spring constant k 119873119873119899119899

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Forc

e (n

N)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration Fo

rce

(nN

)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

Tip-Sample Separation (nm)

BBML - All R

ights Reserve

d

AFM-Based Indentation Parameters

Approach Withdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

Peak Force

Area Between Curves Energy Dissipation

Contact Mechanics Fit for Elastic Modulus (E)

Adhesion Force

Indentation Depth (δ)

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

z

r

δ

R

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119877119877 ∙ 12057512057532

Es Sample modulus νs Poissonrsquos ratio R tip radius of curvature δ load point displacement

Relevant Hertz Assumptions bull Strains are small and within the elastic limit bull Indenter is infinitely stiff (only sample deforms) bull Indentation depth ltlt radius of curvature of indenter bull Surfaces are continuous non-conforming (must be flat) bull Load is normal to surface

BBML - All R

ights Reserve

d

Proper Indentation Model is Essential

δ

r

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119903119903 ∙ 12057512057532

δ

α

119865119865 =2120587120587∙

1198641198641199041199041 minus 1205841205841199041199042

∙ tan120572120572 ∙ 1205751205752

Es Sample modulus νs Poissonrsquos ratio R tip radius α opening angle

Hertz Model Spherical Indenter

depth ltlt radius of curvature

Sneddon Model Conical Indenter

depth ge radius of curvature

BBML - All R

ights Reserve

d

Indentation in OI Tendon Study

x

z

yBrtl+ and WT mice 6 month old males

4 tails per group

2-3 fascicles per tail

25-30 fibrils per tail

4-5 locations per fibril

Indent force 20 nN wet (Sneddon) 50 nN dry (Hertz) BBML - A

ll Rights R

eserved

OI Tendon Differential Effects of Dehydration

Dry 1268 plusmn 718 MPa

Wet 305 plusmn 243 MPa

Dry 1737 plusmn 973 MPa

Wet 301 plusmn 202 MPa

Kemp et al J StructBio 2012 180 (3) 428-438

bull Significant stiffening of fibrils with dehydration

bull More pronounced modulus modulus as a function of disease

BBML - All R

ights Reserve

d

OI Tendon Differential Effects of Dehydration

Kemp et al J StructBio 2012 180 (3) 428-438

Diseased phenotype not present when wet phenotype observed when dried

0

10

20

30

40

50

60

0 20 40 60 80 100 120 140 160 180 200

Gro

up S

ampl

es (

)

Indent Modulus (MPa)

WT WetBrtl Wet

0

2

4

6

8

10

12

14

16

18

20

025

050

075

010

0012

5015

0017

5020

0022

5025

0027

5030

0032

5035

0037

5040

0042

5045

0047

5050

00

Gro

up S

ampl

es (

)Indent Modulus (MPa)

WT DryBrtl Dry

555 WT Indents 305 plusmn 243 MPa

614 Brtl+ Indents 301 plusmn 202 MPa

KS Test p=0129 t Test p=0464 464 WT Indents

1268 plusmn 718 MPa 479 Brtl+ Indents 1737 plusmn 973 MPa

KS Test plt0001 T Test plt0001

BBML - All R

ights Reserve

d

Indentation in Diabetic Tendon (Poster 0495)

x

z

y

Control and Diabetic Rat Tail Tendon

4-5 rats per group

2-3 fascicles per rat

~70 fibrilstail

4-5 locationsfibril

20 nN (Sneddon)

BBML - All R

ights Reserve

d

Multiscale Diabetes Study (Poster 0495)

0

5

10

15

20

25

300 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 gt25

Perc

enta

ge o

f Gro

up S

ampl

es

Elastic Modulus (MPa)

CD (1530 indents)ZDSD (913 indents)

CD (n=5) 381 plusmn 149 MPa

ZDSD (n=3) 706 plusmn 263 MPa

0

100

200

300

400

500

600

700

800

900

0 2 4 6 8 10

Mic

rosc

ale

Mod

ulus

(MPa

)

Nanoscale Modulus (MPa)

plt0001

r2=0531 p=0040

bull Increased indentation modulus in diabetic tendon

bull Strong relationship between nanoscale indent modulus and microscale tensile modulus

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

Proper probe selection for intended application bull Cantilever must be stiff relative to sample bull Sharp probes may damage sample easily dull

R or α must be determined for each probe bull Properties provided by manufacturer are unreliable bull Vary from probe to probe can dull during indent process

Spring constant must be determined for each probe

bull Value provided by manufacturer is inaccurate bull Thermal tuning OK for ldquosoftrdquo probes poor performance

for stiffer probes bull Sader method (geometry) added mass reference

cantilevers

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

Veeco Application Technical note Ben Ohler Practical Advice on the Determination of Cantilever Spring Constants

BBML - All R

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d

Indentation Limitations and Weaknesses

bull Sample processing

bull Sample hydration

bull Proper force range

bull Substrate effects for thin samples

bull Proper modulus model and range of data goodness of fit

Bearing in mind these assumptions and limitations AFM can provide true ldquonanoscalerdquo mechanical properties both in terms of forces and deformations

BBML - All R

ights Reserve

d

A Note on Peak Force QNM for Bruker AFMs bull Mechanical properties mapped pixel-by-pixel at same

resolution as the height image (PeakForce QNM)

Height Modulus BBML - All R

ights Reserve

d

A Note on Peak Force QNM

Correct probe stiffness must be used based on sample Pittenger ndash Veeco Product Bulletin

BBML - All R

ights Reserve

d

A Note on Peak Force QNM Extremely sensitive to calibration (details rarely given) Absolute radius and spring constant known

bull especially important from probe to probe bull radius uarr with indent depth ndash must determine R at depth

you expect to image bull Depth may vary as a function of sample (or at different

regions within the sample)

Relative reference material of ldquoknownrdquo modulus used to determine a ratio of 119896119896 119877119877 bull ldquoknownrdquo modulus may come from tension compression

and not indentation bull R is related to depth indentation depth on sample must

be same as on reference BBML - All R

ights Reserve

d

A Note on Peak Force QNM bull Modulus fit on the fly ndash no ability to monitor fit (or range) bull Local sample topography and sample tilt play a role in

measured parameters bull Assuming all modulus values in an image are correct how to

quantify

512 x 512 pixels = 262144 measurements

n=1 n=262144

n from selected ROIs

BBML - All R

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d

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d

bull Imaging in air on mica ndash substrate effect

bull Same imaging force or indent depth for all

bull For each area were all modulus values pooled

Lamprou et al PLoS ONE 2013 BBML - All R

ights Reserve

d

AFM Applications in Bone Research Other Cool Mechanical Techniques

BBML - All R

ights Reserve

d

ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo Bonds within or between collagen fibrils in bone that rupture to dissipate energy as a toughening mechanism

Thompson et al Nature 2001

Fantner et al Nature Mat 2005

Hansma et al JMNI 2005

Fantner et al Nano Letters 2007 BBML - All R

ights Reserve

d

Other Mechanical Techniques

Jimenez-Palomar J Mech Beh Bio Mat 2012

van der Rijt Macro Bio 2004

Yang J Biomed Mater Res 2007

Yang Biophysical Journal 2008 BBML - All R

ights Reserve

d

Other Cool Techniques bull Piezoresponse Force Microscopy (PFM)

bull Electrochemical AFM (EC-AFM)

bull Force Modulation Microscopy (FMM)

bull Lateral Force Microscopy (LFM)

bull Magnetic Force Microscopy (MFM)

bull Conductive AFM (C-AFM)

bull Force Pulling

bull Cantilever Bending

bull Tensile testing of individual fibrils

bull 3 point bending of fibrils

BBML - All R

ights Reserve

d

Summary Things to rememberhellip

bull Imaging mode imaging medium and probe selection

bull Morphology Measurements bull Sample preparation and hydration state bull Measuring fibril diameter bull Site selection and proper sampling bull Appropriate system calibration

bull Mechanical Measurements

bull Probe selection (stiffness and tip radius) bull Probe calibration (radius angle spring constant) bull Indentation force range and mechanical model bull Hydration in biological samples bull PeakForce QNM

BBML - All R

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d

Acknowledgements BBML

Max Hammond Armando Diaz Gonzalez Zachary Bart Silvia Canelon Creasy Clauser

IU School of Medicine Dr David Burr Dr Matt Allen Dr Stuart Warden Dr Lilian Plotkin

Funding Sources NIHNIDCR 1F32DE018840-01 A1 IUPUI BME Departmental Startup Funds IUPUI Research Support Funds Grant IUPUI Biomechanics and Biomaterials Research Center

NIH NICHD Dr Joan Marini

BBML - All R

ights Reserve

d

Bone Biology and Mechanics Lab (BBML) wwwiupuiedu~bbml Facebook BoneBiologyMechanicsLab

BBML - All R

ights Reserve

d

• Slide Number 1
• General Seminar Outline
• Bonersquos Hierarchical Structure
• Critical Gap in Understanding Collagen
• Methods For Analyzing Collagen Structure
• Methods For Imaging Collagen Structure
• Cryo-TEM
• Atomic Force Microscopy (AFM)
• AFM Image of Pentacene (C22H14)
• AFM Image of Hexabenzocoronene
• Basics of AFM Imaging The Probe
• Basics of AFM Imaging System Components
• van der Waals Potential Energy Curve
• Contact Mode ndash Monitor Deflection
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Peak Force Tapping Mode ndash Monitor Force
• AFM Applications in Bone Research Imaging
• AFM Imaging of Collagen
• Imaging of Osteoclast Activity
• Mineralized collagen deposition in vitro
• Imaging of Canilicular Structure
• AFM Imaging in Bone
• AFM Imaging in Bone
• Removing Mineral in Bone to Expose Collagen
• Removing Mineral in Bone to Expose Collagen
• Demineralization with EDTA
• Demineralization with EDTA
• Demineralization with EDTA
• Issues to Consider Effects of Acid
• Slide Number 32
• Slide Number 33
• Analysis of Collagen Structure in Bone
• Analysis of Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril D-Spacing
• Slide Number 39
• Collagen in Bone Fibril D-Spacing
• Collagen in Bone Fibril D-Spacing
• 2D Fast Fourier Transform Analysis
• 2D Fast Fourier Transform Analysis
• D-Spacing Distribution
• D-Spacing Distribution
• Cumulative Distribution Function
• CDF vs Histogram
• Changes in D-Spacing with Disease
• Changes in D-Spacing with Disease
• Slide Number 50
• Issues to Consider Site Selection and Sampling
• Issues to Consider Site Selection and Sampling
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• AFM calibration ndash Take Away Message
• AFM Applications in Bone ResearchIndentation Mechanics
• AFM-Based Indentation
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Parameters
• Hertz Contact Equation
• Hertz Contact Equation
• Hertz Contact Equation
• Proper Indentation Model is Essential
• Indentation in OI Tendon Study
• OI Tendon Differential Effects of Dehydration
• OI Tendon Differential Effects of Dehydration
• Indentation in Diabetic Tendon (Poster 0495)
• Multiscale Diabetes Study (Poster 0495)
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• A Note on Peak Force QNM for Bruker AFMs
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• Slide Number 83
• Slide Number 84
• AFM Applications in Bone ResearchOther Cool Mechanical Techniques
• ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo
• Other Mechanical Techniques
• Other Cool Techniques
• Summary Things to rememberhellip
• Acknowledgements
• Bone Biology and Mechanics Lab (BBML)

2D Fast Fourier Transform Analysis

Harmonics

Fibril D-Spacing

Erickson B Wallace JM et al Biotechnology Journal 2013

Raw AFM Image 2D FFT Power Spectrum

BBML - All R

ights Reserve

d

2D Fast Fourier Transform Analysis

Harmonics

Fibril D-Spacing

Raw AFM Image 2D FFT Power Spectrum

0

20000

40000

60000

80000

100000

120000

0 001 002 003 004 005 006

Am

plitu

de (

microV2 )

Position (1nm)

D-periodic spacing 0015 (1nm) ~ 67 nm

BBML - All R

ights Reserve

d

D-Spacing Distribution

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Overall Mean 670 plusmn 00 nm

BBML - All R

ights Reserve

d

D-Spacing Distribution

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Overall Mean 670 plusmn 00 nm

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

4 Samples Total of n = 255 fibrils Overall Mean 682 plusmn 12 nm

BBML - All R

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d

Cumulative Distribution Function

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

This population 255 fibrils - Each fibril 1255 = 0392 - Data range 628-716 nm

BBML - All R

ights Reserve

d

CDF vs Histogram 62 63 64 65 66 67 68 69 70 71 72 73 74

0

10

20

30

40

50

60

70

80

90

100

0

5

10

15

20

25

30

35

40

45

50

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

HistoCDF

BBML - All R

ights Reserve

d

Changes in D-Spacing with Disease

0

5

10

15

20

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

0

5

10

15

20

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

Wallace et al Bone 2010 46 1349-1354

KS Test plt0001

Control 680 plusmn 26 nm

Estrogen Dep 659 plusmn 31 nm

t-test on mean p=0048

BBML - All R

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d

Changes in D-Spacing with Disease

Wallace et al Bone 2010 46 1349-1354

0

10

20

30

40

50

60

70

80

90

100

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

KS Test plt0001

BBML - All R

ights Reserve

d

Question Model and Tissue Morphology Publication

Tissue Specificity in Mice Male mouse 8 weeks Femur dentin tail tendon

No differences in bone vs dentin both ∆ vs tendon

Wallace et al Langmuir 2011 26(10) 7349-7354

Gender Effects Male and female mice 16 weeks Femur No differences NA

Estrogen Depletion OVX in sheep 5 years + 2 Radius Mean and population shift Wallace et al Bone

2010 46 1349-1354

Estrogen Depletion OVX in female 5 years + 2 Skin (dermis) Significant population shift Erickson et al Biotech

J 2013 8(1) 1174-126

Osteoporosis and Mechanical Loading

OVX in ulnar loading in rats Ulna

No loading effect population shift with OVX

Warden et al Endocrinology 2013 154(9) 3178-3187

Osteogenesis Imperfecta Brtl+ model male mice 2 months Femur

No mean ∆ widening of distribution

Wallace et al J Stuct Bio 2011 173 146-152

Osteogenesis Imperfecta Oimoim model male mice 12 weeks Femur Mean and population shift Bart et al Conn Tiss

Res 2014 In Press

Osteogenesis Imperfecta and Tissue Hydration

Brtl+ model male mice 6 months Tail Tendon Significant population shift Kemp et al J Stuct Bio

2012 180 428-438

Type 2 Diabetes Male ZDSD rats 30 weeks Tibia Significant population shift Hammond et al Bone

2014 60 26-32

Type 2 Diabetes Female ZDSD rats 32 weeks Tail Tendon Significant population shift

Diaz Gonzalez et al J Biomech 2014 47(3)

681-686

Raloxifene (in vivo and in vitro) Female dog treated with Raloxifene Femur Significant population shift Gallant et al Bone

2014 61 191-200 BBML - All R

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d

Issues to Consider Site Selection and Sampling

KS Test plt0001

0023 of bonersquos length

15 m

m =

150

00 micro

m

Wallace et al Langmuir 2011 26(10) 7349-7354

100 microm x 100 microm

35 microm x 35 microm

BBML - All R

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d

Issues to Consider Site Selection and Sampling

60

62

64

66

68

70

72

74

1 (35)

2 (33)

3 (40)

4 (39)

5 (35)

6 (29)

7 (26)

8 (23)

9 (28)

WT (288)

D-P

erio

dic

Gap

Ove

rlap

Spac

ing

(nm

)

Axial Location (Proximal End = 1 Distal End = 9)

( of Fibrils)

91

23

45

87

6

Wallace et al Langmuir 2011 26(10) 7349-7354

288 fibrils for population n=1 for mean comparisons BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

Wet Tendon Dry Tendon Kemp et al J StructBio 2012 180 (3) 428-438

Collagen and bone exist in a hydrated environment in vivo bull Hydration state can impact morphology and mechanics

BBML - All R

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d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

Nonsignificant mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet (n=255) 682 plusmn 12 nm

WT Dry (n=257) 686 plusmn 08 nm

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

Brtl DryBrtl Wet

KS Test plt0001

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

WT Case 04 nm mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet 682 plusmn 12 nm

WT Dry 686 plusmn 08 nm

Brtl+ Wet 675 plusmn 14 nm

Brtl+ Dry 689 plusmn 10 nm

Brtl+ Case 14 nm increase with drying (plt0001) bull Entire population shifted upward (plt0001)

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT WetBrtl Wet

KS Test plt0001

Brtl+ distribution shifted downward vs WT when wet

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryBrtl Dry

KS Test plt0001

Brtl+ distribution shifted upward vs WT when dry

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider AFM calibration

bull Absolute distance measurements require accurate piezo calibration

bull AFM systems have large scan ranges (150 microm to lt 1 microm)

bull Scanner non-linearities can introduce substantial error over that range

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

ights Reserve

d

Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

BBML - All R

ights Reserve

d

Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

100 nm 10 80 602 1198 100 nm 7 60 827 1439 100 nm 5 40 814 1427 100 nm 35 20 917 1724 100 nm 2 10 937 1774

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

ights Reserve

d

AFM calibration ndash Take Away Message

Calibration using a scan size and feature size comparable to features of interest is highly recommended

Calibration addresses absolute accuracy not precision (differential sensitivity between measurements)

bull Does not limit ability to differentiate between groups using the same AFM with the same calibration parameters

BBML - All R

ights Reserve

d

AFM Applications in Bone Research Indentation Mechanics

BBML - All R

ights Reserve

d

AFM-Based Indentation

In addition to high resolution imaging AFM can be used to extract nanoscale mechanical data

ApproachWithdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Can

tilev

er D

efle

ctio

n (V

olts

)

1 Ramp into a rigid sample ndash measure the slope

Deflection sensitivity x 119899119899119899119899119881119881

2 Calculate cantilever spring constant

3 Hookersquos law takes deflection (volts) and converts to units of force F = kx

Spring constant k 119873119873119899119899

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Forc

e (n

N)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration Fo

rce

(nN

)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

Tip-Sample Separation (nm)

BBML - All R

ights Reserve

d

AFM-Based Indentation Parameters

Approach Withdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

Peak Force

Area Between Curves Energy Dissipation

Contact Mechanics Fit for Elastic Modulus (E)

Adhesion Force

Indentation Depth (δ)

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

z

r

δ

R

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119877119877 ∙ 12057512057532

Es Sample modulus νs Poissonrsquos ratio R tip radius of curvature δ load point displacement

Relevant Hertz Assumptions bull Strains are small and within the elastic limit bull Indenter is infinitely stiff (only sample deforms) bull Indentation depth ltlt radius of curvature of indenter bull Surfaces are continuous non-conforming (must be flat) bull Load is normal to surface

BBML - All R

ights Reserve

d

Proper Indentation Model is Essential

δ

r

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119903119903 ∙ 12057512057532

δ

α

119865119865 =2120587120587∙

1198641198641199041199041 minus 1205841205841199041199042

∙ tan120572120572 ∙ 1205751205752

Es Sample modulus νs Poissonrsquos ratio R tip radius α opening angle

Hertz Model Spherical Indenter

depth ltlt radius of curvature

Sneddon Model Conical Indenter

depth ge radius of curvature

BBML - All R

ights Reserve

d

Indentation in OI Tendon Study

x

z

yBrtl+ and WT mice 6 month old males

4 tails per group

2-3 fascicles per tail

25-30 fibrils per tail

4-5 locations per fibril

Indent force 20 nN wet (Sneddon) 50 nN dry (Hertz) BBML - A

ll Rights R

eserved

OI Tendon Differential Effects of Dehydration

Dry 1268 plusmn 718 MPa

Wet 305 plusmn 243 MPa

Dry 1737 plusmn 973 MPa

Wet 301 plusmn 202 MPa

Kemp et al J StructBio 2012 180 (3) 428-438

bull Significant stiffening of fibrils with dehydration

bull More pronounced modulus modulus as a function of disease

BBML - All R

ights Reserve

d

OI Tendon Differential Effects of Dehydration

Kemp et al J StructBio 2012 180 (3) 428-438

Diseased phenotype not present when wet phenotype observed when dried

0

10

20

30

40

50

60

0 20 40 60 80 100 120 140 160 180 200

Gro

up S

ampl

es (

)

Indent Modulus (MPa)

WT WetBrtl Wet

0

2

4

6

8

10

12

14

16

18

20

025

050

075

010

0012

5015

0017

5020

0022

5025

0027

5030

0032

5035

0037

5040

0042

5045

0047

5050

00

Gro

up S

ampl

es (

)Indent Modulus (MPa)

WT DryBrtl Dry

555 WT Indents 305 plusmn 243 MPa

614 Brtl+ Indents 301 plusmn 202 MPa

KS Test p=0129 t Test p=0464 464 WT Indents

1268 plusmn 718 MPa 479 Brtl+ Indents 1737 plusmn 973 MPa

KS Test plt0001 T Test plt0001

BBML - All R

ights Reserve

d

Indentation in Diabetic Tendon (Poster 0495)

x

z

y

Control and Diabetic Rat Tail Tendon

4-5 rats per group

2-3 fascicles per rat

~70 fibrilstail

4-5 locationsfibril

20 nN (Sneddon)

BBML - All R

ights Reserve

d

Multiscale Diabetes Study (Poster 0495)

0

5

10

15

20

25

300 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 gt25

Perc

enta

ge o

f Gro

up S

ampl

es

Elastic Modulus (MPa)

CD (1530 indents)ZDSD (913 indents)

CD (n=5) 381 plusmn 149 MPa

ZDSD (n=3) 706 plusmn 263 MPa

0

100

200

300

400

500

600

700

800

900

0 2 4 6 8 10

Mic

rosc

ale

Mod

ulus

(MPa

)

Nanoscale Modulus (MPa)

plt0001

r2=0531 p=0040

bull Increased indentation modulus in diabetic tendon

bull Strong relationship between nanoscale indent modulus and microscale tensile modulus

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

Proper probe selection for intended application bull Cantilever must be stiff relative to sample bull Sharp probes may damage sample easily dull

R or α must be determined for each probe bull Properties provided by manufacturer are unreliable bull Vary from probe to probe can dull during indent process

Spring constant must be determined for each probe

bull Value provided by manufacturer is inaccurate bull Thermal tuning OK for ldquosoftrdquo probes poor performance

for stiffer probes bull Sader method (geometry) added mass reference

cantilevers

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

Veeco Application Technical note Ben Ohler Practical Advice on the Determination of Cantilever Spring Constants

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

bull Sample processing

bull Sample hydration

bull Proper force range

bull Substrate effects for thin samples

bull Proper modulus model and range of data goodness of fit

Bearing in mind these assumptions and limitations AFM can provide true ldquonanoscalerdquo mechanical properties both in terms of forces and deformations

BBML - All R

ights Reserve

d

A Note on Peak Force QNM for Bruker AFMs bull Mechanical properties mapped pixel-by-pixel at same

resolution as the height image (PeakForce QNM)

Height Modulus BBML - All R

ights Reserve

d

A Note on Peak Force QNM

Correct probe stiffness must be used based on sample Pittenger ndash Veeco Product Bulletin

BBML - All R

ights Reserve

d

A Note on Peak Force QNM Extremely sensitive to calibration (details rarely given) Absolute radius and spring constant known

bull especially important from probe to probe bull radius uarr with indent depth ndash must determine R at depth

you expect to image bull Depth may vary as a function of sample (or at different

regions within the sample)

Relative reference material of ldquoknownrdquo modulus used to determine a ratio of 119896119896 119877119877 bull ldquoknownrdquo modulus may come from tension compression

and not indentation bull R is related to depth indentation depth on sample must

be same as on reference BBML - All R

ights Reserve

d

A Note on Peak Force QNM bull Modulus fit on the fly ndash no ability to monitor fit (or range) bull Local sample topography and sample tilt play a role in

measured parameters bull Assuming all modulus values in an image are correct how to

quantify

512 x 512 pixels = 262144 measurements

n=1 n=262144

n from selected ROIs

BBML - All R

ights Reserve

d

BBML - All R

ights Reserve

d

bull Imaging in air on mica ndash substrate effect

bull Same imaging force or indent depth for all

bull For each area were all modulus values pooled

Lamprou et al PLoS ONE 2013 BBML - All R

ights Reserve

d

AFM Applications in Bone Research Other Cool Mechanical Techniques

BBML - All R

ights Reserve

d

ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo Bonds within or between collagen fibrils in bone that rupture to dissipate energy as a toughening mechanism

Thompson et al Nature 2001

Fantner et al Nature Mat 2005

Hansma et al JMNI 2005

Fantner et al Nano Letters 2007 BBML - All R

ights Reserve

d

Other Mechanical Techniques

Jimenez-Palomar J Mech Beh Bio Mat 2012

van der Rijt Macro Bio 2004

Yang J Biomed Mater Res 2007

Yang Biophysical Journal 2008 BBML - All R

ights Reserve

d

Other Cool Techniques bull Piezoresponse Force Microscopy (PFM)

bull Electrochemical AFM (EC-AFM)

bull Force Modulation Microscopy (FMM)

bull Lateral Force Microscopy (LFM)

bull Magnetic Force Microscopy (MFM)

bull Conductive AFM (C-AFM)

bull Force Pulling

bull Cantilever Bending

bull Tensile testing of individual fibrils

bull 3 point bending of fibrils

BBML - All R

ights Reserve

d

Summary Things to rememberhellip

bull Imaging mode imaging medium and probe selection

bull Morphology Measurements bull Sample preparation and hydration state bull Measuring fibril diameter bull Site selection and proper sampling bull Appropriate system calibration

bull Mechanical Measurements

bull Probe selection (stiffness and tip radius) bull Probe calibration (radius angle spring constant) bull Indentation force range and mechanical model bull Hydration in biological samples bull PeakForce QNM

BBML - All R

ights Reserve

d

Acknowledgements BBML

Max Hammond Armando Diaz Gonzalez Zachary Bart Silvia Canelon Creasy Clauser

IU School of Medicine Dr David Burr Dr Matt Allen Dr Stuart Warden Dr Lilian Plotkin

Funding Sources NIHNIDCR 1F32DE018840-01 A1 IUPUI BME Departmental Startup Funds IUPUI Research Support Funds Grant IUPUI Biomechanics and Biomaterials Research Center

NIH NICHD Dr Joan Marini

BBML - All R

ights Reserve

d

Bone Biology and Mechanics Lab (BBML) wwwiupuiedu~bbml Facebook BoneBiologyMechanicsLab

BBML - All R

ights Reserve

d

• Slide Number 1
• General Seminar Outline
• Bonersquos Hierarchical Structure
• Critical Gap in Understanding Collagen
• Methods For Analyzing Collagen Structure
• Methods For Imaging Collagen Structure
• Cryo-TEM
• Atomic Force Microscopy (AFM)
• AFM Image of Pentacene (C22H14)
• AFM Image of Hexabenzocoronene
• Basics of AFM Imaging The Probe
• Basics of AFM Imaging System Components
• van der Waals Potential Energy Curve
• Contact Mode ndash Monitor Deflection
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Peak Force Tapping Mode ndash Monitor Force
• AFM Applications in Bone Research Imaging
• AFM Imaging of Collagen
• Imaging of Osteoclast Activity
• Mineralized collagen deposition in vitro
• Imaging of Canilicular Structure
• AFM Imaging in Bone
• AFM Imaging in Bone
• Removing Mineral in Bone to Expose Collagen
• Removing Mineral in Bone to Expose Collagen
• Demineralization with EDTA
• Demineralization with EDTA
• Demineralization with EDTA
• Issues to Consider Effects of Acid
• Slide Number 32
• Slide Number 33
• Analysis of Collagen Structure in Bone
• Analysis of Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril D-Spacing
• Slide Number 39
• Collagen in Bone Fibril D-Spacing
• Collagen in Bone Fibril D-Spacing
• 2D Fast Fourier Transform Analysis
• 2D Fast Fourier Transform Analysis
• D-Spacing Distribution
• D-Spacing Distribution
• Cumulative Distribution Function
• CDF vs Histogram
• Changes in D-Spacing with Disease
• Changes in D-Spacing with Disease
• Slide Number 50
• Issues to Consider Site Selection and Sampling
• Issues to Consider Site Selection and Sampling
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• AFM calibration ndash Take Away Message
• AFM Applications in Bone ResearchIndentation Mechanics
• AFM-Based Indentation
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Parameters
• Hertz Contact Equation
• Hertz Contact Equation
• Hertz Contact Equation
• Proper Indentation Model is Essential
• Indentation in OI Tendon Study
• OI Tendon Differential Effects of Dehydration
• OI Tendon Differential Effects of Dehydration
• Indentation in Diabetic Tendon (Poster 0495)
• Multiscale Diabetes Study (Poster 0495)
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• A Note on Peak Force QNM for Bruker AFMs
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• Slide Number 83
• Slide Number 84
• AFM Applications in Bone ResearchOther Cool Mechanical Techniques
• ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo
• Other Mechanical Techniques
• Other Cool Techniques
• Summary Things to rememberhellip
• Acknowledgements
• Bone Biology and Mechanics Lab (BBML)

2D Fast Fourier Transform Analysis

Harmonics

Fibril D-Spacing

Raw AFM Image 2D FFT Power Spectrum

0

20000

40000

60000

80000

100000

120000

0 001 002 003 004 005 006

Am

plitu

de (

microV2 )

Position (1nm)

D-periodic spacing 0015 (1nm) ~ 67 nm

BBML - All R

ights Reserve

d

D-Spacing Distribution

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Overall Mean 670 plusmn 00 nm

BBML - All R

ights Reserve

d

D-Spacing Distribution

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Overall Mean 670 plusmn 00 nm

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

4 Samples Total of n = 255 fibrils Overall Mean 682 plusmn 12 nm

BBML - All R

ights Reserve

d

Cumulative Distribution Function

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

This population 255 fibrils - Each fibril 1255 = 0392 - Data range 628-716 nm

BBML - All R

ights Reserve

d

CDF vs Histogram 62 63 64 65 66 67 68 69 70 71 72 73 74

0

10

20

30

40

50

60

70

80

90

100

0

5

10

15

20

25

30

35

40

45

50

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

HistoCDF

BBML - All R

ights Reserve

d

Changes in D-Spacing with Disease

0

5

10

15

20

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

0

5

10

15

20

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

Wallace et al Bone 2010 46 1349-1354

KS Test plt0001

Control 680 plusmn 26 nm

Estrogen Dep 659 plusmn 31 nm

t-test on mean p=0048

BBML - All R

ights Reserve

d

Changes in D-Spacing with Disease

Wallace et al Bone 2010 46 1349-1354

0

10

20

30

40

50

60

70

80

90

100

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

KS Test plt0001

BBML - All R

ights Reserve

d

Question Model and Tissue Morphology Publication

Tissue Specificity in Mice Male mouse 8 weeks Femur dentin tail tendon

No differences in bone vs dentin both ∆ vs tendon

Wallace et al Langmuir 2011 26(10) 7349-7354

Gender Effects Male and female mice 16 weeks Femur No differences NA

Estrogen Depletion OVX in sheep 5 years + 2 Radius Mean and population shift Wallace et al Bone

2010 46 1349-1354

Estrogen Depletion OVX in female 5 years + 2 Skin (dermis) Significant population shift Erickson et al Biotech

J 2013 8(1) 1174-126

Osteoporosis and Mechanical Loading

OVX in ulnar loading in rats Ulna

No loading effect population shift with OVX

Warden et al Endocrinology 2013 154(9) 3178-3187

Osteogenesis Imperfecta Brtl+ model male mice 2 months Femur

No mean ∆ widening of distribution

Wallace et al J Stuct Bio 2011 173 146-152

Osteogenesis Imperfecta Oimoim model male mice 12 weeks Femur Mean and population shift Bart et al Conn Tiss

Res 2014 In Press

Osteogenesis Imperfecta and Tissue Hydration

Brtl+ model male mice 6 months Tail Tendon Significant population shift Kemp et al J Stuct Bio

2012 180 428-438

Type 2 Diabetes Male ZDSD rats 30 weeks Tibia Significant population shift Hammond et al Bone

2014 60 26-32

Type 2 Diabetes Female ZDSD rats 32 weeks Tail Tendon Significant population shift

Diaz Gonzalez et al J Biomech 2014 47(3)

681-686

Raloxifene (in vivo and in vitro) Female dog treated with Raloxifene Femur Significant population shift Gallant et al Bone

2014 61 191-200 BBML - All R

ights Reserve

d

Issues to Consider Site Selection and Sampling

KS Test plt0001

0023 of bonersquos length

15 m

m =

150

00 micro

m

Wallace et al Langmuir 2011 26(10) 7349-7354

100 microm x 100 microm

35 microm x 35 microm

BBML - All R

ights Reserve

d

Issues to Consider Site Selection and Sampling

60

62

64

66

68

70

72

74

1 (35)

2 (33)

3 (40)

4 (39)

5 (35)

6 (29)

7 (26)

8 (23)

9 (28)

WT (288)

D-P

erio

dic

Gap

Ove

rlap

Spac

ing

(nm

)

Axial Location (Proximal End = 1 Distal End = 9)

( of Fibrils)

91

23

45

87

6

Wallace et al Langmuir 2011 26(10) 7349-7354

288 fibrils for population n=1 for mean comparisons BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

Wet Tendon Dry Tendon Kemp et al J StructBio 2012 180 (3) 428-438

Collagen and bone exist in a hydrated environment in vivo bull Hydration state can impact morphology and mechanics

BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

Nonsignificant mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet (n=255) 682 plusmn 12 nm

WT Dry (n=257) 686 plusmn 08 nm

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

Brtl DryBrtl Wet

KS Test plt0001

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

WT Case 04 nm mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet 682 plusmn 12 nm

WT Dry 686 plusmn 08 nm

Brtl+ Wet 675 plusmn 14 nm

Brtl+ Dry 689 plusmn 10 nm

Brtl+ Case 14 nm increase with drying (plt0001) bull Entire population shifted upward (plt0001)

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT WetBrtl Wet

KS Test plt0001

Brtl+ distribution shifted downward vs WT when wet

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryBrtl Dry

KS Test plt0001

Brtl+ distribution shifted upward vs WT when dry

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider AFM calibration

bull Absolute distance measurements require accurate piezo calibration

bull AFM systems have large scan ranges (150 microm to lt 1 microm)

bull Scanner non-linearities can introduce substantial error over that range

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

ights Reserve

d

Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

BBML - All R

ights Reserve

d

Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

100 nm 10 80 602 1198 100 nm 7 60 827 1439 100 nm 5 40 814 1427 100 nm 35 20 917 1724 100 nm 2 10 937 1774

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

ights Reserve

d

AFM calibration ndash Take Away Message

Calibration using a scan size and feature size comparable to features of interest is highly recommended

Calibration addresses absolute accuracy not precision (differential sensitivity between measurements)

bull Does not limit ability to differentiate between groups using the same AFM with the same calibration parameters

BBML - All R

ights Reserve

d

AFM Applications in Bone Research Indentation Mechanics

BBML - All R

ights Reserve

d

AFM-Based Indentation

In addition to high resolution imaging AFM can be used to extract nanoscale mechanical data

ApproachWithdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Can

tilev

er D

efle

ctio

n (V

olts

)

1 Ramp into a rigid sample ndash measure the slope

Deflection sensitivity x 119899119899119899119899119881119881

2 Calculate cantilever spring constant

3 Hookersquos law takes deflection (volts) and converts to units of force F = kx

Spring constant k 119873119873119899119899

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Forc

e (n

N)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration Fo

rce

(nN

)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

Tip-Sample Separation (nm)

BBML - All R

ights Reserve

d

AFM-Based Indentation Parameters

Approach Withdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

Peak Force

Area Between Curves Energy Dissipation

Contact Mechanics Fit for Elastic Modulus (E)

Adhesion Force

Indentation Depth (δ)

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

z

r

δ

R

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119877119877 ∙ 12057512057532

Es Sample modulus νs Poissonrsquos ratio R tip radius of curvature δ load point displacement

Relevant Hertz Assumptions bull Strains are small and within the elastic limit bull Indenter is infinitely stiff (only sample deforms) bull Indentation depth ltlt radius of curvature of indenter bull Surfaces are continuous non-conforming (must be flat) bull Load is normal to surface

BBML - All R

ights Reserve

d

Proper Indentation Model is Essential

δ

r

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119903119903 ∙ 12057512057532

δ

α

119865119865 =2120587120587∙

1198641198641199041199041 minus 1205841205841199041199042

∙ tan120572120572 ∙ 1205751205752

Es Sample modulus νs Poissonrsquos ratio R tip radius α opening angle

Hertz Model Spherical Indenter

depth ltlt radius of curvature

Sneddon Model Conical Indenter

depth ge radius of curvature

BBML - All R

ights Reserve

d

Indentation in OI Tendon Study

x

z

yBrtl+ and WT mice 6 month old males

4 tails per group

2-3 fascicles per tail

25-30 fibrils per tail

4-5 locations per fibril

Indent force 20 nN wet (Sneddon) 50 nN dry (Hertz) BBML - A

ll Rights R

eserved

OI Tendon Differential Effects of Dehydration

Dry 1268 plusmn 718 MPa

Wet 305 plusmn 243 MPa

Dry 1737 plusmn 973 MPa

Wet 301 plusmn 202 MPa

Kemp et al J StructBio 2012 180 (3) 428-438

bull Significant stiffening of fibrils with dehydration

bull More pronounced modulus modulus as a function of disease

BBML - All R

ights Reserve

d

OI Tendon Differential Effects of Dehydration

Kemp et al J StructBio 2012 180 (3) 428-438

Diseased phenotype not present when wet phenotype observed when dried

0

10

20

30

40

50

60

0 20 40 60 80 100 120 140 160 180 200

Gro

up S

ampl

es (

)

Indent Modulus (MPa)

WT WetBrtl Wet

0

2

4

6

8

10

12

14

16

18

20

025

050

075

010

0012

5015

0017

5020

0022

5025

0027

5030

0032

5035

0037

5040

0042

5045

0047

5050

00

Gro

up S

ampl

es (

)Indent Modulus (MPa)

WT DryBrtl Dry

555 WT Indents 305 plusmn 243 MPa

614 Brtl+ Indents 301 plusmn 202 MPa

KS Test p=0129 t Test p=0464 464 WT Indents

1268 plusmn 718 MPa 479 Brtl+ Indents 1737 plusmn 973 MPa

KS Test plt0001 T Test plt0001

BBML - All R

ights Reserve

d

Indentation in Diabetic Tendon (Poster 0495)

x

z

y

Control and Diabetic Rat Tail Tendon

4-5 rats per group

2-3 fascicles per rat

~70 fibrilstail

4-5 locationsfibril

20 nN (Sneddon)

BBML - All R

ights Reserve

d

Multiscale Diabetes Study (Poster 0495)

0

5

10

15

20

25

300 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 gt25

Perc

enta

ge o

f Gro

up S

ampl

es

Elastic Modulus (MPa)

CD (1530 indents)ZDSD (913 indents)

CD (n=5) 381 plusmn 149 MPa

ZDSD (n=3) 706 plusmn 263 MPa

0

100

200

300

400

500

600

700

800

900

0 2 4 6 8 10

Mic

rosc

ale

Mod

ulus

(MPa

)

Nanoscale Modulus (MPa)

plt0001

r2=0531 p=0040

bull Increased indentation modulus in diabetic tendon

bull Strong relationship between nanoscale indent modulus and microscale tensile modulus

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

Proper probe selection for intended application bull Cantilever must be stiff relative to sample bull Sharp probes may damage sample easily dull

R or α must be determined for each probe bull Properties provided by manufacturer are unreliable bull Vary from probe to probe can dull during indent process

Spring constant must be determined for each probe

bull Value provided by manufacturer is inaccurate bull Thermal tuning OK for ldquosoftrdquo probes poor performance

for stiffer probes bull Sader method (geometry) added mass reference

cantilevers

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

Veeco Application Technical note Ben Ohler Practical Advice on the Determination of Cantilever Spring Constants

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

bull Sample processing

bull Sample hydration

bull Proper force range

bull Substrate effects for thin samples

bull Proper modulus model and range of data goodness of fit

Bearing in mind these assumptions and limitations AFM can provide true ldquonanoscalerdquo mechanical properties both in terms of forces and deformations

BBML - All R

ights Reserve

d

A Note on Peak Force QNM for Bruker AFMs bull Mechanical properties mapped pixel-by-pixel at same

resolution as the height image (PeakForce QNM)

Height Modulus BBML - All R

ights Reserve

d

A Note on Peak Force QNM

Correct probe stiffness must be used based on sample Pittenger ndash Veeco Product Bulletin

BBML - All R

ights Reserve

d

A Note on Peak Force QNM Extremely sensitive to calibration (details rarely given) Absolute radius and spring constant known

bull especially important from probe to probe bull radius uarr with indent depth ndash must determine R at depth

you expect to image bull Depth may vary as a function of sample (or at different

regions within the sample)

Relative reference material of ldquoknownrdquo modulus used to determine a ratio of 119896119896 119877119877 bull ldquoknownrdquo modulus may come from tension compression

and not indentation bull R is related to depth indentation depth on sample must

be same as on reference BBML - All R

ights Reserve

d

A Note on Peak Force QNM bull Modulus fit on the fly ndash no ability to monitor fit (or range) bull Local sample topography and sample tilt play a role in

measured parameters bull Assuming all modulus values in an image are correct how to

quantify

512 x 512 pixels = 262144 measurements

n=1 n=262144

n from selected ROIs

BBML - All R

ights Reserve

d

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ights Reserve

d

bull Imaging in air on mica ndash substrate effect

bull Same imaging force or indent depth for all

bull For each area were all modulus values pooled

Lamprou et al PLoS ONE 2013 BBML - All R

ights Reserve

d

AFM Applications in Bone Research Other Cool Mechanical Techniques

BBML - All R

ights Reserve

d

ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo Bonds within or between collagen fibrils in bone that rupture to dissipate energy as a toughening mechanism

Thompson et al Nature 2001

Fantner et al Nature Mat 2005

Hansma et al JMNI 2005

Fantner et al Nano Letters 2007 BBML - All R

ights Reserve

d

Other Mechanical Techniques

Jimenez-Palomar J Mech Beh Bio Mat 2012

van der Rijt Macro Bio 2004

Yang J Biomed Mater Res 2007

Yang Biophysical Journal 2008 BBML - All R

ights Reserve

d

Other Cool Techniques bull Piezoresponse Force Microscopy (PFM)

bull Electrochemical AFM (EC-AFM)

bull Force Modulation Microscopy (FMM)

bull Lateral Force Microscopy (LFM)

bull Magnetic Force Microscopy (MFM)

bull Conductive AFM (C-AFM)

bull Force Pulling

bull Cantilever Bending

bull Tensile testing of individual fibrils

bull 3 point bending of fibrils

BBML - All R

ights Reserve

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Summary Things to rememberhellip

bull Imaging mode imaging medium and probe selection

bull Morphology Measurements bull Sample preparation and hydration state bull Measuring fibril diameter bull Site selection and proper sampling bull Appropriate system calibration

bull Mechanical Measurements

bull Probe selection (stiffness and tip radius) bull Probe calibration (radius angle spring constant) bull Indentation force range and mechanical model bull Hydration in biological samples bull PeakForce QNM

BBML - All R

ights Reserve

d

Acknowledgements BBML

Max Hammond Armando Diaz Gonzalez Zachary Bart Silvia Canelon Creasy Clauser

IU School of Medicine Dr David Burr Dr Matt Allen Dr Stuart Warden Dr Lilian Plotkin

Funding Sources NIHNIDCR 1F32DE018840-01 A1 IUPUI BME Departmental Startup Funds IUPUI Research Support Funds Grant IUPUI Biomechanics and Biomaterials Research Center

NIH NICHD Dr Joan Marini

BBML - All R

ights Reserve

d

Bone Biology and Mechanics Lab (BBML) wwwiupuiedu~bbml Facebook BoneBiologyMechanicsLab

BBML - All R

ights Reserve

d

• Slide Number 1
• General Seminar Outline
• Bonersquos Hierarchical Structure
• Critical Gap in Understanding Collagen
• Methods For Analyzing Collagen Structure
• Methods For Imaging Collagen Structure
• Cryo-TEM
• Atomic Force Microscopy (AFM)
• AFM Image of Pentacene (C22H14)
• AFM Image of Hexabenzocoronene
• Basics of AFM Imaging The Probe
• Basics of AFM Imaging System Components
• van der Waals Potential Energy Curve
• Contact Mode ndash Monitor Deflection
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Peak Force Tapping Mode ndash Monitor Force
• AFM Applications in Bone Research Imaging
• AFM Imaging of Collagen
• Imaging of Osteoclast Activity
• Mineralized collagen deposition in vitro
• Imaging of Canilicular Structure
• AFM Imaging in Bone
• AFM Imaging in Bone
• Removing Mineral in Bone to Expose Collagen
• Removing Mineral in Bone to Expose Collagen
• Demineralization with EDTA
• Demineralization with EDTA
• Demineralization with EDTA
• Issues to Consider Effects of Acid
• Slide Number 32
• Slide Number 33
• Analysis of Collagen Structure in Bone
• Analysis of Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril D-Spacing
• Slide Number 39
• Collagen in Bone Fibril D-Spacing
• Collagen in Bone Fibril D-Spacing
• 2D Fast Fourier Transform Analysis
• 2D Fast Fourier Transform Analysis
• D-Spacing Distribution
• D-Spacing Distribution
• Cumulative Distribution Function
• CDF vs Histogram
• Changes in D-Spacing with Disease
• Changes in D-Spacing with Disease
• Slide Number 50
• Issues to Consider Site Selection and Sampling
• Issues to Consider Site Selection and Sampling
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• AFM calibration ndash Take Away Message
• AFM Applications in Bone ResearchIndentation Mechanics
• AFM-Based Indentation
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Parameters
• Hertz Contact Equation
• Hertz Contact Equation
• Hertz Contact Equation
• Proper Indentation Model is Essential
• Indentation in OI Tendon Study
• OI Tendon Differential Effects of Dehydration
• OI Tendon Differential Effects of Dehydration
• Indentation in Diabetic Tendon (Poster 0495)
• Multiscale Diabetes Study (Poster 0495)
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• A Note on Peak Force QNM for Bruker AFMs
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• Slide Number 83
• Slide Number 84
• AFM Applications in Bone ResearchOther Cool Mechanical Techniques
• ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo
• Other Mechanical Techniques
• Other Cool Techniques
• Summary Things to rememberhellip
• Acknowledgements
• Bone Biology and Mechanics Lab (BBML)

D-Spacing Distribution

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Overall Mean 670 plusmn 00 nm

BBML - All R

ights Reserve

d

D-Spacing Distribution

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Overall Mean 670 plusmn 00 nm

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

4 Samples Total of n = 255 fibrils Overall Mean 682 plusmn 12 nm

BBML - All R

ights Reserve

d

Cumulative Distribution Function

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

This population 255 fibrils - Each fibril 1255 = 0392 - Data range 628-716 nm

BBML - All R

ights Reserve

d

CDF vs Histogram 62 63 64 65 66 67 68 69 70 71 72 73 74

0

10

20

30

40

50

60

70

80

90

100

0

5

10

15

20

25

30

35

40

45

50

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

HistoCDF

BBML - All R

ights Reserve

d

Changes in D-Spacing with Disease

0

5

10

15

20

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

0

5

10

15

20

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

Wallace et al Bone 2010 46 1349-1354

KS Test plt0001

Control 680 plusmn 26 nm

Estrogen Dep 659 plusmn 31 nm

t-test on mean p=0048

BBML - All R

ights Reserve

d

Changes in D-Spacing with Disease

Wallace et al Bone 2010 46 1349-1354

0

10

20

30

40

50

60

70

80

90

100

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

KS Test plt0001

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ights Reserve

d

Question Model and Tissue Morphology Publication

Tissue Specificity in Mice Male mouse 8 weeks Femur dentin tail tendon

No differences in bone vs dentin both ∆ vs tendon

Wallace et al Langmuir 2011 26(10) 7349-7354

Gender Effects Male and female mice 16 weeks Femur No differences NA

Estrogen Depletion OVX in sheep 5 years + 2 Radius Mean and population shift Wallace et al Bone

2010 46 1349-1354

Estrogen Depletion OVX in female 5 years + 2 Skin (dermis) Significant population shift Erickson et al Biotech

J 2013 8(1) 1174-126

Osteoporosis and Mechanical Loading

OVX in ulnar loading in rats Ulna

No loading effect population shift with OVX

Warden et al Endocrinology 2013 154(9) 3178-3187

Osteogenesis Imperfecta Brtl+ model male mice 2 months Femur

No mean ∆ widening of distribution

Wallace et al J Stuct Bio 2011 173 146-152

Osteogenesis Imperfecta Oimoim model male mice 12 weeks Femur Mean and population shift Bart et al Conn Tiss

Res 2014 In Press

Osteogenesis Imperfecta and Tissue Hydration

Brtl+ model male mice 6 months Tail Tendon Significant population shift Kemp et al J Stuct Bio

2012 180 428-438

Type 2 Diabetes Male ZDSD rats 30 weeks Tibia Significant population shift Hammond et al Bone

2014 60 26-32

Type 2 Diabetes Female ZDSD rats 32 weeks Tail Tendon Significant population shift

Diaz Gonzalez et al J Biomech 2014 47(3)

681-686

Raloxifene (in vivo and in vitro) Female dog treated with Raloxifene Femur Significant population shift Gallant et al Bone

2014 61 191-200 BBML - All R

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d

Issues to Consider Site Selection and Sampling

KS Test plt0001

0023 of bonersquos length

15 m

m =

150

00 micro

m

Wallace et al Langmuir 2011 26(10) 7349-7354

100 microm x 100 microm

35 microm x 35 microm

BBML - All R

ights Reserve

d

Issues to Consider Site Selection and Sampling

60

62

64

66

68

70

72

74

1 (35)

2 (33)

3 (40)

4 (39)

5 (35)

6 (29)

7 (26)

8 (23)

9 (28)

WT (288)

D-P

erio

dic

Gap

Ove

rlap

Spac

ing

(nm

)

Axial Location (Proximal End = 1 Distal End = 9)

( of Fibrils)

91

23

45

87

6

Wallace et al Langmuir 2011 26(10) 7349-7354

288 fibrils for population n=1 for mean comparisons BBML - All R

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d

Issues to Consider Effects of Hydration

Wet Tendon Dry Tendon Kemp et al J StructBio 2012 180 (3) 428-438

Collagen and bone exist in a hydrated environment in vivo bull Hydration state can impact morphology and mechanics

BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

Nonsignificant mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet (n=255) 682 plusmn 12 nm

WT Dry (n=257) 686 plusmn 08 nm

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

Brtl DryBrtl Wet

KS Test plt0001

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

WT Case 04 nm mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet 682 plusmn 12 nm

WT Dry 686 plusmn 08 nm

Brtl+ Wet 675 plusmn 14 nm

Brtl+ Dry 689 plusmn 10 nm

Brtl+ Case 14 nm increase with drying (plt0001) bull Entire population shifted upward (plt0001)

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

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d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT WetBrtl Wet

KS Test plt0001

Brtl+ distribution shifted downward vs WT when wet

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryBrtl Dry

KS Test plt0001

Brtl+ distribution shifted upward vs WT when dry

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider AFM calibration

bull Absolute distance measurements require accurate piezo calibration

bull AFM systems have large scan ranges (150 microm to lt 1 microm)

bull Scanner non-linearities can introduce substantial error over that range

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

ights Reserve

d

Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

BBML - All R

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Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

100 nm 10 80 602 1198 100 nm 7 60 827 1439 100 nm 5 40 814 1427 100 nm 35 20 917 1724 100 nm 2 10 937 1774

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

ights Reserve

d

AFM calibration ndash Take Away Message

Calibration using a scan size and feature size comparable to features of interest is highly recommended

Calibration addresses absolute accuracy not precision (differential sensitivity between measurements)

bull Does not limit ability to differentiate between groups using the same AFM with the same calibration parameters

BBML - All R

ights Reserve

d

AFM Applications in Bone Research Indentation Mechanics

BBML - All R

ights Reserve

d

AFM-Based Indentation

In addition to high resolution imaging AFM can be used to extract nanoscale mechanical data

ApproachWithdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Can

tilev

er D

efle

ctio

n (V

olts

)

1 Ramp into a rigid sample ndash measure the slope

Deflection sensitivity x 119899119899119899119899119881119881

2 Calculate cantilever spring constant

3 Hookersquos law takes deflection (volts) and converts to units of force F = kx

Spring constant k 119873119873119899119899

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Forc

e (n

N)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration Fo

rce

(nN

)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

Tip-Sample Separation (nm)

BBML - All R

ights Reserve

d

AFM-Based Indentation Parameters

Approach Withdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

Peak Force

Area Between Curves Energy Dissipation

Contact Mechanics Fit for Elastic Modulus (E)

Adhesion Force

Indentation Depth (δ)

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

z

r

δ

R

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119877119877 ∙ 12057512057532

Es Sample modulus νs Poissonrsquos ratio R tip radius of curvature δ load point displacement

Relevant Hertz Assumptions bull Strains are small and within the elastic limit bull Indenter is infinitely stiff (only sample deforms) bull Indentation depth ltlt radius of curvature of indenter bull Surfaces are continuous non-conforming (must be flat) bull Load is normal to surface

BBML - All R

ights Reserve

d

Proper Indentation Model is Essential

δ

r

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119903119903 ∙ 12057512057532

δ

α

119865119865 =2120587120587∙

1198641198641199041199041 minus 1205841205841199041199042

∙ tan120572120572 ∙ 1205751205752

Es Sample modulus νs Poissonrsquos ratio R tip radius α opening angle

Hertz Model Spherical Indenter

depth ltlt radius of curvature

Sneddon Model Conical Indenter

depth ge radius of curvature

BBML - All R

ights Reserve

d

Indentation in OI Tendon Study

x

z

yBrtl+ and WT mice 6 month old males

4 tails per group

2-3 fascicles per tail

25-30 fibrils per tail

4-5 locations per fibril

Indent force 20 nN wet (Sneddon) 50 nN dry (Hertz) BBML - A

ll Rights R

eserved

OI Tendon Differential Effects of Dehydration

Dry 1268 plusmn 718 MPa

Wet 305 plusmn 243 MPa

Dry 1737 plusmn 973 MPa

Wet 301 plusmn 202 MPa

Kemp et al J StructBio 2012 180 (3) 428-438

bull Significant stiffening of fibrils with dehydration

bull More pronounced modulus modulus as a function of disease

BBML - All R

ights Reserve

d

OI Tendon Differential Effects of Dehydration

Kemp et al J StructBio 2012 180 (3) 428-438

Diseased phenotype not present when wet phenotype observed when dried

0

10

20

30

40

50

60

0 20 40 60 80 100 120 140 160 180 200

Gro

up S

ampl

es (

)

Indent Modulus (MPa)

WT WetBrtl Wet

0

2

4

6

8

10

12

14

16

18

20

025

050

075

010

0012

5015

0017

5020

0022

5025

0027

5030

0032

5035

0037

5040

0042

5045

0047

5050

00

Gro

up S

ampl

es (

)Indent Modulus (MPa)

WT DryBrtl Dry

555 WT Indents 305 plusmn 243 MPa

614 Brtl+ Indents 301 plusmn 202 MPa

KS Test p=0129 t Test p=0464 464 WT Indents

1268 plusmn 718 MPa 479 Brtl+ Indents 1737 plusmn 973 MPa

KS Test plt0001 T Test plt0001

BBML - All R

ights Reserve

d

Indentation in Diabetic Tendon (Poster 0495)

x

z

y

Control and Diabetic Rat Tail Tendon

4-5 rats per group

2-3 fascicles per rat

~70 fibrilstail

4-5 locationsfibril

20 nN (Sneddon)

BBML - All R

ights Reserve

d

Multiscale Diabetes Study (Poster 0495)

0

5

10

15

20

25

300 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 gt25

Perc

enta

ge o

f Gro

up S

ampl

es

Elastic Modulus (MPa)

CD (1530 indents)ZDSD (913 indents)

CD (n=5) 381 plusmn 149 MPa

ZDSD (n=3) 706 plusmn 263 MPa

0

100

200

300

400

500

600

700

800

900

0 2 4 6 8 10

Mic

rosc

ale

Mod

ulus

(MPa

)

Nanoscale Modulus (MPa)

plt0001

r2=0531 p=0040

bull Increased indentation modulus in diabetic tendon

bull Strong relationship between nanoscale indent modulus and microscale tensile modulus

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

Proper probe selection for intended application bull Cantilever must be stiff relative to sample bull Sharp probes may damage sample easily dull

R or α must be determined for each probe bull Properties provided by manufacturer are unreliable bull Vary from probe to probe can dull during indent process

Spring constant must be determined for each probe

bull Value provided by manufacturer is inaccurate bull Thermal tuning OK for ldquosoftrdquo probes poor performance

for stiffer probes bull Sader method (geometry) added mass reference

cantilevers

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

Veeco Application Technical note Ben Ohler Practical Advice on the Determination of Cantilever Spring Constants

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

bull Sample processing

bull Sample hydration

bull Proper force range

bull Substrate effects for thin samples

bull Proper modulus model and range of data goodness of fit

Bearing in mind these assumptions and limitations AFM can provide true ldquonanoscalerdquo mechanical properties both in terms of forces and deformations

BBML - All R

ights Reserve

d

A Note on Peak Force QNM for Bruker AFMs bull Mechanical properties mapped pixel-by-pixel at same

resolution as the height image (PeakForce QNM)

Height Modulus BBML - All R

ights Reserve

d

A Note on Peak Force QNM

Correct probe stiffness must be used based on sample Pittenger ndash Veeco Product Bulletin

BBML - All R

ights Reserve

d

A Note on Peak Force QNM Extremely sensitive to calibration (details rarely given) Absolute radius and spring constant known

bull especially important from probe to probe bull radius uarr with indent depth ndash must determine R at depth

you expect to image bull Depth may vary as a function of sample (or at different

regions within the sample)

Relative reference material of ldquoknownrdquo modulus used to determine a ratio of 119896119896 119877119877 bull ldquoknownrdquo modulus may come from tension compression

and not indentation bull R is related to depth indentation depth on sample must

be same as on reference BBML - All R

ights Reserve

d

A Note on Peak Force QNM bull Modulus fit on the fly ndash no ability to monitor fit (or range) bull Local sample topography and sample tilt play a role in

measured parameters bull Assuming all modulus values in an image are correct how to

quantify

512 x 512 pixels = 262144 measurements

n=1 n=262144

n from selected ROIs

BBML - All R

ights Reserve

d

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ights Reserve

d

bull Imaging in air on mica ndash substrate effect

bull Same imaging force or indent depth for all

bull For each area were all modulus values pooled

Lamprou et al PLoS ONE 2013 BBML - All R

ights Reserve

d

AFM Applications in Bone Research Other Cool Mechanical Techniques

BBML - All R

ights Reserve

d

ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo Bonds within or between collagen fibrils in bone that rupture to dissipate energy as a toughening mechanism

Thompson et al Nature 2001

Fantner et al Nature Mat 2005

Hansma et al JMNI 2005

Fantner et al Nano Letters 2007 BBML - All R

ights Reserve

d

Other Mechanical Techniques

Jimenez-Palomar J Mech Beh Bio Mat 2012

van der Rijt Macro Bio 2004

Yang J Biomed Mater Res 2007

Yang Biophysical Journal 2008 BBML - All R

ights Reserve

d

Other Cool Techniques bull Piezoresponse Force Microscopy (PFM)

bull Electrochemical AFM (EC-AFM)

bull Force Modulation Microscopy (FMM)

bull Lateral Force Microscopy (LFM)

bull Magnetic Force Microscopy (MFM)

bull Conductive AFM (C-AFM)

bull Force Pulling

bull Cantilever Bending

bull Tensile testing of individual fibrils

bull 3 point bending of fibrils

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Summary Things to rememberhellip

bull Imaging mode imaging medium and probe selection

bull Morphology Measurements bull Sample preparation and hydration state bull Measuring fibril diameter bull Site selection and proper sampling bull Appropriate system calibration

bull Mechanical Measurements

bull Probe selection (stiffness and tip radius) bull Probe calibration (radius angle spring constant) bull Indentation force range and mechanical model bull Hydration in biological samples bull PeakForce QNM

BBML - All R

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d

Acknowledgements BBML

Max Hammond Armando Diaz Gonzalez Zachary Bart Silvia Canelon Creasy Clauser

IU School of Medicine Dr David Burr Dr Matt Allen Dr Stuart Warden Dr Lilian Plotkin

Funding Sources NIHNIDCR 1F32DE018840-01 A1 IUPUI BME Departmental Startup Funds IUPUI Research Support Funds Grant IUPUI Biomechanics and Biomaterials Research Center

NIH NICHD Dr Joan Marini

BBML - All R

ights Reserve

d

Bone Biology and Mechanics Lab (BBML) wwwiupuiedu~bbml Facebook BoneBiologyMechanicsLab

BBML - All R

ights Reserve

d

• Slide Number 1
• General Seminar Outline
• Bonersquos Hierarchical Structure
• Critical Gap in Understanding Collagen
• Methods For Analyzing Collagen Structure
• Methods For Imaging Collagen Structure
• Cryo-TEM
• Atomic Force Microscopy (AFM)
• AFM Image of Pentacene (C22H14)
• AFM Image of Hexabenzocoronene
• Basics of AFM Imaging The Probe
• Basics of AFM Imaging System Components
• van der Waals Potential Energy Curve
• Contact Mode ndash Monitor Deflection
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Peak Force Tapping Mode ndash Monitor Force
• AFM Applications in Bone Research Imaging
• AFM Imaging of Collagen
• Imaging of Osteoclast Activity
• Mineralized collagen deposition in vitro
• Imaging of Canilicular Structure
• AFM Imaging in Bone
• AFM Imaging in Bone
• Removing Mineral in Bone to Expose Collagen
• Removing Mineral in Bone to Expose Collagen
• Demineralization with EDTA
• Demineralization with EDTA
• Demineralization with EDTA
• Issues to Consider Effects of Acid
• Slide Number 32
• Slide Number 33
• Analysis of Collagen Structure in Bone
• Analysis of Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril D-Spacing
• Slide Number 39
• Collagen in Bone Fibril D-Spacing
• Collagen in Bone Fibril D-Spacing
• 2D Fast Fourier Transform Analysis
• 2D Fast Fourier Transform Analysis
• D-Spacing Distribution
• D-Spacing Distribution
• Cumulative Distribution Function
• CDF vs Histogram
• Changes in D-Spacing with Disease
• Changes in D-Spacing with Disease
• Slide Number 50
• Issues to Consider Site Selection and Sampling
• Issues to Consider Site Selection and Sampling
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• AFM calibration ndash Take Away Message
• AFM Applications in Bone ResearchIndentation Mechanics
• AFM-Based Indentation
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Parameters
• Hertz Contact Equation
• Hertz Contact Equation
• Hertz Contact Equation
• Proper Indentation Model is Essential
• Indentation in OI Tendon Study
• OI Tendon Differential Effects of Dehydration
• OI Tendon Differential Effects of Dehydration
• Indentation in Diabetic Tendon (Poster 0495)
• Multiscale Diabetes Study (Poster 0495)
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• A Note on Peak Force QNM for Bruker AFMs
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• Slide Number 83
• Slide Number 84
• AFM Applications in Bone ResearchOther Cool Mechanical Techniques
• ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo
• Other Mechanical Techniques
• Other Cool Techniques
• Summary Things to rememberhellip
• Acknowledgements
• Bone Biology and Mechanics Lab (BBML)

D-Spacing Distribution

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Overall Mean 670 plusmn 00 nm

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

4 Samples Total of n = 255 fibrils Overall Mean 682 plusmn 12 nm

BBML - All R

ights Reserve

d

Cumulative Distribution Function

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

This population 255 fibrils - Each fibril 1255 = 0392 - Data range 628-716 nm

BBML - All R

ights Reserve

d

CDF vs Histogram 62 63 64 65 66 67 68 69 70 71 72 73 74

0

10

20

30

40

50

60

70

80

90

100

0

5

10

15

20

25

30

35

40

45

50

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

HistoCDF

BBML - All R

ights Reserve

d

Changes in D-Spacing with Disease

0

5

10

15

20

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

0

5

10

15

20

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

Wallace et al Bone 2010 46 1349-1354

KS Test plt0001

Control 680 plusmn 26 nm

Estrogen Dep 659 plusmn 31 nm

t-test on mean p=0048

BBML - All R

ights Reserve

d

Changes in D-Spacing with Disease

Wallace et al Bone 2010 46 1349-1354

0

10

20

30

40

50

60

70

80

90

100

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

KS Test plt0001

BBML - All R

ights Reserve

d

Question Model and Tissue Morphology Publication

Tissue Specificity in Mice Male mouse 8 weeks Femur dentin tail tendon

No differences in bone vs dentin both ∆ vs tendon

Wallace et al Langmuir 2011 26(10) 7349-7354

Gender Effects Male and female mice 16 weeks Femur No differences NA

Estrogen Depletion OVX in sheep 5 years + 2 Radius Mean and population shift Wallace et al Bone

2010 46 1349-1354

Estrogen Depletion OVX in female 5 years + 2 Skin (dermis) Significant population shift Erickson et al Biotech

J 2013 8(1) 1174-126

Osteoporosis and Mechanical Loading

OVX in ulnar loading in rats Ulna

No loading effect population shift with OVX

Warden et al Endocrinology 2013 154(9) 3178-3187

Osteogenesis Imperfecta Brtl+ model male mice 2 months Femur

No mean ∆ widening of distribution

Wallace et al J Stuct Bio 2011 173 146-152

Osteogenesis Imperfecta Oimoim model male mice 12 weeks Femur Mean and population shift Bart et al Conn Tiss

Res 2014 In Press

Osteogenesis Imperfecta and Tissue Hydration

Brtl+ model male mice 6 months Tail Tendon Significant population shift Kemp et al J Stuct Bio

2012 180 428-438

Type 2 Diabetes Male ZDSD rats 30 weeks Tibia Significant population shift Hammond et al Bone

2014 60 26-32

Type 2 Diabetes Female ZDSD rats 32 weeks Tail Tendon Significant population shift

Diaz Gonzalez et al J Biomech 2014 47(3)

681-686

Raloxifene (in vivo and in vitro) Female dog treated with Raloxifene Femur Significant population shift Gallant et al Bone

2014 61 191-200 BBML - All R

ights Reserve

d

Issues to Consider Site Selection and Sampling

KS Test plt0001

0023 of bonersquos length

15 m

m =

150

00 micro

m

Wallace et al Langmuir 2011 26(10) 7349-7354

100 microm x 100 microm

35 microm x 35 microm

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ights Reserve

d

Issues to Consider Site Selection and Sampling

60

62

64

66

68

70

72

74

1 (35)

2 (33)

3 (40)

4 (39)

5 (35)

6 (29)

7 (26)

8 (23)

9 (28)

WT (288)

D-P

erio

dic

Gap

Ove

rlap

Spac

ing

(nm

)

Axial Location (Proximal End = 1 Distal End = 9)

( of Fibrils)

91

23

45

87

6

Wallace et al Langmuir 2011 26(10) 7349-7354

288 fibrils for population n=1 for mean comparisons BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

Wet Tendon Dry Tendon Kemp et al J StructBio 2012 180 (3) 428-438

Collagen and bone exist in a hydrated environment in vivo bull Hydration state can impact morphology and mechanics

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ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

Nonsignificant mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet (n=255) 682 plusmn 12 nm

WT Dry (n=257) 686 plusmn 08 nm

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

Brtl DryBrtl Wet

KS Test plt0001

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

WT Case 04 nm mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet 682 plusmn 12 nm

WT Dry 686 plusmn 08 nm

Brtl+ Wet 675 plusmn 14 nm

Brtl+ Dry 689 plusmn 10 nm

Brtl+ Case 14 nm increase with drying (plt0001) bull Entire population shifted upward (plt0001)

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

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d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT WetBrtl Wet

KS Test plt0001

Brtl+ distribution shifted downward vs WT when wet

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryBrtl Dry

KS Test plt0001

Brtl+ distribution shifted upward vs WT when dry

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

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d

Issues to Consider AFM calibration

bull Absolute distance measurements require accurate piezo calibration

bull AFM systems have large scan ranges (150 microm to lt 1 microm)

bull Scanner non-linearities can introduce substantial error over that range

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

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d

Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

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Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

100 nm 10 80 602 1198 100 nm 7 60 827 1439 100 nm 5 40 814 1427 100 nm 35 20 917 1724 100 nm 2 10 937 1774

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

ights Reserve

d

AFM calibration ndash Take Away Message

Calibration using a scan size and feature size comparable to features of interest is highly recommended

Calibration addresses absolute accuracy not precision (differential sensitivity between measurements)

bull Does not limit ability to differentiate between groups using the same AFM with the same calibration parameters

BBML - All R

ights Reserve

d

AFM Applications in Bone Research Indentation Mechanics

BBML - All R

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d

AFM-Based Indentation

In addition to high resolution imaging AFM can be used to extract nanoscale mechanical data

ApproachWithdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Can

tilev

er D

efle

ctio

n (V

olts

)

1 Ramp into a rigid sample ndash measure the slope

Deflection sensitivity x 119899119899119899119899119881119881

2 Calculate cantilever spring constant

3 Hookersquos law takes deflection (volts) and converts to units of force F = kx

Spring constant k 119873119873119899119899

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Forc

e (n

N)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration Fo

rce

(nN

)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

Tip-Sample Separation (nm)

BBML - All R

ights Reserve

d

AFM-Based Indentation Parameters

Approach Withdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

Peak Force

Area Between Curves Energy Dissipation

Contact Mechanics Fit for Elastic Modulus (E)

Adhesion Force

Indentation Depth (δ)

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

z

r

δ

R

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119877119877 ∙ 12057512057532

Es Sample modulus νs Poissonrsquos ratio R tip radius of curvature δ load point displacement

Relevant Hertz Assumptions bull Strains are small and within the elastic limit bull Indenter is infinitely stiff (only sample deforms) bull Indentation depth ltlt radius of curvature of indenter bull Surfaces are continuous non-conforming (must be flat) bull Load is normal to surface

BBML - All R

ights Reserve

d

Proper Indentation Model is Essential

δ

r

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119903119903 ∙ 12057512057532

δ

α

119865119865 =2120587120587∙

1198641198641199041199041 minus 1205841205841199041199042

∙ tan120572120572 ∙ 1205751205752

Es Sample modulus νs Poissonrsquos ratio R tip radius α opening angle

Hertz Model Spherical Indenter

depth ltlt radius of curvature

Sneddon Model Conical Indenter

depth ge radius of curvature

BBML - All R

ights Reserve

d

Indentation in OI Tendon Study

x

z

yBrtl+ and WT mice 6 month old males

4 tails per group

2-3 fascicles per tail

25-30 fibrils per tail

4-5 locations per fibril

Indent force 20 nN wet (Sneddon) 50 nN dry (Hertz) BBML - A

ll Rights R

eserved

OI Tendon Differential Effects of Dehydration

Dry 1268 plusmn 718 MPa

Wet 305 plusmn 243 MPa

Dry 1737 plusmn 973 MPa

Wet 301 plusmn 202 MPa

Kemp et al J StructBio 2012 180 (3) 428-438

bull Significant stiffening of fibrils with dehydration

bull More pronounced modulus modulus as a function of disease

BBML - All R

ights Reserve

d

OI Tendon Differential Effects of Dehydration

Kemp et al J StructBio 2012 180 (3) 428-438

Diseased phenotype not present when wet phenotype observed when dried

0

10

20

30

40

50

60

0 20 40 60 80 100 120 140 160 180 200

Gro

up S

ampl

es (

)

Indent Modulus (MPa)

WT WetBrtl Wet

0

2

4

6

8

10

12

14

16

18

20

025

050

075

010

0012

5015

0017

5020

0022

5025

0027

5030

0032

5035

0037

5040

0042

5045

0047

5050

00

Gro

up S

ampl

es (

)Indent Modulus (MPa)

WT DryBrtl Dry

555 WT Indents 305 plusmn 243 MPa

614 Brtl+ Indents 301 plusmn 202 MPa

KS Test p=0129 t Test p=0464 464 WT Indents

1268 plusmn 718 MPa 479 Brtl+ Indents 1737 plusmn 973 MPa

KS Test plt0001 T Test plt0001

BBML - All R

ights Reserve

d

Indentation in Diabetic Tendon (Poster 0495)

x

z

y

Control and Diabetic Rat Tail Tendon

4-5 rats per group

2-3 fascicles per rat

~70 fibrilstail

4-5 locationsfibril

20 nN (Sneddon)

BBML - All R

ights Reserve

d

Multiscale Diabetes Study (Poster 0495)

0

5

10

15

20

25

300 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 gt25

Perc

enta

ge o

f Gro

up S

ampl

es

Elastic Modulus (MPa)

CD (1530 indents)ZDSD (913 indents)

CD (n=5) 381 plusmn 149 MPa

ZDSD (n=3) 706 plusmn 263 MPa

0

100

200

300

400

500

600

700

800

900

0 2 4 6 8 10

Mic

rosc

ale

Mod

ulus

(MPa

)

Nanoscale Modulus (MPa)

plt0001

r2=0531 p=0040

bull Increased indentation modulus in diabetic tendon

bull Strong relationship between nanoscale indent modulus and microscale tensile modulus

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

Proper probe selection for intended application bull Cantilever must be stiff relative to sample bull Sharp probes may damage sample easily dull

R or α must be determined for each probe bull Properties provided by manufacturer are unreliable bull Vary from probe to probe can dull during indent process

Spring constant must be determined for each probe

bull Value provided by manufacturer is inaccurate bull Thermal tuning OK for ldquosoftrdquo probes poor performance

for stiffer probes bull Sader method (geometry) added mass reference

cantilevers

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

Veeco Application Technical note Ben Ohler Practical Advice on the Determination of Cantilever Spring Constants

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

bull Sample processing

bull Sample hydration

bull Proper force range

bull Substrate effects for thin samples

bull Proper modulus model and range of data goodness of fit

Bearing in mind these assumptions and limitations AFM can provide true ldquonanoscalerdquo mechanical properties both in terms of forces and deformations

BBML - All R

ights Reserve

d

A Note on Peak Force QNM for Bruker AFMs bull Mechanical properties mapped pixel-by-pixel at same

resolution as the height image (PeakForce QNM)

Height Modulus BBML - All R

ights Reserve

d

A Note on Peak Force QNM

Correct probe stiffness must be used based on sample Pittenger ndash Veeco Product Bulletin

BBML - All R

ights Reserve

d

A Note on Peak Force QNM Extremely sensitive to calibration (details rarely given) Absolute radius and spring constant known

bull especially important from probe to probe bull radius uarr with indent depth ndash must determine R at depth

you expect to image bull Depth may vary as a function of sample (or at different

regions within the sample)

Relative reference material of ldquoknownrdquo modulus used to determine a ratio of 119896119896 119877119877 bull ldquoknownrdquo modulus may come from tension compression

and not indentation bull R is related to depth indentation depth on sample must

be same as on reference BBML - All R

ights Reserve

d

A Note on Peak Force QNM bull Modulus fit on the fly ndash no ability to monitor fit (or range) bull Local sample topography and sample tilt play a role in

measured parameters bull Assuming all modulus values in an image are correct how to

quantify

512 x 512 pixels = 262144 measurements

n=1 n=262144

n from selected ROIs

BBML - All R

ights Reserve

d

BBML - All R

ights Reserve

d

bull Imaging in air on mica ndash substrate effect

bull Same imaging force or indent depth for all

bull For each area were all modulus values pooled

Lamprou et al PLoS ONE 2013 BBML - All R

ights Reserve

d

AFM Applications in Bone Research Other Cool Mechanical Techniques

BBML - All R

ights Reserve

d

ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo Bonds within or between collagen fibrils in bone that rupture to dissipate energy as a toughening mechanism

Thompson et al Nature 2001

Fantner et al Nature Mat 2005

Hansma et al JMNI 2005

Fantner et al Nano Letters 2007 BBML - All R

ights Reserve

d

Other Mechanical Techniques

Jimenez-Palomar J Mech Beh Bio Mat 2012

van der Rijt Macro Bio 2004

Yang J Biomed Mater Res 2007

Yang Biophysical Journal 2008 BBML - All R

ights Reserve

d

Other Cool Techniques bull Piezoresponse Force Microscopy (PFM)

bull Electrochemical AFM (EC-AFM)

bull Force Modulation Microscopy (FMM)

bull Lateral Force Microscopy (LFM)

bull Magnetic Force Microscopy (MFM)

bull Conductive AFM (C-AFM)

bull Force Pulling

bull Cantilever Bending

bull Tensile testing of individual fibrils

bull 3 point bending of fibrils

BBML - All R

ights Reserve

d

Summary Things to rememberhellip

bull Imaging mode imaging medium and probe selection

bull Morphology Measurements bull Sample preparation and hydration state bull Measuring fibril diameter bull Site selection and proper sampling bull Appropriate system calibration

bull Mechanical Measurements

bull Probe selection (stiffness and tip radius) bull Probe calibration (radius angle spring constant) bull Indentation force range and mechanical model bull Hydration in biological samples bull PeakForce QNM

BBML - All R

ights Reserve

d

Acknowledgements BBML

Max Hammond Armando Diaz Gonzalez Zachary Bart Silvia Canelon Creasy Clauser

IU School of Medicine Dr David Burr Dr Matt Allen Dr Stuart Warden Dr Lilian Plotkin

Funding Sources NIHNIDCR 1F32DE018840-01 A1 IUPUI BME Departmental Startup Funds IUPUI Research Support Funds Grant IUPUI Biomechanics and Biomaterials Research Center

NIH NICHD Dr Joan Marini

BBML - All R

ights Reserve

d

Bone Biology and Mechanics Lab (BBML) wwwiupuiedu~bbml Facebook BoneBiologyMechanicsLab

BBML - All R

ights Reserve

d

• Slide Number 1
• General Seminar Outline
• Bonersquos Hierarchical Structure
• Critical Gap in Understanding Collagen
• Methods For Analyzing Collagen Structure
• Methods For Imaging Collagen Structure
• Cryo-TEM
• Atomic Force Microscopy (AFM)
• AFM Image of Pentacene (C22H14)
• AFM Image of Hexabenzocoronene
• Basics of AFM Imaging The Probe
• Basics of AFM Imaging System Components
• van der Waals Potential Energy Curve
• Contact Mode ndash Monitor Deflection
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Peak Force Tapping Mode ndash Monitor Force
• AFM Applications in Bone Research Imaging
• AFM Imaging of Collagen
• Imaging of Osteoclast Activity
• Mineralized collagen deposition in vitro
• Imaging of Canilicular Structure
• AFM Imaging in Bone
• AFM Imaging in Bone
• Removing Mineral in Bone to Expose Collagen
• Removing Mineral in Bone to Expose Collagen
• Demineralization with EDTA
• Demineralization with EDTA
• Demineralization with EDTA
• Issues to Consider Effects of Acid
• Slide Number 32
• Slide Number 33
• Analysis of Collagen Structure in Bone
• Analysis of Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril D-Spacing
• Slide Number 39
• Collagen in Bone Fibril D-Spacing
• Collagen in Bone Fibril D-Spacing
• 2D Fast Fourier Transform Analysis
• 2D Fast Fourier Transform Analysis
• D-Spacing Distribution
• D-Spacing Distribution
• Cumulative Distribution Function
• CDF vs Histogram
• Changes in D-Spacing with Disease
• Changes in D-Spacing with Disease
• Slide Number 50
• Issues to Consider Site Selection and Sampling
• Issues to Consider Site Selection and Sampling
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• AFM calibration ndash Take Away Message
• AFM Applications in Bone ResearchIndentation Mechanics
• AFM-Based Indentation
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Parameters
• Hertz Contact Equation
• Hertz Contact Equation
• Hertz Contact Equation
• Proper Indentation Model is Essential
• Indentation in OI Tendon Study
• OI Tendon Differential Effects of Dehydration
• OI Tendon Differential Effects of Dehydration
• Indentation in Diabetic Tendon (Poster 0495)
• Multiscale Diabetes Study (Poster 0495)
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• A Note on Peak Force QNM for Bruker AFMs
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• Slide Number 83
• Slide Number 84
• AFM Applications in Bone ResearchOther Cool Mechanical Techniques
• ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo
• Other Mechanical Techniques
• Other Cool Techniques
• Summary Things to rememberhellip
• Acknowledgements
• Bone Biology and Mechanics Lab (BBML)

Cumulative Distribution Function

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

This population 255 fibrils - Each fibril 1255 = 0392 - Data range 628-716 nm

BBML - All R

ights Reserve

d

CDF vs Histogram 62 63 64 65 66 67 68 69 70 71 72 73 74

0

10

20

30

40

50

60

70

80

90

100

0

5

10

15

20

25

30

35

40

45

50

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

HistoCDF

BBML - All R

ights Reserve

d

Changes in D-Spacing with Disease

0

5

10

15

20

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

0

5

10

15

20

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

Wallace et al Bone 2010 46 1349-1354

KS Test plt0001

Control 680 plusmn 26 nm

Estrogen Dep 659 plusmn 31 nm

t-test on mean p=0048

BBML - All R

ights Reserve

d

Changes in D-Spacing with Disease

Wallace et al Bone 2010 46 1349-1354

0

10

20

30

40

50

60

70

80

90

100

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

KS Test plt0001

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ights Reserve

d

Question Model and Tissue Morphology Publication

Tissue Specificity in Mice Male mouse 8 weeks Femur dentin tail tendon

No differences in bone vs dentin both ∆ vs tendon

Wallace et al Langmuir 2011 26(10) 7349-7354

Gender Effects Male and female mice 16 weeks Femur No differences NA

Estrogen Depletion OVX in sheep 5 years + 2 Radius Mean and population shift Wallace et al Bone

2010 46 1349-1354

Estrogen Depletion OVX in female 5 years + 2 Skin (dermis) Significant population shift Erickson et al Biotech

J 2013 8(1) 1174-126

Osteoporosis and Mechanical Loading

OVX in ulnar loading in rats Ulna

No loading effect population shift with OVX

Warden et al Endocrinology 2013 154(9) 3178-3187

Osteogenesis Imperfecta Brtl+ model male mice 2 months Femur

No mean ∆ widening of distribution

Wallace et al J Stuct Bio 2011 173 146-152

Osteogenesis Imperfecta Oimoim model male mice 12 weeks Femur Mean and population shift Bart et al Conn Tiss

Res 2014 In Press

Osteogenesis Imperfecta and Tissue Hydration

Brtl+ model male mice 6 months Tail Tendon Significant population shift Kemp et al J Stuct Bio

2012 180 428-438

Type 2 Diabetes Male ZDSD rats 30 weeks Tibia Significant population shift Hammond et al Bone

2014 60 26-32

Type 2 Diabetes Female ZDSD rats 32 weeks Tail Tendon Significant population shift

Diaz Gonzalez et al J Biomech 2014 47(3)

681-686

Raloxifene (in vivo and in vitro) Female dog treated with Raloxifene Femur Significant population shift Gallant et al Bone

2014 61 191-200 BBML - All R

ights Reserve

d

Issues to Consider Site Selection and Sampling

KS Test plt0001

0023 of bonersquos length

15 m

m =

150

00 micro

m

Wallace et al Langmuir 2011 26(10) 7349-7354

100 microm x 100 microm

35 microm x 35 microm

BBML - All R

ights Reserve

d

Issues to Consider Site Selection and Sampling

60

62

64

66

68

70

72

74

1 (35)

2 (33)

3 (40)

4 (39)

5 (35)

6 (29)

7 (26)

8 (23)

9 (28)

WT (288)

D-P

erio

dic

Gap

Ove

rlap

Spac

ing

(nm

)

Axial Location (Proximal End = 1 Distal End = 9)

( of Fibrils)

91

23

45

87

6

Wallace et al Langmuir 2011 26(10) 7349-7354

288 fibrils for population n=1 for mean comparisons BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

Wet Tendon Dry Tendon Kemp et al J StructBio 2012 180 (3) 428-438

Collagen and bone exist in a hydrated environment in vivo bull Hydration state can impact morphology and mechanics

BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

Nonsignificant mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet (n=255) 682 plusmn 12 nm

WT Dry (n=257) 686 plusmn 08 nm

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

Brtl DryBrtl Wet

KS Test plt0001

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

WT Case 04 nm mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet 682 plusmn 12 nm

WT Dry 686 plusmn 08 nm

Brtl+ Wet 675 plusmn 14 nm

Brtl+ Dry 689 plusmn 10 nm

Brtl+ Case 14 nm increase with drying (plt0001) bull Entire population shifted upward (plt0001)

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT WetBrtl Wet

KS Test plt0001

Brtl+ distribution shifted downward vs WT when wet

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryBrtl Dry

KS Test plt0001

Brtl+ distribution shifted upward vs WT when dry

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider AFM calibration

bull Absolute distance measurements require accurate piezo calibration

bull AFM systems have large scan ranges (150 microm to lt 1 microm)

bull Scanner non-linearities can introduce substantial error over that range

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

ights Reserve

d

Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

BBML - All R

ights Reserve

d

Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

100 nm 10 80 602 1198 100 nm 7 60 827 1439 100 nm 5 40 814 1427 100 nm 35 20 917 1724 100 nm 2 10 937 1774

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

ights Reserve

d

AFM calibration ndash Take Away Message

Calibration using a scan size and feature size comparable to features of interest is highly recommended

Calibration addresses absolute accuracy not precision (differential sensitivity between measurements)

bull Does not limit ability to differentiate between groups using the same AFM with the same calibration parameters

BBML - All R

ights Reserve

d

AFM Applications in Bone Research Indentation Mechanics

BBML - All R

ights Reserve

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AFM-Based Indentation

In addition to high resolution imaging AFM can be used to extract nanoscale mechanical data

ApproachWithdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Can

tilev

er D

efle

ctio

n (V

olts

)

1 Ramp into a rigid sample ndash measure the slope

Deflection sensitivity x 119899119899119899119899119881119881

2 Calculate cantilever spring constant

3 Hookersquos law takes deflection (volts) and converts to units of force F = kx

Spring constant k 119873119873119899119899

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Forc

e (n

N)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration Fo

rce

(nN

)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

Tip-Sample Separation (nm)

BBML - All R

ights Reserve

d

AFM-Based Indentation Parameters

Approach Withdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

Peak Force

Area Between Curves Energy Dissipation

Contact Mechanics Fit for Elastic Modulus (E)

Adhesion Force

Indentation Depth (δ)

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

z

r

δ

R

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119877119877 ∙ 12057512057532

Es Sample modulus νs Poissonrsquos ratio R tip radius of curvature δ load point displacement

Relevant Hertz Assumptions bull Strains are small and within the elastic limit bull Indenter is infinitely stiff (only sample deforms) bull Indentation depth ltlt radius of curvature of indenter bull Surfaces are continuous non-conforming (must be flat) bull Load is normal to surface

BBML - All R

ights Reserve

d

Proper Indentation Model is Essential

δ

r

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119903119903 ∙ 12057512057532

δ

α

119865119865 =2120587120587∙

1198641198641199041199041 minus 1205841205841199041199042

∙ tan120572120572 ∙ 1205751205752

Es Sample modulus νs Poissonrsquos ratio R tip radius α opening angle

Hertz Model Spherical Indenter

depth ltlt radius of curvature

Sneddon Model Conical Indenter

depth ge radius of curvature

BBML - All R

ights Reserve

d

Indentation in OI Tendon Study

x

z

yBrtl+ and WT mice 6 month old males

4 tails per group

2-3 fascicles per tail

25-30 fibrils per tail

4-5 locations per fibril

Indent force 20 nN wet (Sneddon) 50 nN dry (Hertz) BBML - A

ll Rights R

eserved

OI Tendon Differential Effects of Dehydration

Dry 1268 plusmn 718 MPa

Wet 305 plusmn 243 MPa

Dry 1737 plusmn 973 MPa

Wet 301 plusmn 202 MPa

Kemp et al J StructBio 2012 180 (3) 428-438

bull Significant stiffening of fibrils with dehydration

bull More pronounced modulus modulus as a function of disease

BBML - All R

ights Reserve

d

OI Tendon Differential Effects of Dehydration

Kemp et al J StructBio 2012 180 (3) 428-438

Diseased phenotype not present when wet phenotype observed when dried

0

10

20

30

40

50

60

0 20 40 60 80 100 120 140 160 180 200

Gro

up S

ampl

es (

)

Indent Modulus (MPa)

WT WetBrtl Wet

0

2

4

6

8

10

12

14

16

18

20

025

050

075

010

0012

5015

0017

5020

0022

5025

0027

5030

0032

5035

0037

5040

0042

5045

0047

5050

00

Gro

up S

ampl

es (

)Indent Modulus (MPa)

WT DryBrtl Dry

555 WT Indents 305 plusmn 243 MPa

614 Brtl+ Indents 301 plusmn 202 MPa

KS Test p=0129 t Test p=0464 464 WT Indents

1268 plusmn 718 MPa 479 Brtl+ Indents 1737 plusmn 973 MPa

KS Test plt0001 T Test plt0001

BBML - All R

ights Reserve

d

Indentation in Diabetic Tendon (Poster 0495)

x

z

y

Control and Diabetic Rat Tail Tendon

4-5 rats per group

2-3 fascicles per rat

~70 fibrilstail

4-5 locationsfibril

20 nN (Sneddon)

BBML - All R

ights Reserve

d

Multiscale Diabetes Study (Poster 0495)

0

5

10

15

20

25

300 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 gt25

Perc

enta

ge o

f Gro

up S

ampl

es

Elastic Modulus (MPa)

CD (1530 indents)ZDSD (913 indents)

CD (n=5) 381 plusmn 149 MPa

ZDSD (n=3) 706 plusmn 263 MPa

0

100

200

300

400

500

600

700

800

900

0 2 4 6 8 10

Mic

rosc

ale

Mod

ulus

(MPa

)

Nanoscale Modulus (MPa)

plt0001

r2=0531 p=0040

bull Increased indentation modulus in diabetic tendon

bull Strong relationship between nanoscale indent modulus and microscale tensile modulus

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

Proper probe selection for intended application bull Cantilever must be stiff relative to sample bull Sharp probes may damage sample easily dull

R or α must be determined for each probe bull Properties provided by manufacturer are unreliable bull Vary from probe to probe can dull during indent process

Spring constant must be determined for each probe

bull Value provided by manufacturer is inaccurate bull Thermal tuning OK for ldquosoftrdquo probes poor performance

for stiffer probes bull Sader method (geometry) added mass reference

cantilevers

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

Veeco Application Technical note Ben Ohler Practical Advice on the Determination of Cantilever Spring Constants

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

bull Sample processing

bull Sample hydration

bull Proper force range

bull Substrate effects for thin samples

bull Proper modulus model and range of data goodness of fit

Bearing in mind these assumptions and limitations AFM can provide true ldquonanoscalerdquo mechanical properties both in terms of forces and deformations

BBML - All R

ights Reserve

d

A Note on Peak Force QNM for Bruker AFMs bull Mechanical properties mapped pixel-by-pixel at same

resolution as the height image (PeakForce QNM)

Height Modulus BBML - All R

ights Reserve

d

A Note on Peak Force QNM

Correct probe stiffness must be used based on sample Pittenger ndash Veeco Product Bulletin

BBML - All R

ights Reserve

d

A Note on Peak Force QNM Extremely sensitive to calibration (details rarely given) Absolute radius and spring constant known

bull especially important from probe to probe bull radius uarr with indent depth ndash must determine R at depth

you expect to image bull Depth may vary as a function of sample (or at different

regions within the sample)

Relative reference material of ldquoknownrdquo modulus used to determine a ratio of 119896119896 119877119877 bull ldquoknownrdquo modulus may come from tension compression

and not indentation bull R is related to depth indentation depth on sample must

be same as on reference BBML - All R

ights Reserve

d

A Note on Peak Force QNM bull Modulus fit on the fly ndash no ability to monitor fit (or range) bull Local sample topography and sample tilt play a role in

measured parameters bull Assuming all modulus values in an image are correct how to

quantify

512 x 512 pixels = 262144 measurements

n=1 n=262144

n from selected ROIs

BBML - All R

ights Reserve

d

BBML - All R

ights Reserve

d

bull Imaging in air on mica ndash substrate effect

bull Same imaging force or indent depth for all

bull For each area were all modulus values pooled

Lamprou et al PLoS ONE 2013 BBML - All R

ights Reserve

d

AFM Applications in Bone Research Other Cool Mechanical Techniques

BBML - All R

ights Reserve

d

ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo Bonds within or between collagen fibrils in bone that rupture to dissipate energy as a toughening mechanism

Thompson et al Nature 2001

Fantner et al Nature Mat 2005

Hansma et al JMNI 2005

Fantner et al Nano Letters 2007 BBML - All R

ights Reserve

d

Other Mechanical Techniques

Jimenez-Palomar J Mech Beh Bio Mat 2012

van der Rijt Macro Bio 2004

Yang J Biomed Mater Res 2007

Yang Biophysical Journal 2008 BBML - All R

ights Reserve

d

Other Cool Techniques bull Piezoresponse Force Microscopy (PFM)

bull Electrochemical AFM (EC-AFM)

bull Force Modulation Microscopy (FMM)

bull Lateral Force Microscopy (LFM)

bull Magnetic Force Microscopy (MFM)

bull Conductive AFM (C-AFM)

bull Force Pulling

bull Cantilever Bending

bull Tensile testing of individual fibrils

bull 3 point bending of fibrils

BBML - All R

ights Reserve

d

Summary Things to rememberhellip

bull Imaging mode imaging medium and probe selection

bull Morphology Measurements bull Sample preparation and hydration state bull Measuring fibril diameter bull Site selection and proper sampling bull Appropriate system calibration

bull Mechanical Measurements

bull Probe selection (stiffness and tip radius) bull Probe calibration (radius angle spring constant) bull Indentation force range and mechanical model bull Hydration in biological samples bull PeakForce QNM

BBML - All R

ights Reserve

d

Acknowledgements BBML

Max Hammond Armando Diaz Gonzalez Zachary Bart Silvia Canelon Creasy Clauser

IU School of Medicine Dr David Burr Dr Matt Allen Dr Stuart Warden Dr Lilian Plotkin

Funding Sources NIHNIDCR 1F32DE018840-01 A1 IUPUI BME Departmental Startup Funds IUPUI Research Support Funds Grant IUPUI Biomechanics and Biomaterials Research Center

NIH NICHD Dr Joan Marini

BBML - All R

ights Reserve

d

Bone Biology and Mechanics Lab (BBML) wwwiupuiedu~bbml Facebook BoneBiologyMechanicsLab

BBML - All R

ights Reserve

d

• Slide Number 1
• General Seminar Outline
• Bonersquos Hierarchical Structure
• Critical Gap in Understanding Collagen
• Methods For Analyzing Collagen Structure
• Methods For Imaging Collagen Structure
• Cryo-TEM
• Atomic Force Microscopy (AFM)
• AFM Image of Pentacene (C22H14)
• AFM Image of Hexabenzocoronene
• Basics of AFM Imaging The Probe
• Basics of AFM Imaging System Components
• van der Waals Potential Energy Curve
• Contact Mode ndash Monitor Deflection
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Peak Force Tapping Mode ndash Monitor Force
• AFM Applications in Bone Research Imaging
• AFM Imaging of Collagen
• Imaging of Osteoclast Activity
• Mineralized collagen deposition in vitro
• Imaging of Canilicular Structure
• AFM Imaging in Bone
• AFM Imaging in Bone
• Removing Mineral in Bone to Expose Collagen
• Removing Mineral in Bone to Expose Collagen
• Demineralization with EDTA
• Demineralization with EDTA
• Demineralization with EDTA
• Issues to Consider Effects of Acid
• Slide Number 32
• Slide Number 33
• Analysis of Collagen Structure in Bone
• Analysis of Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril D-Spacing
• Slide Number 39
• Collagen in Bone Fibril D-Spacing
• Collagen in Bone Fibril D-Spacing
• 2D Fast Fourier Transform Analysis
• 2D Fast Fourier Transform Analysis
• D-Spacing Distribution
• D-Spacing Distribution
• Cumulative Distribution Function
• CDF vs Histogram
• Changes in D-Spacing with Disease
• Changes in D-Spacing with Disease
• Slide Number 50
• Issues to Consider Site Selection and Sampling
• Issues to Consider Site Selection and Sampling
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• AFM calibration ndash Take Away Message
• AFM Applications in Bone ResearchIndentation Mechanics
• AFM-Based Indentation
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Parameters
• Hertz Contact Equation
• Hertz Contact Equation
• Hertz Contact Equation
• Proper Indentation Model is Essential
• Indentation in OI Tendon Study
• OI Tendon Differential Effects of Dehydration
• OI Tendon Differential Effects of Dehydration
• Indentation in Diabetic Tendon (Poster 0495)
• Multiscale Diabetes Study (Poster 0495)
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• A Note on Peak Force QNM for Bruker AFMs
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• Slide Number 83
• Slide Number 84
• AFM Applications in Bone ResearchOther Cool Mechanical Techniques
• ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo
• Other Mechanical Techniques
• Other Cool Techniques
• Summary Things to rememberhellip
• Acknowledgements
• Bone Biology and Mechanics Lab (BBML)

CDF vs Histogram 62 63 64 65 66 67 68 69 70 71 72 73 74

0

10

20

30

40

50

60

70

80

90

100

0

5

10

15

20

25

30

35

40

45

50

62 63 64 65 66 67 68 69 70 71 72 73 74

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

HistoCDF

BBML - All R

ights Reserve

d

Changes in D-Spacing with Disease

0

5

10

15

20

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

0

5

10

15

20

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

Wallace et al Bone 2010 46 1349-1354

KS Test plt0001

Control 680 plusmn 26 nm

Estrogen Dep 659 plusmn 31 nm

t-test on mean p=0048

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Changes in D-Spacing with Disease

Wallace et al Bone 2010 46 1349-1354

0

10

20

30

40

50

60

70

80

90

100

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

KS Test plt0001

BBML - All R

ights Reserve

d

Question Model and Tissue Morphology Publication

Tissue Specificity in Mice Male mouse 8 weeks Femur dentin tail tendon

No differences in bone vs dentin both ∆ vs tendon

Wallace et al Langmuir 2011 26(10) 7349-7354

Gender Effects Male and female mice 16 weeks Femur No differences NA

Estrogen Depletion OVX in sheep 5 years + 2 Radius Mean and population shift Wallace et al Bone

2010 46 1349-1354

Estrogen Depletion OVX in female 5 years + 2 Skin (dermis) Significant population shift Erickson et al Biotech

J 2013 8(1) 1174-126

Osteoporosis and Mechanical Loading

OVX in ulnar loading in rats Ulna

No loading effect population shift with OVX

Warden et al Endocrinology 2013 154(9) 3178-3187

Osteogenesis Imperfecta Brtl+ model male mice 2 months Femur

No mean ∆ widening of distribution

Wallace et al J Stuct Bio 2011 173 146-152

Osteogenesis Imperfecta Oimoim model male mice 12 weeks Femur Mean and population shift Bart et al Conn Tiss

Res 2014 In Press

Osteogenesis Imperfecta and Tissue Hydration

Brtl+ model male mice 6 months Tail Tendon Significant population shift Kemp et al J Stuct Bio

2012 180 428-438

Type 2 Diabetes Male ZDSD rats 30 weeks Tibia Significant population shift Hammond et al Bone

2014 60 26-32

Type 2 Diabetes Female ZDSD rats 32 weeks Tail Tendon Significant population shift

Diaz Gonzalez et al J Biomech 2014 47(3)

681-686

Raloxifene (in vivo and in vitro) Female dog treated with Raloxifene Femur Significant population shift Gallant et al Bone

2014 61 191-200 BBML - All R

ights Reserve

d

Issues to Consider Site Selection and Sampling

KS Test plt0001

0023 of bonersquos length

15 m

m =

150

00 micro

m

Wallace et al Langmuir 2011 26(10) 7349-7354

100 microm x 100 microm

35 microm x 35 microm

BBML - All R

ights Reserve

d

Issues to Consider Site Selection and Sampling

60

62

64

66

68

70

72

74

1 (35)

2 (33)

3 (40)

4 (39)

5 (35)

6 (29)

7 (26)

8 (23)

9 (28)

WT (288)

D-P

erio

dic

Gap

Ove

rlap

Spac

ing

(nm

)

Axial Location (Proximal End = 1 Distal End = 9)

( of Fibrils)

91

23

45

87

6

Wallace et al Langmuir 2011 26(10) 7349-7354

288 fibrils for population n=1 for mean comparisons BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

Wet Tendon Dry Tendon Kemp et al J StructBio 2012 180 (3) 428-438

Collagen and bone exist in a hydrated environment in vivo bull Hydration state can impact morphology and mechanics

BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

Nonsignificant mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet (n=255) 682 plusmn 12 nm

WT Dry (n=257) 686 plusmn 08 nm

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

Brtl DryBrtl Wet

KS Test plt0001

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

WT Case 04 nm mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet 682 plusmn 12 nm

WT Dry 686 plusmn 08 nm

Brtl+ Wet 675 plusmn 14 nm

Brtl+ Dry 689 plusmn 10 nm

Brtl+ Case 14 nm increase with drying (plt0001) bull Entire population shifted upward (plt0001)

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

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Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT WetBrtl Wet

KS Test plt0001

Brtl+ distribution shifted downward vs WT when wet

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryBrtl Dry

KS Test plt0001

Brtl+ distribution shifted upward vs WT when dry

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider AFM calibration

bull Absolute distance measurements require accurate piezo calibration

bull AFM systems have large scan ranges (150 microm to lt 1 microm)

bull Scanner non-linearities can introduce substantial error over that range

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

ights Reserve

d

Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

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d

Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

100 nm 10 80 602 1198 100 nm 7 60 827 1439 100 nm 5 40 814 1427 100 nm 35 20 917 1724 100 nm 2 10 937 1774

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

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d

AFM calibration ndash Take Away Message

Calibration using a scan size and feature size comparable to features of interest is highly recommended

Calibration addresses absolute accuracy not precision (differential sensitivity between measurements)

bull Does not limit ability to differentiate between groups using the same AFM with the same calibration parameters

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d

AFM Applications in Bone Research Indentation Mechanics

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ights Reserve

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AFM-Based Indentation

In addition to high resolution imaging AFM can be used to extract nanoscale mechanical data

ApproachWithdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

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d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Can

tilev

er D

efle

ctio

n (V

olts

)

1 Ramp into a rigid sample ndash measure the slope

Deflection sensitivity x 119899119899119899119899119881119881

2 Calculate cantilever spring constant

3 Hookersquos law takes deflection (volts) and converts to units of force F = kx

Spring constant k 119873119873119899119899

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ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Forc

e (n

N)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

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ights Reserve

d

AFM-Based Indentation Cantilever Calibration Fo

rce

(nN

)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

Tip-Sample Separation (nm)

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ights Reserve

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AFM-Based Indentation Parameters

Approach Withdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

Peak Force

Area Between Curves Energy Dissipation

Contact Mechanics Fit for Elastic Modulus (E)

Adhesion Force

Indentation Depth (δ)

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ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

z

r

δ

R

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119877119877 ∙ 12057512057532

Es Sample modulus νs Poissonrsquos ratio R tip radius of curvature δ load point displacement

Relevant Hertz Assumptions bull Strains are small and within the elastic limit bull Indenter is infinitely stiff (only sample deforms) bull Indentation depth ltlt radius of curvature of indenter bull Surfaces are continuous non-conforming (must be flat) bull Load is normal to surface

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ights Reserve

d

Proper Indentation Model is Essential

δ

r

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119903119903 ∙ 12057512057532

δ

α

119865119865 =2120587120587∙

1198641198641199041199041 minus 1205841205841199041199042

∙ tan120572120572 ∙ 1205751205752

Es Sample modulus νs Poissonrsquos ratio R tip radius α opening angle

Hertz Model Spherical Indenter

depth ltlt radius of curvature

Sneddon Model Conical Indenter

depth ge radius of curvature

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ights Reserve

d

Indentation in OI Tendon Study

x

z

yBrtl+ and WT mice 6 month old males

4 tails per group

2-3 fascicles per tail

25-30 fibrils per tail

4-5 locations per fibril

Indent force 20 nN wet (Sneddon) 50 nN dry (Hertz) BBML - A

ll Rights R

eserved

OI Tendon Differential Effects of Dehydration

Dry 1268 plusmn 718 MPa

Wet 305 plusmn 243 MPa

Dry 1737 plusmn 973 MPa

Wet 301 plusmn 202 MPa

Kemp et al J StructBio 2012 180 (3) 428-438

bull Significant stiffening of fibrils with dehydration

bull More pronounced modulus modulus as a function of disease

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OI Tendon Differential Effects of Dehydration

Kemp et al J StructBio 2012 180 (3) 428-438

Diseased phenotype not present when wet phenotype observed when dried

0

10

20

30

40

50

60

0 20 40 60 80 100 120 140 160 180 200

Gro

up S

ampl

es (

)

Indent Modulus (MPa)

WT WetBrtl Wet

0

2

4

6

8

10

12

14

16

18

20

025

050

075

010

0012

5015

0017

5020

0022

5025

0027

5030

0032

5035

0037

5040

0042

5045

0047

5050

00

Gro

up S

ampl

es (

)Indent Modulus (MPa)

WT DryBrtl Dry

555 WT Indents 305 plusmn 243 MPa

614 Brtl+ Indents 301 plusmn 202 MPa

KS Test p=0129 t Test p=0464 464 WT Indents

1268 plusmn 718 MPa 479 Brtl+ Indents 1737 plusmn 973 MPa

KS Test plt0001 T Test plt0001

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ights Reserve

d

Indentation in Diabetic Tendon (Poster 0495)

x

z

y

Control and Diabetic Rat Tail Tendon

4-5 rats per group

2-3 fascicles per rat

~70 fibrilstail

4-5 locationsfibril

20 nN (Sneddon)

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Multiscale Diabetes Study (Poster 0495)

0

5

10

15

20

25

300 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 gt25

Perc

enta

ge o

f Gro

up S

ampl

es

Elastic Modulus (MPa)

CD (1530 indents)ZDSD (913 indents)

CD (n=5) 381 plusmn 149 MPa

ZDSD (n=3) 706 plusmn 263 MPa

0

100

200

300

400

500

600

700

800

900

0 2 4 6 8 10

Mic

rosc

ale

Mod

ulus

(MPa

)

Nanoscale Modulus (MPa)

plt0001

r2=0531 p=0040

bull Increased indentation modulus in diabetic tendon

bull Strong relationship between nanoscale indent modulus and microscale tensile modulus

BBML - All R

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d

Indentation Limitations and Weaknesses

Proper probe selection for intended application bull Cantilever must be stiff relative to sample bull Sharp probes may damage sample easily dull

R or α must be determined for each probe bull Properties provided by manufacturer are unreliable bull Vary from probe to probe can dull during indent process

Spring constant must be determined for each probe

bull Value provided by manufacturer is inaccurate bull Thermal tuning OK for ldquosoftrdquo probes poor performance

for stiffer probes bull Sader method (geometry) added mass reference

cantilevers

BBML - All R

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d

Indentation Limitations and Weaknesses

Veeco Application Technical note Ben Ohler Practical Advice on the Determination of Cantilever Spring Constants

BBML - All R

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d

Indentation Limitations and Weaknesses

bull Sample processing

bull Sample hydration

bull Proper force range

bull Substrate effects for thin samples

bull Proper modulus model and range of data goodness of fit

Bearing in mind these assumptions and limitations AFM can provide true ldquonanoscalerdquo mechanical properties both in terms of forces and deformations

BBML - All R

ights Reserve

d

A Note on Peak Force QNM for Bruker AFMs bull Mechanical properties mapped pixel-by-pixel at same

resolution as the height image (PeakForce QNM)

Height Modulus BBML - All R

ights Reserve

d

A Note on Peak Force QNM

Correct probe stiffness must be used based on sample Pittenger ndash Veeco Product Bulletin

BBML - All R

ights Reserve

d

A Note on Peak Force QNM Extremely sensitive to calibration (details rarely given) Absolute radius and spring constant known

bull especially important from probe to probe bull radius uarr with indent depth ndash must determine R at depth

you expect to image bull Depth may vary as a function of sample (or at different

regions within the sample)

Relative reference material of ldquoknownrdquo modulus used to determine a ratio of 119896119896 119877119877 bull ldquoknownrdquo modulus may come from tension compression

and not indentation bull R is related to depth indentation depth on sample must

be same as on reference BBML - All R

ights Reserve

d

A Note on Peak Force QNM bull Modulus fit on the fly ndash no ability to monitor fit (or range) bull Local sample topography and sample tilt play a role in

measured parameters bull Assuming all modulus values in an image are correct how to

quantify

512 x 512 pixels = 262144 measurements

n=1 n=262144

n from selected ROIs

BBML - All R

ights Reserve

d

BBML - All R

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d

bull Imaging in air on mica ndash substrate effect

bull Same imaging force or indent depth for all

bull For each area were all modulus values pooled

Lamprou et al PLoS ONE 2013 BBML - All R

ights Reserve

d

AFM Applications in Bone Research Other Cool Mechanical Techniques

BBML - All R

ights Reserve

d

ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo Bonds within or between collagen fibrils in bone that rupture to dissipate energy as a toughening mechanism

Thompson et al Nature 2001

Fantner et al Nature Mat 2005

Hansma et al JMNI 2005

Fantner et al Nano Letters 2007 BBML - All R

ights Reserve

d

Other Mechanical Techniques

Jimenez-Palomar J Mech Beh Bio Mat 2012

van der Rijt Macro Bio 2004

Yang J Biomed Mater Res 2007

Yang Biophysical Journal 2008 BBML - All R

ights Reserve

d

Other Cool Techniques bull Piezoresponse Force Microscopy (PFM)

bull Electrochemical AFM (EC-AFM)

bull Force Modulation Microscopy (FMM)

bull Lateral Force Microscopy (LFM)

bull Magnetic Force Microscopy (MFM)

bull Conductive AFM (C-AFM)

bull Force Pulling

bull Cantilever Bending

bull Tensile testing of individual fibrils

bull 3 point bending of fibrils

BBML - All R

ights Reserve

d

Summary Things to rememberhellip

bull Imaging mode imaging medium and probe selection

bull Morphology Measurements bull Sample preparation and hydration state bull Measuring fibril diameter bull Site selection and proper sampling bull Appropriate system calibration

bull Mechanical Measurements

bull Probe selection (stiffness and tip radius) bull Probe calibration (radius angle spring constant) bull Indentation force range and mechanical model bull Hydration in biological samples bull PeakForce QNM

BBML - All R

ights Reserve

d

Acknowledgements BBML

Max Hammond Armando Diaz Gonzalez Zachary Bart Silvia Canelon Creasy Clauser

IU School of Medicine Dr David Burr Dr Matt Allen Dr Stuart Warden Dr Lilian Plotkin

Funding Sources NIHNIDCR 1F32DE018840-01 A1 IUPUI BME Departmental Startup Funds IUPUI Research Support Funds Grant IUPUI Biomechanics and Biomaterials Research Center

NIH NICHD Dr Joan Marini

BBML - All R

ights Reserve

d

Bone Biology and Mechanics Lab (BBML) wwwiupuiedu~bbml Facebook BoneBiologyMechanicsLab

BBML - All R

ights Reserve

d

• Slide Number 1
• General Seminar Outline
• Bonersquos Hierarchical Structure
• Critical Gap in Understanding Collagen
• Methods For Analyzing Collagen Structure
• Methods For Imaging Collagen Structure
• Cryo-TEM
• Atomic Force Microscopy (AFM)
• AFM Image of Pentacene (C22H14)
• AFM Image of Hexabenzocoronene
• Basics of AFM Imaging The Probe
• Basics of AFM Imaging System Components
• van der Waals Potential Energy Curve
• Contact Mode ndash Monitor Deflection
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Peak Force Tapping Mode ndash Monitor Force
• AFM Applications in Bone Research Imaging
• AFM Imaging of Collagen
• Imaging of Osteoclast Activity
• Mineralized collagen deposition in vitro
• Imaging of Canilicular Structure
• AFM Imaging in Bone
• AFM Imaging in Bone
• Removing Mineral in Bone to Expose Collagen
• Removing Mineral in Bone to Expose Collagen
• Demineralization with EDTA
• Demineralization with EDTA
• Demineralization with EDTA
• Issues to Consider Effects of Acid
• Slide Number 32
• Slide Number 33
• Analysis of Collagen Structure in Bone
• Analysis of Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril D-Spacing
• Slide Number 39
• Collagen in Bone Fibril D-Spacing
• Collagen in Bone Fibril D-Spacing
• 2D Fast Fourier Transform Analysis
• 2D Fast Fourier Transform Analysis
• D-Spacing Distribution
• D-Spacing Distribution
• Cumulative Distribution Function
• CDF vs Histogram
• Changes in D-Spacing with Disease
• Changes in D-Spacing with Disease
• Slide Number 50
• Issues to Consider Site Selection and Sampling
• Issues to Consider Site Selection and Sampling
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• AFM calibration ndash Take Away Message
• AFM Applications in Bone ResearchIndentation Mechanics
• AFM-Based Indentation
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Parameters
• Hertz Contact Equation
• Hertz Contact Equation
• Hertz Contact Equation
• Proper Indentation Model is Essential
• Indentation in OI Tendon Study
• OI Tendon Differential Effects of Dehydration
• OI Tendon Differential Effects of Dehydration
• Indentation in Diabetic Tendon (Poster 0495)
• Multiscale Diabetes Study (Poster 0495)
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• A Note on Peak Force QNM for Bruker AFMs
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• Slide Number 83
• Slide Number 84
• AFM Applications in Bone ResearchOther Cool Mechanical Techniques
• ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo
• Other Mechanical Techniques
• Other Cool Techniques
• Summary Things to rememberhellip
• Acknowledgements
• Bone Biology and Mechanics Lab (BBML)

Changes in D-Spacing with Disease

0

5

10

15

20

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

0

5

10

15

20

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Gro

up S

ampl

es (

)

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

Wallace et al Bone 2010 46 1349-1354

KS Test plt0001

Control 680 plusmn 26 nm

Estrogen Dep 659 plusmn 31 nm

t-test on mean p=0048

BBML - All R

ights Reserve

d

Changes in D-Spacing with Disease

Wallace et al Bone 2010 46 1349-1354

0

10

20

30

40

50

60

70

80

90

100

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

KS Test plt0001

BBML - All R

ights Reserve

d

Question Model and Tissue Morphology Publication

Tissue Specificity in Mice Male mouse 8 weeks Femur dentin tail tendon

No differences in bone vs dentin both ∆ vs tendon

Wallace et al Langmuir 2011 26(10) 7349-7354

Gender Effects Male and female mice 16 weeks Femur No differences NA

Estrogen Depletion OVX in sheep 5 years + 2 Radius Mean and population shift Wallace et al Bone

2010 46 1349-1354

Estrogen Depletion OVX in female 5 years + 2 Skin (dermis) Significant population shift Erickson et al Biotech

J 2013 8(1) 1174-126

Osteoporosis and Mechanical Loading

OVX in ulnar loading in rats Ulna

No loading effect population shift with OVX

Warden et al Endocrinology 2013 154(9) 3178-3187

Osteogenesis Imperfecta Brtl+ model male mice 2 months Femur

No mean ∆ widening of distribution

Wallace et al J Stuct Bio 2011 173 146-152

Osteogenesis Imperfecta Oimoim model male mice 12 weeks Femur Mean and population shift Bart et al Conn Tiss

Res 2014 In Press

Osteogenesis Imperfecta and Tissue Hydration

Brtl+ model male mice 6 months Tail Tendon Significant population shift Kemp et al J Stuct Bio

2012 180 428-438

Type 2 Diabetes Male ZDSD rats 30 weeks Tibia Significant population shift Hammond et al Bone

2014 60 26-32

Type 2 Diabetes Female ZDSD rats 32 weeks Tail Tendon Significant population shift

Diaz Gonzalez et al J Biomech 2014 47(3)

681-686

Raloxifene (in vivo and in vitro) Female dog treated with Raloxifene Femur Significant population shift Gallant et al Bone

2014 61 191-200 BBML - All R

ights Reserve

d

Issues to Consider Site Selection and Sampling

KS Test plt0001

0023 of bonersquos length

15 m

m =

150

00 micro

m

Wallace et al Langmuir 2011 26(10) 7349-7354

100 microm x 100 microm

35 microm x 35 microm

BBML - All R

ights Reserve

d

Issues to Consider Site Selection and Sampling

60

62

64

66

68

70

72

74

1 (35)

2 (33)

3 (40)

4 (39)

5 (35)

6 (29)

7 (26)

8 (23)

9 (28)

WT (288)

D-P

erio

dic

Gap

Ove

rlap

Spac

ing

(nm

)

Axial Location (Proximal End = 1 Distal End = 9)

( of Fibrils)

91

23

45

87

6

Wallace et al Langmuir 2011 26(10) 7349-7354

288 fibrils for population n=1 for mean comparisons BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

Wet Tendon Dry Tendon Kemp et al J StructBio 2012 180 (3) 428-438

Collagen and bone exist in a hydrated environment in vivo bull Hydration state can impact morphology and mechanics

BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

Nonsignificant mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet (n=255) 682 plusmn 12 nm

WT Dry (n=257) 686 plusmn 08 nm

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

Brtl DryBrtl Wet

KS Test plt0001

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

WT Case 04 nm mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet 682 plusmn 12 nm

WT Dry 686 plusmn 08 nm

Brtl+ Wet 675 plusmn 14 nm

Brtl+ Dry 689 plusmn 10 nm

Brtl+ Case 14 nm increase with drying (plt0001) bull Entire population shifted upward (plt0001)

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT WetBrtl Wet

KS Test plt0001

Brtl+ distribution shifted downward vs WT when wet

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryBrtl Dry

KS Test plt0001

Brtl+ distribution shifted upward vs WT when dry

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider AFM calibration

bull Absolute distance measurements require accurate piezo calibration

bull AFM systems have large scan ranges (150 microm to lt 1 microm)

bull Scanner non-linearities can introduce substantial error over that range

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

ights Reserve

d

Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

BBML - All R

ights Reserve

d

Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

100 nm 10 80 602 1198 100 nm 7 60 827 1439 100 nm 5 40 814 1427 100 nm 35 20 917 1724 100 nm 2 10 937 1774

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

ights Reserve

d

AFM calibration ndash Take Away Message

Calibration using a scan size and feature size comparable to features of interest is highly recommended

Calibration addresses absolute accuracy not precision (differential sensitivity between measurements)

bull Does not limit ability to differentiate between groups using the same AFM with the same calibration parameters

BBML - All R

ights Reserve

d

AFM Applications in Bone Research Indentation Mechanics

BBML - All R

ights Reserve

d

AFM-Based Indentation

In addition to high resolution imaging AFM can be used to extract nanoscale mechanical data

ApproachWithdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Can

tilev

er D

efle

ctio

n (V

olts

)

1 Ramp into a rigid sample ndash measure the slope

Deflection sensitivity x 119899119899119899119899119881119881

2 Calculate cantilever spring constant

3 Hookersquos law takes deflection (volts) and converts to units of force F = kx

Spring constant k 119873119873119899119899

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Forc

e (n

N)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration Fo

rce

(nN

)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

Tip-Sample Separation (nm)

BBML - All R

ights Reserve

d

AFM-Based Indentation Parameters

Approach Withdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

Peak Force

Area Between Curves Energy Dissipation

Contact Mechanics Fit for Elastic Modulus (E)

Adhesion Force

Indentation Depth (δ)

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

z

r

δ

R

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119877119877 ∙ 12057512057532

Es Sample modulus νs Poissonrsquos ratio R tip radius of curvature δ load point displacement

Relevant Hertz Assumptions bull Strains are small and within the elastic limit bull Indenter is infinitely stiff (only sample deforms) bull Indentation depth ltlt radius of curvature of indenter bull Surfaces are continuous non-conforming (must be flat) bull Load is normal to surface

BBML - All R

ights Reserve

d

Proper Indentation Model is Essential

δ

r

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119903119903 ∙ 12057512057532

δ

α

119865119865 =2120587120587∙

1198641198641199041199041 minus 1205841205841199041199042

∙ tan120572120572 ∙ 1205751205752

Es Sample modulus νs Poissonrsquos ratio R tip radius α opening angle

Hertz Model Spherical Indenter

depth ltlt radius of curvature

Sneddon Model Conical Indenter

depth ge radius of curvature

BBML - All R

ights Reserve

d

Indentation in OI Tendon Study

x

z

yBrtl+ and WT mice 6 month old males

4 tails per group

2-3 fascicles per tail

25-30 fibrils per tail

4-5 locations per fibril

Indent force 20 nN wet (Sneddon) 50 nN dry (Hertz) BBML - A

ll Rights R

eserved

OI Tendon Differential Effects of Dehydration

Dry 1268 plusmn 718 MPa

Wet 305 plusmn 243 MPa

Dry 1737 plusmn 973 MPa

Wet 301 plusmn 202 MPa

Kemp et al J StructBio 2012 180 (3) 428-438

bull Significant stiffening of fibrils with dehydration

bull More pronounced modulus modulus as a function of disease

BBML - All R

ights Reserve

d

OI Tendon Differential Effects of Dehydration

Kemp et al J StructBio 2012 180 (3) 428-438

Diseased phenotype not present when wet phenotype observed when dried

0

10

20

30

40

50

60

0 20 40 60 80 100 120 140 160 180 200

Gro

up S

ampl

es (

)

Indent Modulus (MPa)

WT WetBrtl Wet

0

2

4

6

8

10

12

14

16

18

20

025

050

075

010

0012

5015

0017

5020

0022

5025

0027

5030

0032

5035

0037

5040

0042

5045

0047

5050

00

Gro

up S

ampl

es (

)Indent Modulus (MPa)

WT DryBrtl Dry

555 WT Indents 305 plusmn 243 MPa

614 Brtl+ Indents 301 plusmn 202 MPa

KS Test p=0129 t Test p=0464 464 WT Indents

1268 plusmn 718 MPa 479 Brtl+ Indents 1737 plusmn 973 MPa

KS Test plt0001 T Test plt0001

BBML - All R

ights Reserve

d

Indentation in Diabetic Tendon (Poster 0495)

x

z

y

Control and Diabetic Rat Tail Tendon

4-5 rats per group

2-3 fascicles per rat

~70 fibrilstail

4-5 locationsfibril

20 nN (Sneddon)

BBML - All R

ights Reserve

d

Multiscale Diabetes Study (Poster 0495)

0

5

10

15

20

25

300 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 gt25

Perc

enta

ge o

f Gro

up S

ampl

es

Elastic Modulus (MPa)

CD (1530 indents)ZDSD (913 indents)

CD (n=5) 381 plusmn 149 MPa

ZDSD (n=3) 706 plusmn 263 MPa

0

100

200

300

400

500

600

700

800

900

0 2 4 6 8 10

Mic

rosc

ale

Mod

ulus

(MPa

)

Nanoscale Modulus (MPa)

plt0001

r2=0531 p=0040

bull Increased indentation modulus in diabetic tendon

bull Strong relationship between nanoscale indent modulus and microscale tensile modulus

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

Proper probe selection for intended application bull Cantilever must be stiff relative to sample bull Sharp probes may damage sample easily dull

R or α must be determined for each probe bull Properties provided by manufacturer are unreliable bull Vary from probe to probe can dull during indent process

Spring constant must be determined for each probe

bull Value provided by manufacturer is inaccurate bull Thermal tuning OK for ldquosoftrdquo probes poor performance

for stiffer probes bull Sader method (geometry) added mass reference

cantilevers

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

Veeco Application Technical note Ben Ohler Practical Advice on the Determination of Cantilever Spring Constants

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

bull Sample processing

bull Sample hydration

bull Proper force range

bull Substrate effects for thin samples

bull Proper modulus model and range of data goodness of fit

Bearing in mind these assumptions and limitations AFM can provide true ldquonanoscalerdquo mechanical properties both in terms of forces and deformations

BBML - All R

ights Reserve

d

A Note on Peak Force QNM for Bruker AFMs bull Mechanical properties mapped pixel-by-pixel at same

resolution as the height image (PeakForce QNM)

Height Modulus BBML - All R

ights Reserve

d

A Note on Peak Force QNM

Correct probe stiffness must be used based on sample Pittenger ndash Veeco Product Bulletin

BBML - All R

ights Reserve

d

A Note on Peak Force QNM Extremely sensitive to calibration (details rarely given) Absolute radius and spring constant known

bull especially important from probe to probe bull radius uarr with indent depth ndash must determine R at depth

you expect to image bull Depth may vary as a function of sample (or at different

regions within the sample)

Relative reference material of ldquoknownrdquo modulus used to determine a ratio of 119896119896 119877119877 bull ldquoknownrdquo modulus may come from tension compression

and not indentation bull R is related to depth indentation depth on sample must

be same as on reference BBML - All R

ights Reserve

d

A Note on Peak Force QNM bull Modulus fit on the fly ndash no ability to monitor fit (or range) bull Local sample topography and sample tilt play a role in

measured parameters bull Assuming all modulus values in an image are correct how to

quantify

512 x 512 pixels = 262144 measurements

n=1 n=262144

n from selected ROIs

BBML - All R

ights Reserve

d

BBML - All R

ights Reserve

d

bull Imaging in air on mica ndash substrate effect

bull Same imaging force or indent depth for all

bull For each area were all modulus values pooled

Lamprou et al PLoS ONE 2013 BBML - All R

ights Reserve

d

AFM Applications in Bone Research Other Cool Mechanical Techniques

BBML - All R

ights Reserve

d

ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo Bonds within or between collagen fibrils in bone that rupture to dissipate energy as a toughening mechanism

Thompson et al Nature 2001

Fantner et al Nature Mat 2005

Hansma et al JMNI 2005

Fantner et al Nano Letters 2007 BBML - All R

ights Reserve

d

Other Mechanical Techniques

Jimenez-Palomar J Mech Beh Bio Mat 2012

van der Rijt Macro Bio 2004

Yang J Biomed Mater Res 2007

Yang Biophysical Journal 2008 BBML - All R

ights Reserve

d

Other Cool Techniques bull Piezoresponse Force Microscopy (PFM)

bull Electrochemical AFM (EC-AFM)

bull Force Modulation Microscopy (FMM)

bull Lateral Force Microscopy (LFM)

bull Magnetic Force Microscopy (MFM)

bull Conductive AFM (C-AFM)

bull Force Pulling

bull Cantilever Bending

bull Tensile testing of individual fibrils

bull 3 point bending of fibrils

BBML - All R

ights Reserve

d

Summary Things to rememberhellip

bull Imaging mode imaging medium and probe selection

bull Morphology Measurements bull Sample preparation and hydration state bull Measuring fibril diameter bull Site selection and proper sampling bull Appropriate system calibration

bull Mechanical Measurements

bull Probe selection (stiffness and tip radius) bull Probe calibration (radius angle spring constant) bull Indentation force range and mechanical model bull Hydration in biological samples bull PeakForce QNM

BBML - All R

ights Reserve

d

Acknowledgements BBML

Max Hammond Armando Diaz Gonzalez Zachary Bart Silvia Canelon Creasy Clauser

IU School of Medicine Dr David Burr Dr Matt Allen Dr Stuart Warden Dr Lilian Plotkin

Funding Sources NIHNIDCR 1F32DE018840-01 A1 IUPUI BME Departmental Startup Funds IUPUI Research Support Funds Grant IUPUI Biomechanics and Biomaterials Research Center

NIH NICHD Dr Joan Marini

BBML - All R

ights Reserve

d

Bone Biology and Mechanics Lab (BBML) wwwiupuiedu~bbml Facebook BoneBiologyMechanicsLab

BBML - All R

ights Reserve

d

• Slide Number 1
• General Seminar Outline
• Bonersquos Hierarchical Structure
• Critical Gap in Understanding Collagen
• Methods For Analyzing Collagen Structure
• Methods For Imaging Collagen Structure
• Cryo-TEM
• Atomic Force Microscopy (AFM)
• AFM Image of Pentacene (C22H14)
• AFM Image of Hexabenzocoronene
• Basics of AFM Imaging The Probe
• Basics of AFM Imaging System Components
• van der Waals Potential Energy Curve
• Contact Mode ndash Monitor Deflection
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Peak Force Tapping Mode ndash Monitor Force
• AFM Applications in Bone Research Imaging
• AFM Imaging of Collagen
• Imaging of Osteoclast Activity
• Mineralized collagen deposition in vitro
• Imaging of Canilicular Structure
• AFM Imaging in Bone
• AFM Imaging in Bone
• Removing Mineral in Bone to Expose Collagen
• Removing Mineral in Bone to Expose Collagen
• Demineralization with EDTA
• Demineralization with EDTA
• Demineralization with EDTA
• Issues to Consider Effects of Acid
• Slide Number 32
• Slide Number 33
• Analysis of Collagen Structure in Bone
• Analysis of Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril D-Spacing
• Slide Number 39
• Collagen in Bone Fibril D-Spacing
• Collagen in Bone Fibril D-Spacing
• 2D Fast Fourier Transform Analysis
• 2D Fast Fourier Transform Analysis
• D-Spacing Distribution
• D-Spacing Distribution
• Cumulative Distribution Function
• CDF vs Histogram
• Changes in D-Spacing with Disease
• Changes in D-Spacing with Disease
• Slide Number 50
• Issues to Consider Site Selection and Sampling
• Issues to Consider Site Selection and Sampling
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• AFM calibration ndash Take Away Message
• AFM Applications in Bone ResearchIndentation Mechanics
• AFM-Based Indentation
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Parameters
• Hertz Contact Equation
• Hertz Contact Equation
• Hertz Contact Equation
• Proper Indentation Model is Essential
• Indentation in OI Tendon Study
• OI Tendon Differential Effects of Dehydration
• OI Tendon Differential Effects of Dehydration
• Indentation in Diabetic Tendon (Poster 0495)
• Multiscale Diabetes Study (Poster 0495)
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• A Note on Peak Force QNM for Bruker AFMs
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• Slide Number 83
• Slide Number 84
• AFM Applications in Bone ResearchOther Cool Mechanical Techniques
• ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo
• Other Mechanical Techniques
• Other Cool Techniques
• Summary Things to rememberhellip
• Acknowledgements
• Bone Biology and Mechanics Lab (BBML)

Changes in D-Spacing with Disease

Wallace et al Bone 2010 46 1349-1354

0

10

20

30

40

50

60

70

80

90

100

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

Control n=6 182 FibrilsEstrogen Depleted n=5 168 fibrils

KS Test plt0001

BBML - All R

ights Reserve

d

Question Model and Tissue Morphology Publication

Tissue Specificity in Mice Male mouse 8 weeks Femur dentin tail tendon

No differences in bone vs dentin both ∆ vs tendon

Wallace et al Langmuir 2011 26(10) 7349-7354

Gender Effects Male and female mice 16 weeks Femur No differences NA

Estrogen Depletion OVX in sheep 5 years + 2 Radius Mean and population shift Wallace et al Bone

2010 46 1349-1354

Estrogen Depletion OVX in female 5 years + 2 Skin (dermis) Significant population shift Erickson et al Biotech

J 2013 8(1) 1174-126

Osteoporosis and Mechanical Loading

OVX in ulnar loading in rats Ulna

No loading effect population shift with OVX

Warden et al Endocrinology 2013 154(9) 3178-3187

Osteogenesis Imperfecta Brtl+ model male mice 2 months Femur

No mean ∆ widening of distribution

Wallace et al J Stuct Bio 2011 173 146-152

Osteogenesis Imperfecta Oimoim model male mice 12 weeks Femur Mean and population shift Bart et al Conn Tiss

Res 2014 In Press

Osteogenesis Imperfecta and Tissue Hydration

Brtl+ model male mice 6 months Tail Tendon Significant population shift Kemp et al J Stuct Bio

2012 180 428-438

Type 2 Diabetes Male ZDSD rats 30 weeks Tibia Significant population shift Hammond et al Bone

2014 60 26-32

Type 2 Diabetes Female ZDSD rats 32 weeks Tail Tendon Significant population shift

Diaz Gonzalez et al J Biomech 2014 47(3)

681-686

Raloxifene (in vivo and in vitro) Female dog treated with Raloxifene Femur Significant population shift Gallant et al Bone

2014 61 191-200 BBML - All R

ights Reserve

d

Issues to Consider Site Selection and Sampling

KS Test plt0001

0023 of bonersquos length

15 m

m =

150

00 micro

m

Wallace et al Langmuir 2011 26(10) 7349-7354

100 microm x 100 microm

35 microm x 35 microm

BBML - All R

ights Reserve

d

Issues to Consider Site Selection and Sampling

60

62

64

66

68

70

72

74

1 (35)

2 (33)

3 (40)

4 (39)

5 (35)

6 (29)

7 (26)

8 (23)

9 (28)

WT (288)

D-P

erio

dic

Gap

Ove

rlap

Spac

ing

(nm

)

Axial Location (Proximal End = 1 Distal End = 9)

( of Fibrils)

91

23

45

87

6

Wallace et al Langmuir 2011 26(10) 7349-7354

288 fibrils for population n=1 for mean comparisons BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

Wet Tendon Dry Tendon Kemp et al J StructBio 2012 180 (3) 428-438

Collagen and bone exist in a hydrated environment in vivo bull Hydration state can impact morphology and mechanics

BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

Nonsignificant mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet (n=255) 682 plusmn 12 nm

WT Dry (n=257) 686 plusmn 08 nm

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

Brtl DryBrtl Wet

KS Test plt0001

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

WT Case 04 nm mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet 682 plusmn 12 nm

WT Dry 686 plusmn 08 nm

Brtl+ Wet 675 plusmn 14 nm

Brtl+ Dry 689 plusmn 10 nm

Brtl+ Case 14 nm increase with drying (plt0001) bull Entire population shifted upward (plt0001)

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT WetBrtl Wet

KS Test plt0001

Brtl+ distribution shifted downward vs WT when wet

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryBrtl Dry

KS Test plt0001

Brtl+ distribution shifted upward vs WT when dry

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider AFM calibration

bull Absolute distance measurements require accurate piezo calibration

bull AFM systems have large scan ranges (150 microm to lt 1 microm)

bull Scanner non-linearities can introduce substantial error over that range

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

ights Reserve

d

Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

BBML - All R

ights Reserve

d

Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

100 nm 10 80 602 1198 100 nm 7 60 827 1439 100 nm 5 40 814 1427 100 nm 35 20 917 1724 100 nm 2 10 937 1774

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

ights Reserve

d

AFM calibration ndash Take Away Message

Calibration using a scan size and feature size comparable to features of interest is highly recommended

Calibration addresses absolute accuracy not precision (differential sensitivity between measurements)

bull Does not limit ability to differentiate between groups using the same AFM with the same calibration parameters

BBML - All R

ights Reserve

d

AFM Applications in Bone Research Indentation Mechanics

BBML - All R

ights Reserve

d

AFM-Based Indentation

In addition to high resolution imaging AFM can be used to extract nanoscale mechanical data

ApproachWithdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Can

tilev

er D

efle

ctio

n (V

olts

)

1 Ramp into a rigid sample ndash measure the slope

Deflection sensitivity x 119899119899119899119899119881119881

2 Calculate cantilever spring constant

3 Hookersquos law takes deflection (volts) and converts to units of force F = kx

Spring constant k 119873119873119899119899

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Forc

e (n

N)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration Fo

rce

(nN

)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

Tip-Sample Separation (nm)

BBML - All R

ights Reserve

d

AFM-Based Indentation Parameters

Approach Withdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

Peak Force

Area Between Curves Energy Dissipation

Contact Mechanics Fit for Elastic Modulus (E)

Adhesion Force

Indentation Depth (δ)

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

z

r

δ

R

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119877119877 ∙ 12057512057532

Es Sample modulus νs Poissonrsquos ratio R tip radius of curvature δ load point displacement

Relevant Hertz Assumptions bull Strains are small and within the elastic limit bull Indenter is infinitely stiff (only sample deforms) bull Indentation depth ltlt radius of curvature of indenter bull Surfaces are continuous non-conforming (must be flat) bull Load is normal to surface

BBML - All R

ights Reserve

d

Proper Indentation Model is Essential

δ

r

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119903119903 ∙ 12057512057532

δ

α

119865119865 =2120587120587∙

1198641198641199041199041 minus 1205841205841199041199042

∙ tan120572120572 ∙ 1205751205752

Es Sample modulus νs Poissonrsquos ratio R tip radius α opening angle

Hertz Model Spherical Indenter

depth ltlt radius of curvature

Sneddon Model Conical Indenter

depth ge radius of curvature

BBML - All R

ights Reserve

d

Indentation in OI Tendon Study

x

z

yBrtl+ and WT mice 6 month old males

4 tails per group

2-3 fascicles per tail

25-30 fibrils per tail

4-5 locations per fibril

Indent force 20 nN wet (Sneddon) 50 nN dry (Hertz) BBML - A

ll Rights R

eserved

OI Tendon Differential Effects of Dehydration

Dry 1268 plusmn 718 MPa

Wet 305 plusmn 243 MPa

Dry 1737 plusmn 973 MPa

Wet 301 plusmn 202 MPa

Kemp et al J StructBio 2012 180 (3) 428-438

bull Significant stiffening of fibrils with dehydration

bull More pronounced modulus modulus as a function of disease

BBML - All R

ights Reserve

d

OI Tendon Differential Effects of Dehydration

Kemp et al J StructBio 2012 180 (3) 428-438

Diseased phenotype not present when wet phenotype observed when dried

0

10

20

30

40

50

60

0 20 40 60 80 100 120 140 160 180 200

Gro

up S

ampl

es (

)

Indent Modulus (MPa)

WT WetBrtl Wet

0

2

4

6

8

10

12

14

16

18

20

025

050

075

010

0012

5015

0017

5020

0022

5025

0027

5030

0032

5035

0037

5040

0042

5045

0047

5050

00

Gro

up S

ampl

es (

)Indent Modulus (MPa)

WT DryBrtl Dry

555 WT Indents 305 plusmn 243 MPa

614 Brtl+ Indents 301 plusmn 202 MPa

KS Test p=0129 t Test p=0464 464 WT Indents

1268 plusmn 718 MPa 479 Brtl+ Indents 1737 plusmn 973 MPa

KS Test plt0001 T Test plt0001

BBML - All R

ights Reserve

d

Indentation in Diabetic Tendon (Poster 0495)

x

z

y

Control and Diabetic Rat Tail Tendon

4-5 rats per group

2-3 fascicles per rat

~70 fibrilstail

4-5 locationsfibril

20 nN (Sneddon)

BBML - All R

ights Reserve

d

Multiscale Diabetes Study (Poster 0495)

0

5

10

15

20

25

300 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 gt25

Perc

enta

ge o

f Gro

up S

ampl

es

Elastic Modulus (MPa)

CD (1530 indents)ZDSD (913 indents)

CD (n=5) 381 plusmn 149 MPa

ZDSD (n=3) 706 plusmn 263 MPa

0

100

200

300

400

500

600

700

800

900

0 2 4 6 8 10

Mic

rosc

ale

Mod

ulus

(MPa

)

Nanoscale Modulus (MPa)

plt0001

r2=0531 p=0040

bull Increased indentation modulus in diabetic tendon

bull Strong relationship between nanoscale indent modulus and microscale tensile modulus

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

Proper probe selection for intended application bull Cantilever must be stiff relative to sample bull Sharp probes may damage sample easily dull

R or α must be determined for each probe bull Properties provided by manufacturer are unreliable bull Vary from probe to probe can dull during indent process

Spring constant must be determined for each probe

bull Value provided by manufacturer is inaccurate bull Thermal tuning OK for ldquosoftrdquo probes poor performance

for stiffer probes bull Sader method (geometry) added mass reference

cantilevers

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

Veeco Application Technical note Ben Ohler Practical Advice on the Determination of Cantilever Spring Constants

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

bull Sample processing

bull Sample hydration

bull Proper force range

bull Substrate effects for thin samples

bull Proper modulus model and range of data goodness of fit

Bearing in mind these assumptions and limitations AFM can provide true ldquonanoscalerdquo mechanical properties both in terms of forces and deformations

BBML - All R

ights Reserve

d

A Note on Peak Force QNM for Bruker AFMs bull Mechanical properties mapped pixel-by-pixel at same

resolution as the height image (PeakForce QNM)

Height Modulus BBML - All R

ights Reserve

d

A Note on Peak Force QNM

Correct probe stiffness must be used based on sample Pittenger ndash Veeco Product Bulletin

BBML - All R

ights Reserve

d

A Note on Peak Force QNM Extremely sensitive to calibration (details rarely given) Absolute radius and spring constant known

bull especially important from probe to probe bull radius uarr with indent depth ndash must determine R at depth

you expect to image bull Depth may vary as a function of sample (or at different

regions within the sample)

Relative reference material of ldquoknownrdquo modulus used to determine a ratio of 119896119896 119877119877 bull ldquoknownrdquo modulus may come from tension compression

and not indentation bull R is related to depth indentation depth on sample must

be same as on reference BBML - All R

ights Reserve

d

A Note on Peak Force QNM bull Modulus fit on the fly ndash no ability to monitor fit (or range) bull Local sample topography and sample tilt play a role in

measured parameters bull Assuming all modulus values in an image are correct how to

quantify

512 x 512 pixels = 262144 measurements

n=1 n=262144

n from selected ROIs

BBML - All R

ights Reserve

d

BBML - All R

ights Reserve

d

bull Imaging in air on mica ndash substrate effect

bull Same imaging force or indent depth for all

bull For each area were all modulus values pooled

Lamprou et al PLoS ONE 2013 BBML - All R

ights Reserve

d

AFM Applications in Bone Research Other Cool Mechanical Techniques

BBML - All R

ights Reserve

d

ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo Bonds within or between collagen fibrils in bone that rupture to dissipate energy as a toughening mechanism

Thompson et al Nature 2001

Fantner et al Nature Mat 2005

Hansma et al JMNI 2005

Fantner et al Nano Letters 2007 BBML - All R

ights Reserve

d

Other Mechanical Techniques

Jimenez-Palomar J Mech Beh Bio Mat 2012

van der Rijt Macro Bio 2004

Yang J Biomed Mater Res 2007

Yang Biophysical Journal 2008 BBML - All R

ights Reserve

d

Other Cool Techniques bull Piezoresponse Force Microscopy (PFM)

bull Electrochemical AFM (EC-AFM)

bull Force Modulation Microscopy (FMM)

bull Lateral Force Microscopy (LFM)

bull Magnetic Force Microscopy (MFM)

bull Conductive AFM (C-AFM)

bull Force Pulling

bull Cantilever Bending

bull Tensile testing of individual fibrils

bull 3 point bending of fibrils

BBML - All R

ights Reserve

d

Summary Things to rememberhellip

bull Imaging mode imaging medium and probe selection

bull Morphology Measurements bull Sample preparation and hydration state bull Measuring fibril diameter bull Site selection and proper sampling bull Appropriate system calibration

bull Mechanical Measurements

bull Probe selection (stiffness and tip radius) bull Probe calibration (radius angle spring constant) bull Indentation force range and mechanical model bull Hydration in biological samples bull PeakForce QNM

BBML - All R

ights Reserve

d

Acknowledgements BBML

Max Hammond Armando Diaz Gonzalez Zachary Bart Silvia Canelon Creasy Clauser

IU School of Medicine Dr David Burr Dr Matt Allen Dr Stuart Warden Dr Lilian Plotkin

Funding Sources NIHNIDCR 1F32DE018840-01 A1 IUPUI BME Departmental Startup Funds IUPUI Research Support Funds Grant IUPUI Biomechanics and Biomaterials Research Center

NIH NICHD Dr Joan Marini

BBML - All R

ights Reserve

d

Bone Biology and Mechanics Lab (BBML) wwwiupuiedu~bbml Facebook BoneBiologyMechanicsLab

BBML - All R

ights Reserve

d

• Slide Number 1
• General Seminar Outline
• Bonersquos Hierarchical Structure
• Critical Gap in Understanding Collagen
• Methods For Analyzing Collagen Structure
• Methods For Imaging Collagen Structure
• Cryo-TEM
• Atomic Force Microscopy (AFM)
• AFM Image of Pentacene (C22H14)
• AFM Image of Hexabenzocoronene
• Basics of AFM Imaging The Probe
• Basics of AFM Imaging System Components
• van der Waals Potential Energy Curve
• Contact Mode ndash Monitor Deflection
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Peak Force Tapping Mode ndash Monitor Force
• AFM Applications in Bone Research Imaging
• AFM Imaging of Collagen
• Imaging of Osteoclast Activity
• Mineralized collagen deposition in vitro
• Imaging of Canilicular Structure
• AFM Imaging in Bone
• AFM Imaging in Bone
• Removing Mineral in Bone to Expose Collagen
• Removing Mineral in Bone to Expose Collagen
• Demineralization with EDTA
• Demineralization with EDTA
• Demineralization with EDTA
• Issues to Consider Effects of Acid
• Slide Number 32
• Slide Number 33
• Analysis of Collagen Structure in Bone
• Analysis of Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril D-Spacing
• Slide Number 39
• Collagen in Bone Fibril D-Spacing
• Collagen in Bone Fibril D-Spacing
• 2D Fast Fourier Transform Analysis
• 2D Fast Fourier Transform Analysis
• D-Spacing Distribution
• D-Spacing Distribution
• Cumulative Distribution Function
• CDF vs Histogram
• Changes in D-Spacing with Disease
• Changes in D-Spacing with Disease
• Slide Number 50
• Issues to Consider Site Selection and Sampling
• Issues to Consider Site Selection and Sampling
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• AFM calibration ndash Take Away Message
• AFM Applications in Bone ResearchIndentation Mechanics
• AFM-Based Indentation
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Parameters
• Hertz Contact Equation
• Hertz Contact Equation
• Hertz Contact Equation
• Proper Indentation Model is Essential
• Indentation in OI Tendon Study
• OI Tendon Differential Effects of Dehydration
• OI Tendon Differential Effects of Dehydration
• Indentation in Diabetic Tendon (Poster 0495)
• Multiscale Diabetes Study (Poster 0495)
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• A Note on Peak Force QNM for Bruker AFMs
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• Slide Number 83
• Slide Number 84
• AFM Applications in Bone ResearchOther Cool Mechanical Techniques
• ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo
• Other Mechanical Techniques
• Other Cool Techniques
• Summary Things to rememberhellip
• Acknowledgements
• Bone Biology and Mechanics Lab (BBML)

Question Model and Tissue Morphology Publication

Tissue Specificity in Mice Male mouse 8 weeks Femur dentin tail tendon

No differences in bone vs dentin both ∆ vs tendon

Wallace et al Langmuir 2011 26(10) 7349-7354

Gender Effects Male and female mice 16 weeks Femur No differences NA

Estrogen Depletion OVX in sheep 5 years + 2 Radius Mean and population shift Wallace et al Bone

2010 46 1349-1354

Estrogen Depletion OVX in female 5 years + 2 Skin (dermis) Significant population shift Erickson et al Biotech

J 2013 8(1) 1174-126

Osteoporosis and Mechanical Loading

OVX in ulnar loading in rats Ulna

No loading effect population shift with OVX

Warden et al Endocrinology 2013 154(9) 3178-3187

Osteogenesis Imperfecta Brtl+ model male mice 2 months Femur

No mean ∆ widening of distribution

Wallace et al J Stuct Bio 2011 173 146-152

Osteogenesis Imperfecta Oimoim model male mice 12 weeks Femur Mean and population shift Bart et al Conn Tiss

Res 2014 In Press

Osteogenesis Imperfecta and Tissue Hydration

Brtl+ model male mice 6 months Tail Tendon Significant population shift Kemp et al J Stuct Bio

2012 180 428-438

Type 2 Diabetes Male ZDSD rats 30 weeks Tibia Significant population shift Hammond et al Bone

2014 60 26-32

Type 2 Diabetes Female ZDSD rats 32 weeks Tail Tendon Significant population shift

Diaz Gonzalez et al J Biomech 2014 47(3)

681-686

Raloxifene (in vivo and in vitro) Female dog treated with Raloxifene Femur Significant population shift Gallant et al Bone

2014 61 191-200 BBML - All R

ights Reserve

d

Issues to Consider Site Selection and Sampling

KS Test plt0001

0023 of bonersquos length

15 m

m =

150

00 micro

m

Wallace et al Langmuir 2011 26(10) 7349-7354

100 microm x 100 microm

35 microm x 35 microm

BBML - All R

ights Reserve

d

Issues to Consider Site Selection and Sampling

60

62

64

66

68

70

72

74

1 (35)

2 (33)

3 (40)

4 (39)

5 (35)

6 (29)

7 (26)

8 (23)

9 (28)

WT (288)

D-P

erio

dic

Gap

Ove

rlap

Spac

ing

(nm

)

Axial Location (Proximal End = 1 Distal End = 9)

( of Fibrils)

91

23

45

87

6

Wallace et al Langmuir 2011 26(10) 7349-7354

288 fibrils for population n=1 for mean comparisons BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

Wet Tendon Dry Tendon Kemp et al J StructBio 2012 180 (3) 428-438

Collagen and bone exist in a hydrated environment in vivo bull Hydration state can impact morphology and mechanics

BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

Nonsignificant mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet (n=255) 682 plusmn 12 nm

WT Dry (n=257) 686 plusmn 08 nm

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

Brtl DryBrtl Wet

KS Test plt0001

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

WT Case 04 nm mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet 682 plusmn 12 nm

WT Dry 686 plusmn 08 nm

Brtl+ Wet 675 plusmn 14 nm

Brtl+ Dry 689 plusmn 10 nm

Brtl+ Case 14 nm increase with drying (plt0001) bull Entire population shifted upward (plt0001)

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT WetBrtl Wet

KS Test plt0001

Brtl+ distribution shifted downward vs WT when wet

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryBrtl Dry

KS Test plt0001

Brtl+ distribution shifted upward vs WT when dry

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider AFM calibration

bull Absolute distance measurements require accurate piezo calibration

bull AFM systems have large scan ranges (150 microm to lt 1 microm)

bull Scanner non-linearities can introduce substantial error over that range

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

ights Reserve

d

Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

BBML - All R

ights Reserve

d

Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

100 nm 10 80 602 1198 100 nm 7 60 827 1439 100 nm 5 40 814 1427 100 nm 35 20 917 1724 100 nm 2 10 937 1774

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

ights Reserve

d

AFM calibration ndash Take Away Message

Calibration using a scan size and feature size comparable to features of interest is highly recommended

Calibration addresses absolute accuracy not precision (differential sensitivity between measurements)

bull Does not limit ability to differentiate between groups using the same AFM with the same calibration parameters

BBML - All R

ights Reserve

d

AFM Applications in Bone Research Indentation Mechanics

BBML - All R

ights Reserve

d

AFM-Based Indentation

In addition to high resolution imaging AFM can be used to extract nanoscale mechanical data

ApproachWithdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Can

tilev

er D

efle

ctio

n (V

olts

)

1 Ramp into a rigid sample ndash measure the slope

Deflection sensitivity x 119899119899119899119899119881119881

2 Calculate cantilever spring constant

3 Hookersquos law takes deflection (volts) and converts to units of force F = kx

Spring constant k 119873119873119899119899

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Forc

e (n

N)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration Fo

rce

(nN

)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

Tip-Sample Separation (nm)

BBML - All R

ights Reserve

d

AFM-Based Indentation Parameters

Approach Withdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

Peak Force

Area Between Curves Energy Dissipation

Contact Mechanics Fit for Elastic Modulus (E)

Adhesion Force

Indentation Depth (δ)

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

z

r

δ

R

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119877119877 ∙ 12057512057532

Es Sample modulus νs Poissonrsquos ratio R tip radius of curvature δ load point displacement

Relevant Hertz Assumptions bull Strains are small and within the elastic limit bull Indenter is infinitely stiff (only sample deforms) bull Indentation depth ltlt radius of curvature of indenter bull Surfaces are continuous non-conforming (must be flat) bull Load is normal to surface

BBML - All R

ights Reserve

d

Proper Indentation Model is Essential

δ

r

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119903119903 ∙ 12057512057532

δ

α

119865119865 =2120587120587∙

1198641198641199041199041 minus 1205841205841199041199042

∙ tan120572120572 ∙ 1205751205752

Es Sample modulus νs Poissonrsquos ratio R tip radius α opening angle

Hertz Model Spherical Indenter

depth ltlt radius of curvature

Sneddon Model Conical Indenter

depth ge radius of curvature

BBML - All R

ights Reserve

d

Indentation in OI Tendon Study

x

z

yBrtl+ and WT mice 6 month old males

4 tails per group

2-3 fascicles per tail

25-30 fibrils per tail

4-5 locations per fibril

Indent force 20 nN wet (Sneddon) 50 nN dry (Hertz) BBML - A

ll Rights R

eserved

OI Tendon Differential Effects of Dehydration

Dry 1268 plusmn 718 MPa

Wet 305 plusmn 243 MPa

Dry 1737 plusmn 973 MPa

Wet 301 plusmn 202 MPa

Kemp et al J StructBio 2012 180 (3) 428-438

bull Significant stiffening of fibrils with dehydration

bull More pronounced modulus modulus as a function of disease

BBML - All R

ights Reserve

d

OI Tendon Differential Effects of Dehydration

Kemp et al J StructBio 2012 180 (3) 428-438

Diseased phenotype not present when wet phenotype observed when dried

0

10

20

30

40

50

60

0 20 40 60 80 100 120 140 160 180 200

Gro

up S

ampl

es (

)

Indent Modulus (MPa)

WT WetBrtl Wet

0

2

4

6

8

10

12

14

16

18

20

025

050

075

010

0012

5015

0017

5020

0022

5025

0027

5030

0032

5035

0037

5040

0042

5045

0047

5050

00

Gro

up S

ampl

es (

)Indent Modulus (MPa)

WT DryBrtl Dry

555 WT Indents 305 plusmn 243 MPa

614 Brtl+ Indents 301 plusmn 202 MPa

KS Test p=0129 t Test p=0464 464 WT Indents

1268 plusmn 718 MPa 479 Brtl+ Indents 1737 plusmn 973 MPa

KS Test plt0001 T Test plt0001

BBML - All R

ights Reserve

d

Indentation in Diabetic Tendon (Poster 0495)

x

z

y

Control and Diabetic Rat Tail Tendon

4-5 rats per group

2-3 fascicles per rat

~70 fibrilstail

4-5 locationsfibril

20 nN (Sneddon)

BBML - All R

ights Reserve

d

Multiscale Diabetes Study (Poster 0495)

0

5

10

15

20

25

300 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 gt25

Perc

enta

ge o

f Gro

up S

ampl

es

Elastic Modulus (MPa)

CD (1530 indents)ZDSD (913 indents)

CD (n=5) 381 plusmn 149 MPa

ZDSD (n=3) 706 plusmn 263 MPa

0

100

200

300

400

500

600

700

800

900

0 2 4 6 8 10

Mic

rosc

ale

Mod

ulus

(MPa

)

Nanoscale Modulus (MPa)

plt0001

r2=0531 p=0040

bull Increased indentation modulus in diabetic tendon

bull Strong relationship between nanoscale indent modulus and microscale tensile modulus

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

Proper probe selection for intended application bull Cantilever must be stiff relative to sample bull Sharp probes may damage sample easily dull

R or α must be determined for each probe bull Properties provided by manufacturer are unreliable bull Vary from probe to probe can dull during indent process

Spring constant must be determined for each probe

bull Value provided by manufacturer is inaccurate bull Thermal tuning OK for ldquosoftrdquo probes poor performance

for stiffer probes bull Sader method (geometry) added mass reference

cantilevers

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

Veeco Application Technical note Ben Ohler Practical Advice on the Determination of Cantilever Spring Constants

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

bull Sample processing

bull Sample hydration

bull Proper force range

bull Substrate effects for thin samples

bull Proper modulus model and range of data goodness of fit

Bearing in mind these assumptions and limitations AFM can provide true ldquonanoscalerdquo mechanical properties both in terms of forces and deformations

BBML - All R

ights Reserve

d

A Note on Peak Force QNM for Bruker AFMs bull Mechanical properties mapped pixel-by-pixel at same

resolution as the height image (PeakForce QNM)

Height Modulus BBML - All R

ights Reserve

d

A Note on Peak Force QNM

Correct probe stiffness must be used based on sample Pittenger ndash Veeco Product Bulletin

BBML - All R

ights Reserve

d

A Note on Peak Force QNM Extremely sensitive to calibration (details rarely given) Absolute radius and spring constant known

bull especially important from probe to probe bull radius uarr with indent depth ndash must determine R at depth

you expect to image bull Depth may vary as a function of sample (or at different

regions within the sample)

Relative reference material of ldquoknownrdquo modulus used to determine a ratio of 119896119896 119877119877 bull ldquoknownrdquo modulus may come from tension compression

and not indentation bull R is related to depth indentation depth on sample must

be same as on reference BBML - All R

ights Reserve

d

A Note on Peak Force QNM bull Modulus fit on the fly ndash no ability to monitor fit (or range) bull Local sample topography and sample tilt play a role in

measured parameters bull Assuming all modulus values in an image are correct how to

quantify

512 x 512 pixels = 262144 measurements

n=1 n=262144

n from selected ROIs

BBML - All R

ights Reserve

d

BBML - All R

ights Reserve

d

bull Imaging in air on mica ndash substrate effect

bull Same imaging force or indent depth for all

bull For each area were all modulus values pooled

Lamprou et al PLoS ONE 2013 BBML - All R

ights Reserve

d

AFM Applications in Bone Research Other Cool Mechanical Techniques

BBML - All R

ights Reserve

d

ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo Bonds within or between collagen fibrils in bone that rupture to dissipate energy as a toughening mechanism

Thompson et al Nature 2001

Fantner et al Nature Mat 2005

Hansma et al JMNI 2005

Fantner et al Nano Letters 2007 BBML - All R

ights Reserve

d

Other Mechanical Techniques

Jimenez-Palomar J Mech Beh Bio Mat 2012

van der Rijt Macro Bio 2004

Yang J Biomed Mater Res 2007

Yang Biophysical Journal 2008 BBML - All R

ights Reserve

d

Other Cool Techniques bull Piezoresponse Force Microscopy (PFM)

bull Electrochemical AFM (EC-AFM)

bull Force Modulation Microscopy (FMM)

bull Lateral Force Microscopy (LFM)

bull Magnetic Force Microscopy (MFM)

bull Conductive AFM (C-AFM)

bull Force Pulling

bull Cantilever Bending

bull Tensile testing of individual fibrils

bull 3 point bending of fibrils

BBML - All R

ights Reserve

d

Summary Things to rememberhellip

bull Imaging mode imaging medium and probe selection

bull Morphology Measurements bull Sample preparation and hydration state bull Measuring fibril diameter bull Site selection and proper sampling bull Appropriate system calibration

bull Mechanical Measurements

bull Probe selection (stiffness and tip radius) bull Probe calibration (radius angle spring constant) bull Indentation force range and mechanical model bull Hydration in biological samples bull PeakForce QNM

BBML - All R

ights Reserve

d

Acknowledgements BBML

Max Hammond Armando Diaz Gonzalez Zachary Bart Silvia Canelon Creasy Clauser

IU School of Medicine Dr David Burr Dr Matt Allen Dr Stuart Warden Dr Lilian Plotkin

Funding Sources NIHNIDCR 1F32DE018840-01 A1 IUPUI BME Departmental Startup Funds IUPUI Research Support Funds Grant IUPUI Biomechanics and Biomaterials Research Center

NIH NICHD Dr Joan Marini

BBML - All R

ights Reserve

d

Bone Biology and Mechanics Lab (BBML) wwwiupuiedu~bbml Facebook BoneBiologyMechanicsLab

BBML - All R

ights Reserve

d

• Slide Number 1
• General Seminar Outline
• Bonersquos Hierarchical Structure
• Critical Gap in Understanding Collagen
• Methods For Analyzing Collagen Structure
• Methods For Imaging Collagen Structure
• Cryo-TEM
• Atomic Force Microscopy (AFM)
• AFM Image of Pentacene (C22H14)
• AFM Image of Hexabenzocoronene
• Basics of AFM Imaging The Probe
• Basics of AFM Imaging System Components
• van der Waals Potential Energy Curve
• Contact Mode ndash Monitor Deflection
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Peak Force Tapping Mode ndash Monitor Force
• AFM Applications in Bone Research Imaging
• AFM Imaging of Collagen
• Imaging of Osteoclast Activity
• Mineralized collagen deposition in vitro
• Imaging of Canilicular Structure
• AFM Imaging in Bone
• AFM Imaging in Bone
• Removing Mineral in Bone to Expose Collagen
• Removing Mineral in Bone to Expose Collagen
• Demineralization with EDTA
• Demineralization with EDTA
• Demineralization with EDTA
• Issues to Consider Effects of Acid
• Slide Number 32
• Slide Number 33
• Analysis of Collagen Structure in Bone
• Analysis of Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril D-Spacing
• Slide Number 39
• Collagen in Bone Fibril D-Spacing
• Collagen in Bone Fibril D-Spacing
• 2D Fast Fourier Transform Analysis
• 2D Fast Fourier Transform Analysis
• D-Spacing Distribution
• D-Spacing Distribution
• Cumulative Distribution Function
• CDF vs Histogram
• Changes in D-Spacing with Disease
• Changes in D-Spacing with Disease
• Slide Number 50
• Issues to Consider Site Selection and Sampling
• Issues to Consider Site Selection and Sampling
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• AFM calibration ndash Take Away Message
• AFM Applications in Bone ResearchIndentation Mechanics
• AFM-Based Indentation
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Parameters
• Hertz Contact Equation
• Hertz Contact Equation
• Hertz Contact Equation
• Proper Indentation Model is Essential
• Indentation in OI Tendon Study
• OI Tendon Differential Effects of Dehydration
• OI Tendon Differential Effects of Dehydration
• Indentation in Diabetic Tendon (Poster 0495)
• Multiscale Diabetes Study (Poster 0495)
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• A Note on Peak Force QNM for Bruker AFMs
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• Slide Number 83
• Slide Number 84
• AFM Applications in Bone ResearchOther Cool Mechanical Techniques
• ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo
• Other Mechanical Techniques
• Other Cool Techniques
• Summary Things to rememberhellip
• Acknowledgements
• Bone Biology and Mechanics Lab (BBML)

Issues to Consider Site Selection and Sampling

KS Test plt0001

0023 of bonersquos length

15 m

m =

150

00 micro

m

Wallace et al Langmuir 2011 26(10) 7349-7354

100 microm x 100 microm

35 microm x 35 microm

BBML - All R

ights Reserve

d

Issues to Consider Site Selection and Sampling

60

62

64

66

68

70

72

74

1 (35)

2 (33)

3 (40)

4 (39)

5 (35)

6 (29)

7 (26)

8 (23)

9 (28)

WT (288)

D-P

erio

dic

Gap

Ove

rlap

Spac

ing

(nm

)

Axial Location (Proximal End = 1 Distal End = 9)

( of Fibrils)

91

23

45

87

6

Wallace et al Langmuir 2011 26(10) 7349-7354

288 fibrils for population n=1 for mean comparisons BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

Wet Tendon Dry Tendon Kemp et al J StructBio 2012 180 (3) 428-438

Collagen and bone exist in a hydrated environment in vivo bull Hydration state can impact morphology and mechanics

BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

Nonsignificant mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet (n=255) 682 plusmn 12 nm

WT Dry (n=257) 686 plusmn 08 nm

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

Brtl DryBrtl Wet

KS Test plt0001

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

WT Case 04 nm mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet 682 plusmn 12 nm

WT Dry 686 plusmn 08 nm

Brtl+ Wet 675 plusmn 14 nm

Brtl+ Dry 689 plusmn 10 nm

Brtl+ Case 14 nm increase with drying (plt0001) bull Entire population shifted upward (plt0001)

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT WetBrtl Wet

KS Test plt0001

Brtl+ distribution shifted downward vs WT when wet

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryBrtl Dry

KS Test plt0001

Brtl+ distribution shifted upward vs WT when dry

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider AFM calibration

bull Absolute distance measurements require accurate piezo calibration

bull AFM systems have large scan ranges (150 microm to lt 1 microm)

bull Scanner non-linearities can introduce substantial error over that range

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

ights Reserve

d

Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

BBML - All R

ights Reserve

d

Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

100 nm 10 80 602 1198 100 nm 7 60 827 1439 100 nm 5 40 814 1427 100 nm 35 20 917 1724 100 nm 2 10 937 1774

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

ights Reserve

d

AFM calibration ndash Take Away Message

Calibration using a scan size and feature size comparable to features of interest is highly recommended

Calibration addresses absolute accuracy not precision (differential sensitivity between measurements)

bull Does not limit ability to differentiate between groups using the same AFM with the same calibration parameters

BBML - All R

ights Reserve

d

AFM Applications in Bone Research Indentation Mechanics

BBML - All R

ights Reserve

d

AFM-Based Indentation

In addition to high resolution imaging AFM can be used to extract nanoscale mechanical data

ApproachWithdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Can

tilev

er D

efle

ctio

n (V

olts

)

1 Ramp into a rigid sample ndash measure the slope

Deflection sensitivity x 119899119899119899119899119881119881

2 Calculate cantilever spring constant

3 Hookersquos law takes deflection (volts) and converts to units of force F = kx

Spring constant k 119873119873119899119899

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Forc

e (n

N)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration Fo

rce

(nN

)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

Tip-Sample Separation (nm)

BBML - All R

ights Reserve

d

AFM-Based Indentation Parameters

Approach Withdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

Peak Force

Area Between Curves Energy Dissipation

Contact Mechanics Fit for Elastic Modulus (E)

Adhesion Force

Indentation Depth (δ)

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

z

r

δ

R

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119877119877 ∙ 12057512057532

Es Sample modulus νs Poissonrsquos ratio R tip radius of curvature δ load point displacement

Relevant Hertz Assumptions bull Strains are small and within the elastic limit bull Indenter is infinitely stiff (only sample deforms) bull Indentation depth ltlt radius of curvature of indenter bull Surfaces are continuous non-conforming (must be flat) bull Load is normal to surface

BBML - All R

ights Reserve

d

Proper Indentation Model is Essential

δ

r

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119903119903 ∙ 12057512057532

δ

α

119865119865 =2120587120587∙

1198641198641199041199041 minus 1205841205841199041199042

∙ tan120572120572 ∙ 1205751205752

Es Sample modulus νs Poissonrsquos ratio R tip radius α opening angle

Hertz Model Spherical Indenter

depth ltlt radius of curvature

Sneddon Model Conical Indenter

depth ge radius of curvature

BBML - All R

ights Reserve

d

Indentation in OI Tendon Study

x

z

yBrtl+ and WT mice 6 month old males

4 tails per group

2-3 fascicles per tail

25-30 fibrils per tail

4-5 locations per fibril

Indent force 20 nN wet (Sneddon) 50 nN dry (Hertz) BBML - A

ll Rights R

eserved

OI Tendon Differential Effects of Dehydration

Dry 1268 plusmn 718 MPa

Wet 305 plusmn 243 MPa

Dry 1737 plusmn 973 MPa

Wet 301 plusmn 202 MPa

Kemp et al J StructBio 2012 180 (3) 428-438

bull Significant stiffening of fibrils with dehydration

bull More pronounced modulus modulus as a function of disease

BBML - All R

ights Reserve

d

OI Tendon Differential Effects of Dehydration

Kemp et al J StructBio 2012 180 (3) 428-438

Diseased phenotype not present when wet phenotype observed when dried

0

10

20

30

40

50

60

0 20 40 60 80 100 120 140 160 180 200

Gro

up S

ampl

es (

)

Indent Modulus (MPa)

WT WetBrtl Wet

0

2

4

6

8

10

12

14

16

18

20

025

050

075

010

0012

5015

0017

5020

0022

5025

0027

5030

0032

5035

0037

5040

0042

5045

0047

5050

00

Gro

up S

ampl

es (

)Indent Modulus (MPa)

WT DryBrtl Dry

555 WT Indents 305 plusmn 243 MPa

614 Brtl+ Indents 301 plusmn 202 MPa

KS Test p=0129 t Test p=0464 464 WT Indents

1268 plusmn 718 MPa 479 Brtl+ Indents 1737 plusmn 973 MPa

KS Test plt0001 T Test plt0001

BBML - All R

ights Reserve

d

Indentation in Diabetic Tendon (Poster 0495)

x

z

y

Control and Diabetic Rat Tail Tendon

4-5 rats per group

2-3 fascicles per rat

~70 fibrilstail

4-5 locationsfibril

20 nN (Sneddon)

BBML - All R

ights Reserve

d

Multiscale Diabetes Study (Poster 0495)

0

5

10

15

20

25

300 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 gt25

Perc

enta

ge o

f Gro

up S

ampl

es

Elastic Modulus (MPa)

CD (1530 indents)ZDSD (913 indents)

CD (n=5) 381 plusmn 149 MPa

ZDSD (n=3) 706 plusmn 263 MPa

0

100

200

300

400

500

600

700

800

900

0 2 4 6 8 10

Mic

rosc

ale

Mod

ulus

(MPa

)

Nanoscale Modulus (MPa)

plt0001

r2=0531 p=0040

bull Increased indentation modulus in diabetic tendon

bull Strong relationship between nanoscale indent modulus and microscale tensile modulus

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

Proper probe selection for intended application bull Cantilever must be stiff relative to sample bull Sharp probes may damage sample easily dull

R or α must be determined for each probe bull Properties provided by manufacturer are unreliable bull Vary from probe to probe can dull during indent process

Spring constant must be determined for each probe

bull Value provided by manufacturer is inaccurate bull Thermal tuning OK for ldquosoftrdquo probes poor performance

for stiffer probes bull Sader method (geometry) added mass reference

cantilevers

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

Veeco Application Technical note Ben Ohler Practical Advice on the Determination of Cantilever Spring Constants

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

bull Sample processing

bull Sample hydration

bull Proper force range

bull Substrate effects for thin samples

bull Proper modulus model and range of data goodness of fit

Bearing in mind these assumptions and limitations AFM can provide true ldquonanoscalerdquo mechanical properties both in terms of forces and deformations

BBML - All R

ights Reserve

d

A Note on Peak Force QNM for Bruker AFMs bull Mechanical properties mapped pixel-by-pixel at same

resolution as the height image (PeakForce QNM)

Height Modulus BBML - All R

ights Reserve

d

A Note on Peak Force QNM

Correct probe stiffness must be used based on sample Pittenger ndash Veeco Product Bulletin

BBML - All R

ights Reserve

d

A Note on Peak Force QNM Extremely sensitive to calibration (details rarely given) Absolute radius and spring constant known

bull especially important from probe to probe bull radius uarr with indent depth ndash must determine R at depth

you expect to image bull Depth may vary as a function of sample (or at different

regions within the sample)

Relative reference material of ldquoknownrdquo modulus used to determine a ratio of 119896119896 119877119877 bull ldquoknownrdquo modulus may come from tension compression

and not indentation bull R is related to depth indentation depth on sample must

be same as on reference BBML - All R

ights Reserve

d

A Note on Peak Force QNM bull Modulus fit on the fly ndash no ability to monitor fit (or range) bull Local sample topography and sample tilt play a role in

measured parameters bull Assuming all modulus values in an image are correct how to

quantify

512 x 512 pixels = 262144 measurements

n=1 n=262144

n from selected ROIs

BBML - All R

ights Reserve

d

BBML - All R

ights Reserve

d

bull Imaging in air on mica ndash substrate effect

bull Same imaging force or indent depth for all

bull For each area were all modulus values pooled

Lamprou et al PLoS ONE 2013 BBML - All R

ights Reserve

d

AFM Applications in Bone Research Other Cool Mechanical Techniques

BBML - All R

ights Reserve

d

ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo Bonds within or between collagen fibrils in bone that rupture to dissipate energy as a toughening mechanism

Thompson et al Nature 2001

Fantner et al Nature Mat 2005

Hansma et al JMNI 2005

Fantner et al Nano Letters 2007 BBML - All R

ights Reserve

d

Other Mechanical Techniques

Jimenez-Palomar J Mech Beh Bio Mat 2012

van der Rijt Macro Bio 2004

Yang J Biomed Mater Res 2007

Yang Biophysical Journal 2008 BBML - All R

ights Reserve

d

Other Cool Techniques bull Piezoresponse Force Microscopy (PFM)

bull Electrochemical AFM (EC-AFM)

bull Force Modulation Microscopy (FMM)

bull Lateral Force Microscopy (LFM)

bull Magnetic Force Microscopy (MFM)

bull Conductive AFM (C-AFM)

bull Force Pulling

bull Cantilever Bending

bull Tensile testing of individual fibrils

bull 3 point bending of fibrils

BBML - All R

ights Reserve

d

Summary Things to rememberhellip

bull Imaging mode imaging medium and probe selection

bull Morphology Measurements bull Sample preparation and hydration state bull Measuring fibril diameter bull Site selection and proper sampling bull Appropriate system calibration

bull Mechanical Measurements

bull Probe selection (stiffness and tip radius) bull Probe calibration (radius angle spring constant) bull Indentation force range and mechanical model bull Hydration in biological samples bull PeakForce QNM

BBML - All R

ights Reserve

d

Acknowledgements BBML

Max Hammond Armando Diaz Gonzalez Zachary Bart Silvia Canelon Creasy Clauser

IU School of Medicine Dr David Burr Dr Matt Allen Dr Stuart Warden Dr Lilian Plotkin

Funding Sources NIHNIDCR 1F32DE018840-01 A1 IUPUI BME Departmental Startup Funds IUPUI Research Support Funds Grant IUPUI Biomechanics and Biomaterials Research Center

NIH NICHD Dr Joan Marini

BBML - All R

ights Reserve

d

Bone Biology and Mechanics Lab (BBML) wwwiupuiedu~bbml Facebook BoneBiologyMechanicsLab

BBML - All R

ights Reserve

d

• Slide Number 1
• General Seminar Outline
• Bonersquos Hierarchical Structure
• Critical Gap in Understanding Collagen
• Methods For Analyzing Collagen Structure
• Methods For Imaging Collagen Structure
• Cryo-TEM
• Atomic Force Microscopy (AFM)
• AFM Image of Pentacene (C22H14)
• AFM Image of Hexabenzocoronene
• Basics of AFM Imaging The Probe
• Basics of AFM Imaging System Components
• van der Waals Potential Energy Curve
• Contact Mode ndash Monitor Deflection
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Peak Force Tapping Mode ndash Monitor Force
• AFM Applications in Bone Research Imaging
• AFM Imaging of Collagen
• Imaging of Osteoclast Activity
• Mineralized collagen deposition in vitro
• Imaging of Canilicular Structure
• AFM Imaging in Bone
• AFM Imaging in Bone
• Removing Mineral in Bone to Expose Collagen
• Removing Mineral in Bone to Expose Collagen
• Demineralization with EDTA
• Demineralization with EDTA
• Demineralization with EDTA
• Issues to Consider Effects of Acid
• Slide Number 32
• Slide Number 33
• Analysis of Collagen Structure in Bone
• Analysis of Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril D-Spacing
• Slide Number 39
• Collagen in Bone Fibril D-Spacing
• Collagen in Bone Fibril D-Spacing
• 2D Fast Fourier Transform Analysis
• 2D Fast Fourier Transform Analysis
• D-Spacing Distribution
• D-Spacing Distribution
• Cumulative Distribution Function
• CDF vs Histogram
• Changes in D-Spacing with Disease
• Changes in D-Spacing with Disease
• Slide Number 50
• Issues to Consider Site Selection and Sampling
• Issues to Consider Site Selection and Sampling
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• AFM calibration ndash Take Away Message
• AFM Applications in Bone ResearchIndentation Mechanics
• AFM-Based Indentation
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Parameters
• Hertz Contact Equation
• Hertz Contact Equation
• Hertz Contact Equation
• Proper Indentation Model is Essential
• Indentation in OI Tendon Study
• OI Tendon Differential Effects of Dehydration
• OI Tendon Differential Effects of Dehydration
• Indentation in Diabetic Tendon (Poster 0495)
• Multiscale Diabetes Study (Poster 0495)
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• A Note on Peak Force QNM for Bruker AFMs
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• Slide Number 83
• Slide Number 84
• AFM Applications in Bone ResearchOther Cool Mechanical Techniques
• ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo
• Other Mechanical Techniques
• Other Cool Techniques
• Summary Things to rememberhellip
• Acknowledgements
• Bone Biology and Mechanics Lab (BBML)

Issues to Consider Site Selection and Sampling

60

62

64

66

68

70

72

74

1 (35)

2 (33)

3 (40)

4 (39)

5 (35)

6 (29)

7 (26)

8 (23)

9 (28)

WT (288)

D-P

erio

dic

Gap

Ove

rlap

Spac

ing

(nm

)

Axial Location (Proximal End = 1 Distal End = 9)

( of Fibrils)

91

23

45

87

6

Wallace et al Langmuir 2011 26(10) 7349-7354

288 fibrils for population n=1 for mean comparisons BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

Wet Tendon Dry Tendon Kemp et al J StructBio 2012 180 (3) 428-438

Collagen and bone exist in a hydrated environment in vivo bull Hydration state can impact morphology and mechanics

BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

Nonsignificant mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet (n=255) 682 plusmn 12 nm

WT Dry (n=257) 686 plusmn 08 nm

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

Brtl DryBrtl Wet

KS Test plt0001

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

WT Case 04 nm mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet 682 plusmn 12 nm

WT Dry 686 plusmn 08 nm

Brtl+ Wet 675 plusmn 14 nm

Brtl+ Dry 689 plusmn 10 nm

Brtl+ Case 14 nm increase with drying (plt0001) bull Entire population shifted upward (plt0001)

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT WetBrtl Wet

KS Test plt0001

Brtl+ distribution shifted downward vs WT when wet

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryBrtl Dry

KS Test plt0001

Brtl+ distribution shifted upward vs WT when dry

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider AFM calibration

bull Absolute distance measurements require accurate piezo calibration

bull AFM systems have large scan ranges (150 microm to lt 1 microm)

bull Scanner non-linearities can introduce substantial error over that range

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

ights Reserve

d

Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

BBML - All R

ights Reserve

d

Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

100 nm 10 80 602 1198 100 nm 7 60 827 1439 100 nm 5 40 814 1427 100 nm 35 20 917 1724 100 nm 2 10 937 1774

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

ights Reserve

d

AFM calibration ndash Take Away Message

Calibration using a scan size and feature size comparable to features of interest is highly recommended

Calibration addresses absolute accuracy not precision (differential sensitivity between measurements)

bull Does not limit ability to differentiate between groups using the same AFM with the same calibration parameters

BBML - All R

ights Reserve

d

AFM Applications in Bone Research Indentation Mechanics

BBML - All R

ights Reserve

d

AFM-Based Indentation

In addition to high resolution imaging AFM can be used to extract nanoscale mechanical data

ApproachWithdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Can

tilev

er D

efle

ctio

n (V

olts

)

1 Ramp into a rigid sample ndash measure the slope

Deflection sensitivity x 119899119899119899119899119881119881

2 Calculate cantilever spring constant

3 Hookersquos law takes deflection (volts) and converts to units of force F = kx

Spring constant k 119873119873119899119899

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Forc

e (n

N)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration Fo

rce

(nN

)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

Tip-Sample Separation (nm)

BBML - All R

ights Reserve

d

AFM-Based Indentation Parameters

Approach Withdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

Peak Force

Area Between Curves Energy Dissipation

Contact Mechanics Fit for Elastic Modulus (E)

Adhesion Force

Indentation Depth (δ)

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

z

r

δ

R

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119877119877 ∙ 12057512057532

Es Sample modulus νs Poissonrsquos ratio R tip radius of curvature δ load point displacement

Relevant Hertz Assumptions bull Strains are small and within the elastic limit bull Indenter is infinitely stiff (only sample deforms) bull Indentation depth ltlt radius of curvature of indenter bull Surfaces are continuous non-conforming (must be flat) bull Load is normal to surface

BBML - All R

ights Reserve

d

Proper Indentation Model is Essential

δ

r

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119903119903 ∙ 12057512057532

δ

α

119865119865 =2120587120587∙

1198641198641199041199041 minus 1205841205841199041199042

∙ tan120572120572 ∙ 1205751205752

Es Sample modulus νs Poissonrsquos ratio R tip radius α opening angle

Hertz Model Spherical Indenter

depth ltlt radius of curvature

Sneddon Model Conical Indenter

depth ge radius of curvature

BBML - All R

ights Reserve

d

Indentation in OI Tendon Study

x

z

yBrtl+ and WT mice 6 month old males

4 tails per group

2-3 fascicles per tail

25-30 fibrils per tail

4-5 locations per fibril

Indent force 20 nN wet (Sneddon) 50 nN dry (Hertz) BBML - A

ll Rights R

eserved

OI Tendon Differential Effects of Dehydration

Dry 1268 plusmn 718 MPa

Wet 305 plusmn 243 MPa

Dry 1737 plusmn 973 MPa

Wet 301 plusmn 202 MPa

Kemp et al J StructBio 2012 180 (3) 428-438

bull Significant stiffening of fibrils with dehydration

bull More pronounced modulus modulus as a function of disease

BBML - All R

ights Reserve

d

OI Tendon Differential Effects of Dehydration

Kemp et al J StructBio 2012 180 (3) 428-438

Diseased phenotype not present when wet phenotype observed when dried

0

10

20

30

40

50

60

0 20 40 60 80 100 120 140 160 180 200

Gro

up S

ampl

es (

)

Indent Modulus (MPa)

WT WetBrtl Wet

0

2

4

6

8

10

12

14

16

18

20

025

050

075

010

0012

5015

0017

5020

0022

5025

0027

5030

0032

5035

0037

5040

0042

5045

0047

5050

00

Gro

up S

ampl

es (

)Indent Modulus (MPa)

WT DryBrtl Dry

555 WT Indents 305 plusmn 243 MPa

614 Brtl+ Indents 301 plusmn 202 MPa

KS Test p=0129 t Test p=0464 464 WT Indents

1268 plusmn 718 MPa 479 Brtl+ Indents 1737 plusmn 973 MPa

KS Test plt0001 T Test plt0001

BBML - All R

ights Reserve

d

Indentation in Diabetic Tendon (Poster 0495)

x

z

y

Control and Diabetic Rat Tail Tendon

4-5 rats per group

2-3 fascicles per rat

~70 fibrilstail

4-5 locationsfibril

20 nN (Sneddon)

BBML - All R

ights Reserve

d

Multiscale Diabetes Study (Poster 0495)

0

5

10

15

20

25

300 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 gt25

Perc

enta

ge o

f Gro

up S

ampl

es

Elastic Modulus (MPa)

CD (1530 indents)ZDSD (913 indents)

CD (n=5) 381 plusmn 149 MPa

ZDSD (n=3) 706 plusmn 263 MPa

0

100

200

300

400

500

600

700

800

900

0 2 4 6 8 10

Mic

rosc

ale

Mod

ulus

(MPa

)

Nanoscale Modulus (MPa)

plt0001

r2=0531 p=0040

bull Increased indentation modulus in diabetic tendon

bull Strong relationship between nanoscale indent modulus and microscale tensile modulus

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

Proper probe selection for intended application bull Cantilever must be stiff relative to sample bull Sharp probes may damage sample easily dull

R or α must be determined for each probe bull Properties provided by manufacturer are unreliable bull Vary from probe to probe can dull during indent process

Spring constant must be determined for each probe

bull Value provided by manufacturer is inaccurate bull Thermal tuning OK for ldquosoftrdquo probes poor performance

for stiffer probes bull Sader method (geometry) added mass reference

cantilevers

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

Veeco Application Technical note Ben Ohler Practical Advice on the Determination of Cantilever Spring Constants

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

bull Sample processing

bull Sample hydration

bull Proper force range

bull Substrate effects for thin samples

bull Proper modulus model and range of data goodness of fit

Bearing in mind these assumptions and limitations AFM can provide true ldquonanoscalerdquo mechanical properties both in terms of forces and deformations

BBML - All R

ights Reserve

d

A Note on Peak Force QNM for Bruker AFMs bull Mechanical properties mapped pixel-by-pixel at same

resolution as the height image (PeakForce QNM)

Height Modulus BBML - All R

ights Reserve

d

A Note on Peak Force QNM

Correct probe stiffness must be used based on sample Pittenger ndash Veeco Product Bulletin

BBML - All R

ights Reserve

d

A Note on Peak Force QNM Extremely sensitive to calibration (details rarely given) Absolute radius and spring constant known

bull especially important from probe to probe bull radius uarr with indent depth ndash must determine R at depth

you expect to image bull Depth may vary as a function of sample (or at different

regions within the sample)

Relative reference material of ldquoknownrdquo modulus used to determine a ratio of 119896119896 119877119877 bull ldquoknownrdquo modulus may come from tension compression

and not indentation bull R is related to depth indentation depth on sample must

be same as on reference BBML - All R

ights Reserve

d

A Note on Peak Force QNM bull Modulus fit on the fly ndash no ability to monitor fit (or range) bull Local sample topography and sample tilt play a role in

measured parameters bull Assuming all modulus values in an image are correct how to

quantify

512 x 512 pixels = 262144 measurements

n=1 n=262144

n from selected ROIs

BBML - All R

ights Reserve

d

BBML - All R

ights Reserve

d

bull Imaging in air on mica ndash substrate effect

bull Same imaging force or indent depth for all

bull For each area were all modulus values pooled

Lamprou et al PLoS ONE 2013 BBML - All R

ights Reserve

d

AFM Applications in Bone Research Other Cool Mechanical Techniques

BBML - All R

ights Reserve

d

ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo Bonds within or between collagen fibrils in bone that rupture to dissipate energy as a toughening mechanism

Thompson et al Nature 2001

Fantner et al Nature Mat 2005

Hansma et al JMNI 2005

Fantner et al Nano Letters 2007 BBML - All R

ights Reserve

d

Other Mechanical Techniques

Jimenez-Palomar J Mech Beh Bio Mat 2012

van der Rijt Macro Bio 2004

Yang J Biomed Mater Res 2007

Yang Biophysical Journal 2008 BBML - All R

ights Reserve

d

Other Cool Techniques bull Piezoresponse Force Microscopy (PFM)

bull Electrochemical AFM (EC-AFM)

bull Force Modulation Microscopy (FMM)

bull Lateral Force Microscopy (LFM)

bull Magnetic Force Microscopy (MFM)

bull Conductive AFM (C-AFM)

bull Force Pulling

bull Cantilever Bending

bull Tensile testing of individual fibrils

bull 3 point bending of fibrils

BBML - All R

ights Reserve

d

Summary Things to rememberhellip

bull Imaging mode imaging medium and probe selection

bull Morphology Measurements bull Sample preparation and hydration state bull Measuring fibril diameter bull Site selection and proper sampling bull Appropriate system calibration

bull Mechanical Measurements

bull Probe selection (stiffness and tip radius) bull Probe calibration (radius angle spring constant) bull Indentation force range and mechanical model bull Hydration in biological samples bull PeakForce QNM

BBML - All R

ights Reserve

d

Acknowledgements BBML

Max Hammond Armando Diaz Gonzalez Zachary Bart Silvia Canelon Creasy Clauser

IU School of Medicine Dr David Burr Dr Matt Allen Dr Stuart Warden Dr Lilian Plotkin

Funding Sources NIHNIDCR 1F32DE018840-01 A1 IUPUI BME Departmental Startup Funds IUPUI Research Support Funds Grant IUPUI Biomechanics and Biomaterials Research Center

NIH NICHD Dr Joan Marini

BBML - All R

ights Reserve

d

Bone Biology and Mechanics Lab (BBML) wwwiupuiedu~bbml Facebook BoneBiologyMechanicsLab

BBML - All R

ights Reserve

d

• Slide Number 1
• General Seminar Outline
• Bonersquos Hierarchical Structure
• Critical Gap in Understanding Collagen
• Methods For Analyzing Collagen Structure
• Methods For Imaging Collagen Structure
• Cryo-TEM
• Atomic Force Microscopy (AFM)
• AFM Image of Pentacene (C22H14)
• AFM Image of Hexabenzocoronene
• Basics of AFM Imaging The Probe
• Basics of AFM Imaging System Components
• van der Waals Potential Energy Curve
• Contact Mode ndash Monitor Deflection
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Peak Force Tapping Mode ndash Monitor Force
• AFM Applications in Bone Research Imaging
• AFM Imaging of Collagen
• Imaging of Osteoclast Activity
• Mineralized collagen deposition in vitro
• Imaging of Canilicular Structure
• AFM Imaging in Bone
• AFM Imaging in Bone
• Removing Mineral in Bone to Expose Collagen
• Removing Mineral in Bone to Expose Collagen
• Demineralization with EDTA
• Demineralization with EDTA
• Demineralization with EDTA
• Issues to Consider Effects of Acid
• Slide Number 32
• Slide Number 33
• Analysis of Collagen Structure in Bone
• Analysis of Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril D-Spacing
• Slide Number 39
• Collagen in Bone Fibril D-Spacing
• Collagen in Bone Fibril D-Spacing
• 2D Fast Fourier Transform Analysis
• 2D Fast Fourier Transform Analysis
• D-Spacing Distribution
• D-Spacing Distribution
• Cumulative Distribution Function
• CDF vs Histogram
• Changes in D-Spacing with Disease
• Changes in D-Spacing with Disease
• Slide Number 50
• Issues to Consider Site Selection and Sampling
• Issues to Consider Site Selection and Sampling
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• AFM calibration ndash Take Away Message
• AFM Applications in Bone ResearchIndentation Mechanics
• AFM-Based Indentation
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Parameters
• Hertz Contact Equation
• Hertz Contact Equation
• Hertz Contact Equation
• Proper Indentation Model is Essential
• Indentation in OI Tendon Study
• OI Tendon Differential Effects of Dehydration
• OI Tendon Differential Effects of Dehydration
• Indentation in Diabetic Tendon (Poster 0495)
• Multiscale Diabetes Study (Poster 0495)
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• A Note on Peak Force QNM for Bruker AFMs
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• Slide Number 83
• Slide Number 84
• AFM Applications in Bone ResearchOther Cool Mechanical Techniques
• ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo
• Other Mechanical Techniques
• Other Cool Techniques
• Summary Things to rememberhellip
• Acknowledgements
• Bone Biology and Mechanics Lab (BBML)

Issues to Consider Effects of Hydration

Wet Tendon Dry Tendon Kemp et al J StructBio 2012 180 (3) 428-438

Collagen and bone exist in a hydrated environment in vivo bull Hydration state can impact morphology and mechanics

BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

Nonsignificant mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet (n=255) 682 plusmn 12 nm

WT Dry (n=257) 686 plusmn 08 nm

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

Brtl DryBrtl Wet

KS Test plt0001

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

WT Case 04 nm mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet 682 plusmn 12 nm

WT Dry 686 plusmn 08 nm

Brtl+ Wet 675 plusmn 14 nm

Brtl+ Dry 689 plusmn 10 nm

Brtl+ Case 14 nm increase with drying (plt0001) bull Entire population shifted upward (plt0001)

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT WetBrtl Wet

KS Test plt0001

Brtl+ distribution shifted downward vs WT when wet

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryBrtl Dry

KS Test plt0001

Brtl+ distribution shifted upward vs WT when dry

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider AFM calibration

bull Absolute distance measurements require accurate piezo calibration

bull AFM systems have large scan ranges (150 microm to lt 1 microm)

bull Scanner non-linearities can introduce substantial error over that range

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

ights Reserve

d

Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

BBML - All R

ights Reserve

d

Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

100 nm 10 80 602 1198 100 nm 7 60 827 1439 100 nm 5 40 814 1427 100 nm 35 20 917 1724 100 nm 2 10 937 1774

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

ights Reserve

d

AFM calibration ndash Take Away Message

Calibration using a scan size and feature size comparable to features of interest is highly recommended

Calibration addresses absolute accuracy not precision (differential sensitivity between measurements)

bull Does not limit ability to differentiate between groups using the same AFM with the same calibration parameters

BBML - All R

ights Reserve

d

AFM Applications in Bone Research Indentation Mechanics

BBML - All R

ights Reserve

d

AFM-Based Indentation

In addition to high resolution imaging AFM can be used to extract nanoscale mechanical data

ApproachWithdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Can

tilev

er D

efle

ctio

n (V

olts

)

1 Ramp into a rigid sample ndash measure the slope

Deflection sensitivity x 119899119899119899119899119881119881

2 Calculate cantilever spring constant

3 Hookersquos law takes deflection (volts) and converts to units of force F = kx

Spring constant k 119873119873119899119899

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Forc

e (n

N)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration Fo

rce

(nN

)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

Tip-Sample Separation (nm)

BBML - All R

ights Reserve

d

AFM-Based Indentation Parameters

Approach Withdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

Peak Force

Area Between Curves Energy Dissipation

Contact Mechanics Fit for Elastic Modulus (E)

Adhesion Force

Indentation Depth (δ)

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

z

r

δ

R

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119877119877 ∙ 12057512057532

Es Sample modulus νs Poissonrsquos ratio R tip radius of curvature δ load point displacement

Relevant Hertz Assumptions bull Strains are small and within the elastic limit bull Indenter is infinitely stiff (only sample deforms) bull Indentation depth ltlt radius of curvature of indenter bull Surfaces are continuous non-conforming (must be flat) bull Load is normal to surface

BBML - All R

ights Reserve

d

Proper Indentation Model is Essential

δ

r

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119903119903 ∙ 12057512057532

δ

α

119865119865 =2120587120587∙

1198641198641199041199041 minus 1205841205841199041199042

∙ tan120572120572 ∙ 1205751205752

Es Sample modulus νs Poissonrsquos ratio R tip radius α opening angle

Hertz Model Spherical Indenter

depth ltlt radius of curvature

Sneddon Model Conical Indenter

depth ge radius of curvature

BBML - All R

ights Reserve

d

Indentation in OI Tendon Study

x

z

yBrtl+ and WT mice 6 month old males

4 tails per group

2-3 fascicles per tail

25-30 fibrils per tail

4-5 locations per fibril

Indent force 20 nN wet (Sneddon) 50 nN dry (Hertz) BBML - A

ll Rights R

eserved

OI Tendon Differential Effects of Dehydration

Dry 1268 plusmn 718 MPa

Wet 305 plusmn 243 MPa

Dry 1737 plusmn 973 MPa

Wet 301 plusmn 202 MPa

Kemp et al J StructBio 2012 180 (3) 428-438

bull Significant stiffening of fibrils with dehydration

bull More pronounced modulus modulus as a function of disease

BBML - All R

ights Reserve

d

OI Tendon Differential Effects of Dehydration

Kemp et al J StructBio 2012 180 (3) 428-438

Diseased phenotype not present when wet phenotype observed when dried

0

10

20

30

40

50

60

0 20 40 60 80 100 120 140 160 180 200

Gro

up S

ampl

es (

)

Indent Modulus (MPa)

WT WetBrtl Wet

0

2

4

6

8

10

12

14

16

18

20

025

050

075

010

0012

5015

0017

5020

0022

5025

0027

5030

0032

5035

0037

5040

0042

5045

0047

5050

00

Gro

up S

ampl

es (

)Indent Modulus (MPa)

WT DryBrtl Dry

555 WT Indents 305 plusmn 243 MPa

614 Brtl+ Indents 301 plusmn 202 MPa

KS Test p=0129 t Test p=0464 464 WT Indents

1268 plusmn 718 MPa 479 Brtl+ Indents 1737 plusmn 973 MPa

KS Test plt0001 T Test plt0001

BBML - All R

ights Reserve

d

Indentation in Diabetic Tendon (Poster 0495)

x

z

y

Control and Diabetic Rat Tail Tendon

4-5 rats per group

2-3 fascicles per rat

~70 fibrilstail

4-5 locationsfibril

20 nN (Sneddon)

BBML - All R

ights Reserve

d

Multiscale Diabetes Study (Poster 0495)

0

5

10

15

20

25

300 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 gt25

Perc

enta

ge o

f Gro

up S

ampl

es

Elastic Modulus (MPa)

CD (1530 indents)ZDSD (913 indents)

CD (n=5) 381 plusmn 149 MPa

ZDSD (n=3) 706 plusmn 263 MPa

0

100

200

300

400

500

600

700

800

900

0 2 4 6 8 10

Mic

rosc

ale

Mod

ulus

(MPa

)

Nanoscale Modulus (MPa)

plt0001

r2=0531 p=0040

bull Increased indentation modulus in diabetic tendon

bull Strong relationship between nanoscale indent modulus and microscale tensile modulus

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

Proper probe selection for intended application bull Cantilever must be stiff relative to sample bull Sharp probes may damage sample easily dull

R or α must be determined for each probe bull Properties provided by manufacturer are unreliable bull Vary from probe to probe can dull during indent process

Spring constant must be determined for each probe

bull Value provided by manufacturer is inaccurate bull Thermal tuning OK for ldquosoftrdquo probes poor performance

for stiffer probes bull Sader method (geometry) added mass reference

cantilevers

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

Veeco Application Technical note Ben Ohler Practical Advice on the Determination of Cantilever Spring Constants

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

bull Sample processing

bull Sample hydration

bull Proper force range

bull Substrate effects for thin samples

bull Proper modulus model and range of data goodness of fit

Bearing in mind these assumptions and limitations AFM can provide true ldquonanoscalerdquo mechanical properties both in terms of forces and deformations

BBML - All R

ights Reserve

d

A Note on Peak Force QNM for Bruker AFMs bull Mechanical properties mapped pixel-by-pixel at same

resolution as the height image (PeakForce QNM)

Height Modulus BBML - All R

ights Reserve

d

A Note on Peak Force QNM

Correct probe stiffness must be used based on sample Pittenger ndash Veeco Product Bulletin

BBML - All R

ights Reserve

d

A Note on Peak Force QNM Extremely sensitive to calibration (details rarely given) Absolute radius and spring constant known

bull especially important from probe to probe bull radius uarr with indent depth ndash must determine R at depth

you expect to image bull Depth may vary as a function of sample (or at different

regions within the sample)

Relative reference material of ldquoknownrdquo modulus used to determine a ratio of 119896119896 119877119877 bull ldquoknownrdquo modulus may come from tension compression

and not indentation bull R is related to depth indentation depth on sample must

be same as on reference BBML - All R

ights Reserve

d

A Note on Peak Force QNM bull Modulus fit on the fly ndash no ability to monitor fit (or range) bull Local sample topography and sample tilt play a role in

measured parameters bull Assuming all modulus values in an image are correct how to

quantify

512 x 512 pixels = 262144 measurements

n=1 n=262144

n from selected ROIs

BBML - All R

ights Reserve

d

BBML - All R

ights Reserve

d

bull Imaging in air on mica ndash substrate effect

bull Same imaging force or indent depth for all

bull For each area were all modulus values pooled

Lamprou et al PLoS ONE 2013 BBML - All R

ights Reserve

d

AFM Applications in Bone Research Other Cool Mechanical Techniques

BBML - All R

ights Reserve

d

ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo Bonds within or between collagen fibrils in bone that rupture to dissipate energy as a toughening mechanism

Thompson et al Nature 2001

Fantner et al Nature Mat 2005

Hansma et al JMNI 2005

Fantner et al Nano Letters 2007 BBML - All R

ights Reserve

d

Other Mechanical Techniques

Jimenez-Palomar J Mech Beh Bio Mat 2012

van der Rijt Macro Bio 2004

Yang J Biomed Mater Res 2007

Yang Biophysical Journal 2008 BBML - All R

ights Reserve

d

Other Cool Techniques bull Piezoresponse Force Microscopy (PFM)

bull Electrochemical AFM (EC-AFM)

bull Force Modulation Microscopy (FMM)

bull Lateral Force Microscopy (LFM)

bull Magnetic Force Microscopy (MFM)

bull Conductive AFM (C-AFM)

bull Force Pulling

bull Cantilever Bending

bull Tensile testing of individual fibrils

bull 3 point bending of fibrils

BBML - All R

ights Reserve

d

Summary Things to rememberhellip

bull Imaging mode imaging medium and probe selection

bull Morphology Measurements bull Sample preparation and hydration state bull Measuring fibril diameter bull Site selection and proper sampling bull Appropriate system calibration

bull Mechanical Measurements

bull Probe selection (stiffness and tip radius) bull Probe calibration (radius angle spring constant) bull Indentation force range and mechanical model bull Hydration in biological samples bull PeakForce QNM

BBML - All R

ights Reserve

d

Acknowledgements BBML

Max Hammond Armando Diaz Gonzalez Zachary Bart Silvia Canelon Creasy Clauser

IU School of Medicine Dr David Burr Dr Matt Allen Dr Stuart Warden Dr Lilian Plotkin

Funding Sources NIHNIDCR 1F32DE018840-01 A1 IUPUI BME Departmental Startup Funds IUPUI Research Support Funds Grant IUPUI Biomechanics and Biomaterials Research Center

NIH NICHD Dr Joan Marini

BBML - All R

ights Reserve

d

Bone Biology and Mechanics Lab (BBML) wwwiupuiedu~bbml Facebook BoneBiologyMechanicsLab

BBML - All R

ights Reserve

d

• Slide Number 1
• General Seminar Outline
• Bonersquos Hierarchical Structure
• Critical Gap in Understanding Collagen
• Methods For Analyzing Collagen Structure
• Methods For Imaging Collagen Structure
• Cryo-TEM
• Atomic Force Microscopy (AFM)
• AFM Image of Pentacene (C22H14)
• AFM Image of Hexabenzocoronene
• Basics of AFM Imaging The Probe
• Basics of AFM Imaging System Components
• van der Waals Potential Energy Curve
• Contact Mode ndash Monitor Deflection
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Peak Force Tapping Mode ndash Monitor Force
• AFM Applications in Bone Research Imaging
• AFM Imaging of Collagen
• Imaging of Osteoclast Activity
• Mineralized collagen deposition in vitro
• Imaging of Canilicular Structure
• AFM Imaging in Bone
• AFM Imaging in Bone
• Removing Mineral in Bone to Expose Collagen
• Removing Mineral in Bone to Expose Collagen
• Demineralization with EDTA
• Demineralization with EDTA
• Demineralization with EDTA
• Issues to Consider Effects of Acid
• Slide Number 32
• Slide Number 33
• Analysis of Collagen Structure in Bone
• Analysis of Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril D-Spacing
• Slide Number 39
• Collagen in Bone Fibril D-Spacing
• Collagen in Bone Fibril D-Spacing
• 2D Fast Fourier Transform Analysis
• 2D Fast Fourier Transform Analysis
• D-Spacing Distribution
• D-Spacing Distribution
• Cumulative Distribution Function
• CDF vs Histogram
• Changes in D-Spacing with Disease
• Changes in D-Spacing with Disease
• Slide Number 50
• Issues to Consider Site Selection and Sampling
• Issues to Consider Site Selection and Sampling
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• AFM calibration ndash Take Away Message
• AFM Applications in Bone ResearchIndentation Mechanics
• AFM-Based Indentation
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Parameters
• Hertz Contact Equation
• Hertz Contact Equation
• Hertz Contact Equation
• Proper Indentation Model is Essential
• Indentation in OI Tendon Study
• OI Tendon Differential Effects of Dehydration
• OI Tendon Differential Effects of Dehydration
• Indentation in Diabetic Tendon (Poster 0495)
• Multiscale Diabetes Study (Poster 0495)
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• A Note on Peak Force QNM for Bruker AFMs
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• Slide Number 83
• Slide Number 84
• AFM Applications in Bone ResearchOther Cool Mechanical Techniques
• ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo
• Other Mechanical Techniques
• Other Cool Techniques
• Summary Things to rememberhellip
• Acknowledgements
• Bone Biology and Mechanics Lab (BBML)

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

Nonsignificant mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet (n=255) 682 plusmn 12 nm

WT Dry (n=257) 686 plusmn 08 nm

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

Brtl DryBrtl Wet

KS Test plt0001

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

WT Case 04 nm mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet 682 plusmn 12 nm

WT Dry 686 plusmn 08 nm

Brtl+ Wet 675 plusmn 14 nm

Brtl+ Dry 689 plusmn 10 nm

Brtl+ Case 14 nm increase with drying (plt0001) bull Entire population shifted upward (plt0001)

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT WetBrtl Wet

KS Test plt0001

Brtl+ distribution shifted downward vs WT when wet

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryBrtl Dry

KS Test plt0001

Brtl+ distribution shifted upward vs WT when dry

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider AFM calibration

bull Absolute distance measurements require accurate piezo calibration

bull AFM systems have large scan ranges (150 microm to lt 1 microm)

bull Scanner non-linearities can introduce substantial error over that range

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

ights Reserve

d

Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

BBML - All R

ights Reserve

d

Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

100 nm 10 80 602 1198 100 nm 7 60 827 1439 100 nm 5 40 814 1427 100 nm 35 20 917 1724 100 nm 2 10 937 1774

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

ights Reserve

d

AFM calibration ndash Take Away Message

Calibration using a scan size and feature size comparable to features of interest is highly recommended

Calibration addresses absolute accuracy not precision (differential sensitivity between measurements)

bull Does not limit ability to differentiate between groups using the same AFM with the same calibration parameters

BBML - All R

ights Reserve

d

AFM Applications in Bone Research Indentation Mechanics

BBML - All R

ights Reserve

d

AFM-Based Indentation

In addition to high resolution imaging AFM can be used to extract nanoscale mechanical data

ApproachWithdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Can

tilev

er D

efle

ctio

n (V

olts

)

1 Ramp into a rigid sample ndash measure the slope

Deflection sensitivity x 119899119899119899119899119881119881

2 Calculate cantilever spring constant

3 Hookersquos law takes deflection (volts) and converts to units of force F = kx

Spring constant k 119873119873119899119899

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Forc

e (n

N)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration Fo

rce

(nN

)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

Tip-Sample Separation (nm)

BBML - All R

ights Reserve

d

AFM-Based Indentation Parameters

Approach Withdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

Peak Force

Area Between Curves Energy Dissipation

Contact Mechanics Fit for Elastic Modulus (E)

Adhesion Force

Indentation Depth (δ)

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

z

r

δ

R

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119877119877 ∙ 12057512057532

Es Sample modulus νs Poissonrsquos ratio R tip radius of curvature δ load point displacement

Relevant Hertz Assumptions bull Strains are small and within the elastic limit bull Indenter is infinitely stiff (only sample deforms) bull Indentation depth ltlt radius of curvature of indenter bull Surfaces are continuous non-conforming (must be flat) bull Load is normal to surface

BBML - All R

ights Reserve

d

Proper Indentation Model is Essential

δ

r

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119903119903 ∙ 12057512057532

δ

α

119865119865 =2120587120587∙

1198641198641199041199041 minus 1205841205841199041199042

∙ tan120572120572 ∙ 1205751205752

Es Sample modulus νs Poissonrsquos ratio R tip radius α opening angle

Hertz Model Spherical Indenter

depth ltlt radius of curvature

Sneddon Model Conical Indenter

depth ge radius of curvature

BBML - All R

ights Reserve

d

Indentation in OI Tendon Study

x

z

yBrtl+ and WT mice 6 month old males

4 tails per group

2-3 fascicles per tail

25-30 fibrils per tail

4-5 locations per fibril

Indent force 20 nN wet (Sneddon) 50 nN dry (Hertz) BBML - A

ll Rights R

eserved

OI Tendon Differential Effects of Dehydration

Dry 1268 plusmn 718 MPa

Wet 305 plusmn 243 MPa

Dry 1737 plusmn 973 MPa

Wet 301 plusmn 202 MPa

Kemp et al J StructBio 2012 180 (3) 428-438

bull Significant stiffening of fibrils with dehydration

bull More pronounced modulus modulus as a function of disease

BBML - All R

ights Reserve

d

OI Tendon Differential Effects of Dehydration

Kemp et al J StructBio 2012 180 (3) 428-438

Diseased phenotype not present when wet phenotype observed when dried

0

10

20

30

40

50

60

0 20 40 60 80 100 120 140 160 180 200

Gro

up S

ampl

es (

)

Indent Modulus (MPa)

WT WetBrtl Wet

0

2

4

6

8

10

12

14

16

18

20

025

050

075

010

0012

5015

0017

5020

0022

5025

0027

5030

0032

5035

0037

5040

0042

5045

0047

5050

00

Gro

up S

ampl

es (

)Indent Modulus (MPa)

WT DryBrtl Dry

555 WT Indents 305 plusmn 243 MPa

614 Brtl+ Indents 301 plusmn 202 MPa

KS Test p=0129 t Test p=0464 464 WT Indents

1268 plusmn 718 MPa 479 Brtl+ Indents 1737 plusmn 973 MPa

KS Test plt0001 T Test plt0001

BBML - All R

ights Reserve

d

Indentation in Diabetic Tendon (Poster 0495)

x

z

y

Control and Diabetic Rat Tail Tendon

4-5 rats per group

2-3 fascicles per rat

~70 fibrilstail

4-5 locationsfibril

20 nN (Sneddon)

BBML - All R

ights Reserve

d

Multiscale Diabetes Study (Poster 0495)

0

5

10

15

20

25

300 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 gt25

Perc

enta

ge o

f Gro

up S

ampl

es

Elastic Modulus (MPa)

CD (1530 indents)ZDSD (913 indents)

CD (n=5) 381 plusmn 149 MPa

ZDSD (n=3) 706 plusmn 263 MPa

0

100

200

300

400

500

600

700

800

900

0 2 4 6 8 10

Mic

rosc

ale

Mod

ulus

(MPa

)

Nanoscale Modulus (MPa)

plt0001

r2=0531 p=0040

bull Increased indentation modulus in diabetic tendon

bull Strong relationship between nanoscale indent modulus and microscale tensile modulus

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

Proper probe selection for intended application bull Cantilever must be stiff relative to sample bull Sharp probes may damage sample easily dull

R or α must be determined for each probe bull Properties provided by manufacturer are unreliable bull Vary from probe to probe can dull during indent process

Spring constant must be determined for each probe

bull Value provided by manufacturer is inaccurate bull Thermal tuning OK for ldquosoftrdquo probes poor performance

for stiffer probes bull Sader method (geometry) added mass reference

cantilevers

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

Veeco Application Technical note Ben Ohler Practical Advice on the Determination of Cantilever Spring Constants

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

bull Sample processing

bull Sample hydration

bull Proper force range

bull Substrate effects for thin samples

bull Proper modulus model and range of data goodness of fit

Bearing in mind these assumptions and limitations AFM can provide true ldquonanoscalerdquo mechanical properties both in terms of forces and deformations

BBML - All R

ights Reserve

d

A Note on Peak Force QNM for Bruker AFMs bull Mechanical properties mapped pixel-by-pixel at same

resolution as the height image (PeakForce QNM)

Height Modulus BBML - All R

ights Reserve

d

A Note on Peak Force QNM

Correct probe stiffness must be used based on sample Pittenger ndash Veeco Product Bulletin

BBML - All R

ights Reserve

d

A Note on Peak Force QNM Extremely sensitive to calibration (details rarely given) Absolute radius and spring constant known

bull especially important from probe to probe bull radius uarr with indent depth ndash must determine R at depth

you expect to image bull Depth may vary as a function of sample (or at different

regions within the sample)

Relative reference material of ldquoknownrdquo modulus used to determine a ratio of 119896119896 119877119877 bull ldquoknownrdquo modulus may come from tension compression

and not indentation bull R is related to depth indentation depth on sample must

be same as on reference BBML - All R

ights Reserve

d

A Note on Peak Force QNM bull Modulus fit on the fly ndash no ability to monitor fit (or range) bull Local sample topography and sample tilt play a role in

measured parameters bull Assuming all modulus values in an image are correct how to

quantify

512 x 512 pixels = 262144 measurements

n=1 n=262144

n from selected ROIs

BBML - All R

ights Reserve

d

BBML - All R

ights Reserve

d

bull Imaging in air on mica ndash substrate effect

bull Same imaging force or indent depth for all

bull For each area were all modulus values pooled

Lamprou et al PLoS ONE 2013 BBML - All R

ights Reserve

d

AFM Applications in Bone Research Other Cool Mechanical Techniques

BBML - All R

ights Reserve

d

ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo Bonds within or between collagen fibrils in bone that rupture to dissipate energy as a toughening mechanism

Thompson et al Nature 2001

Fantner et al Nature Mat 2005

Hansma et al JMNI 2005

Fantner et al Nano Letters 2007 BBML - All R

ights Reserve

d

Other Mechanical Techniques

Jimenez-Palomar J Mech Beh Bio Mat 2012

van der Rijt Macro Bio 2004

Yang J Biomed Mater Res 2007

Yang Biophysical Journal 2008 BBML - All R

ights Reserve

d

Other Cool Techniques bull Piezoresponse Force Microscopy (PFM)

bull Electrochemical AFM (EC-AFM)

bull Force Modulation Microscopy (FMM)

bull Lateral Force Microscopy (LFM)

bull Magnetic Force Microscopy (MFM)

bull Conductive AFM (C-AFM)

bull Force Pulling

bull Cantilever Bending

bull Tensile testing of individual fibrils

bull 3 point bending of fibrils

BBML - All R

ights Reserve

d

Summary Things to rememberhellip

bull Imaging mode imaging medium and probe selection

bull Morphology Measurements bull Sample preparation and hydration state bull Measuring fibril diameter bull Site selection and proper sampling bull Appropriate system calibration

bull Mechanical Measurements

bull Probe selection (stiffness and tip radius) bull Probe calibration (radius angle spring constant) bull Indentation force range and mechanical model bull Hydration in biological samples bull PeakForce QNM

BBML - All R

ights Reserve

d

Acknowledgements BBML

Max Hammond Armando Diaz Gonzalez Zachary Bart Silvia Canelon Creasy Clauser

IU School of Medicine Dr David Burr Dr Matt Allen Dr Stuart Warden Dr Lilian Plotkin

Funding Sources NIHNIDCR 1F32DE018840-01 A1 IUPUI BME Departmental Startup Funds IUPUI Research Support Funds Grant IUPUI Biomechanics and Biomaterials Research Center

NIH NICHD Dr Joan Marini

BBML - All R

ights Reserve

d

Bone Biology and Mechanics Lab (BBML) wwwiupuiedu~bbml Facebook BoneBiologyMechanicsLab

BBML - All R

ights Reserve

d

• Slide Number 1
• General Seminar Outline
• Bonersquos Hierarchical Structure
• Critical Gap in Understanding Collagen
• Methods For Analyzing Collagen Structure
• Methods For Imaging Collagen Structure
• Cryo-TEM
• Atomic Force Microscopy (AFM)
• AFM Image of Pentacene (C22H14)
• AFM Image of Hexabenzocoronene
• Basics of AFM Imaging The Probe
• Basics of AFM Imaging System Components
• van der Waals Potential Energy Curve
• Contact Mode ndash Monitor Deflection
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Peak Force Tapping Mode ndash Monitor Force
• AFM Applications in Bone Research Imaging
• AFM Imaging of Collagen
• Imaging of Osteoclast Activity
• Mineralized collagen deposition in vitro
• Imaging of Canilicular Structure
• AFM Imaging in Bone
• AFM Imaging in Bone
• Removing Mineral in Bone to Expose Collagen
• Removing Mineral in Bone to Expose Collagen
• Demineralization with EDTA
• Demineralization with EDTA
• Demineralization with EDTA
• Issues to Consider Effects of Acid
• Slide Number 32
• Slide Number 33
• Analysis of Collagen Structure in Bone
• Analysis of Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril D-Spacing
• Slide Number 39
• Collagen in Bone Fibril D-Spacing
• Collagen in Bone Fibril D-Spacing
• 2D Fast Fourier Transform Analysis
• 2D Fast Fourier Transform Analysis
• D-Spacing Distribution
• D-Spacing Distribution
• Cumulative Distribution Function
• CDF vs Histogram
• Changes in D-Spacing with Disease
• Changes in D-Spacing with Disease
• Slide Number 50
• Issues to Consider Site Selection and Sampling
• Issues to Consider Site Selection and Sampling
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• AFM calibration ndash Take Away Message
• AFM Applications in Bone ResearchIndentation Mechanics
• AFM-Based Indentation
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Parameters
• Hertz Contact Equation
• Hertz Contact Equation
• Hertz Contact Equation
• Proper Indentation Model is Essential
• Indentation in OI Tendon Study
• OI Tendon Differential Effects of Dehydration
• OI Tendon Differential Effects of Dehydration
• Indentation in Diabetic Tendon (Poster 0495)
• Multiscale Diabetes Study (Poster 0495)
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• A Note on Peak Force QNM for Bruker AFMs
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• Slide Number 83
• Slide Number 84
• AFM Applications in Bone ResearchOther Cool Mechanical Techniques
• ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo
• Other Mechanical Techniques
• Other Cool Techniques
• Summary Things to rememberhellip
• Acknowledgements
• Bone Biology and Mechanics Lab (BBML)

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryWT Wet

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

Brtl DryBrtl Wet

KS Test plt0001

0

10

20

30

40

50

62 64 66 68 70 72 74

Gro

up S

ampl

es (

)

D-Spacing (nm)

WT Case 04 nm mean increase with drying (p=0200) bull Loss of fibrils lt 66 nm population more uniform (plt0001)

WT Wet 682 plusmn 12 nm

WT Dry 686 plusmn 08 nm

Brtl+ Wet 675 plusmn 14 nm

Brtl+ Dry 689 plusmn 10 nm

Brtl+ Case 14 nm increase with drying (plt0001) bull Entire population shifted upward (plt0001)

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT WetBrtl Wet

KS Test plt0001

Brtl+ distribution shifted downward vs WT when wet

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryBrtl Dry

KS Test plt0001

Brtl+ distribution shifted upward vs WT when dry

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider AFM calibration

bull Absolute distance measurements require accurate piezo calibration

bull AFM systems have large scan ranges (150 microm to lt 1 microm)

bull Scanner non-linearities can introduce substantial error over that range

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

ights Reserve

d

Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

BBML - All R

ights Reserve

d

Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

100 nm 10 80 602 1198 100 nm 7 60 827 1439 100 nm 5 40 814 1427 100 nm 35 20 917 1724 100 nm 2 10 937 1774

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

ights Reserve

d

AFM calibration ndash Take Away Message

Calibration using a scan size and feature size comparable to features of interest is highly recommended

Calibration addresses absolute accuracy not precision (differential sensitivity between measurements)

bull Does not limit ability to differentiate between groups using the same AFM with the same calibration parameters

BBML - All R

ights Reserve

d

AFM Applications in Bone Research Indentation Mechanics

BBML - All R

ights Reserve

d

AFM-Based Indentation

In addition to high resolution imaging AFM can be used to extract nanoscale mechanical data

ApproachWithdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Can

tilev

er D

efle

ctio

n (V

olts

)

1 Ramp into a rigid sample ndash measure the slope

Deflection sensitivity x 119899119899119899119899119881119881

2 Calculate cantilever spring constant

3 Hookersquos law takes deflection (volts) and converts to units of force F = kx

Spring constant k 119873119873119899119899

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Forc

e (n

N)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration Fo

rce

(nN

)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

Tip-Sample Separation (nm)

BBML - All R

ights Reserve

d

AFM-Based Indentation Parameters

Approach Withdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

Peak Force

Area Between Curves Energy Dissipation

Contact Mechanics Fit for Elastic Modulus (E)

Adhesion Force

Indentation Depth (δ)

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

z

r

δ

R

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119877119877 ∙ 12057512057532

Es Sample modulus νs Poissonrsquos ratio R tip radius of curvature δ load point displacement

Relevant Hertz Assumptions bull Strains are small and within the elastic limit bull Indenter is infinitely stiff (only sample deforms) bull Indentation depth ltlt radius of curvature of indenter bull Surfaces are continuous non-conforming (must be flat) bull Load is normal to surface

BBML - All R

ights Reserve

d

Proper Indentation Model is Essential

δ

r

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119903119903 ∙ 12057512057532

δ

α

119865119865 =2120587120587∙

1198641198641199041199041 minus 1205841205841199041199042

∙ tan120572120572 ∙ 1205751205752

Es Sample modulus νs Poissonrsquos ratio R tip radius α opening angle

Hertz Model Spherical Indenter

depth ltlt radius of curvature

Sneddon Model Conical Indenter

depth ge radius of curvature

BBML - All R

ights Reserve

d

Indentation in OI Tendon Study

x

z

yBrtl+ and WT mice 6 month old males

4 tails per group

2-3 fascicles per tail

25-30 fibrils per tail

4-5 locations per fibril

Indent force 20 nN wet (Sneddon) 50 nN dry (Hertz) BBML - A

ll Rights R

eserved

OI Tendon Differential Effects of Dehydration

Dry 1268 plusmn 718 MPa

Wet 305 plusmn 243 MPa

Dry 1737 plusmn 973 MPa

Wet 301 plusmn 202 MPa

Kemp et al J StructBio 2012 180 (3) 428-438

bull Significant stiffening of fibrils with dehydration

bull More pronounced modulus modulus as a function of disease

BBML - All R

ights Reserve

d

OI Tendon Differential Effects of Dehydration

Kemp et al J StructBio 2012 180 (3) 428-438

Diseased phenotype not present when wet phenotype observed when dried

0

10

20

30

40

50

60

0 20 40 60 80 100 120 140 160 180 200

Gro

up S

ampl

es (

)

Indent Modulus (MPa)

WT WetBrtl Wet

0

2

4

6

8

10

12

14

16

18

20

025

050

075

010

0012

5015

0017

5020

0022

5025

0027

5030

0032

5035

0037

5040

0042

5045

0047

5050

00

Gro

up S

ampl

es (

)Indent Modulus (MPa)

WT DryBrtl Dry

555 WT Indents 305 plusmn 243 MPa

614 Brtl+ Indents 301 plusmn 202 MPa

KS Test p=0129 t Test p=0464 464 WT Indents

1268 plusmn 718 MPa 479 Brtl+ Indents 1737 plusmn 973 MPa

KS Test plt0001 T Test plt0001

BBML - All R

ights Reserve

d

Indentation in Diabetic Tendon (Poster 0495)

x

z

y

Control and Diabetic Rat Tail Tendon

4-5 rats per group

2-3 fascicles per rat

~70 fibrilstail

4-5 locationsfibril

20 nN (Sneddon)

BBML - All R

ights Reserve

d

Multiscale Diabetes Study (Poster 0495)

0

5

10

15

20

25

300 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 gt25

Perc

enta

ge o

f Gro

up S

ampl

es

Elastic Modulus (MPa)

CD (1530 indents)ZDSD (913 indents)

CD (n=5) 381 plusmn 149 MPa

ZDSD (n=3) 706 plusmn 263 MPa

0

100

200

300

400

500

600

700

800

900

0 2 4 6 8 10

Mic

rosc

ale

Mod

ulus

(MPa

)

Nanoscale Modulus (MPa)

plt0001

r2=0531 p=0040

bull Increased indentation modulus in diabetic tendon

bull Strong relationship between nanoscale indent modulus and microscale tensile modulus

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

Proper probe selection for intended application bull Cantilever must be stiff relative to sample bull Sharp probes may damage sample easily dull

R or α must be determined for each probe bull Properties provided by manufacturer are unreliable bull Vary from probe to probe can dull during indent process

Spring constant must be determined for each probe

bull Value provided by manufacturer is inaccurate bull Thermal tuning OK for ldquosoftrdquo probes poor performance

for stiffer probes bull Sader method (geometry) added mass reference

cantilevers

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

Veeco Application Technical note Ben Ohler Practical Advice on the Determination of Cantilever Spring Constants

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

bull Sample processing

bull Sample hydration

bull Proper force range

bull Substrate effects for thin samples

bull Proper modulus model and range of data goodness of fit

Bearing in mind these assumptions and limitations AFM can provide true ldquonanoscalerdquo mechanical properties both in terms of forces and deformations

BBML - All R

ights Reserve

d

A Note on Peak Force QNM for Bruker AFMs bull Mechanical properties mapped pixel-by-pixel at same

resolution as the height image (PeakForce QNM)

Height Modulus BBML - All R

ights Reserve

d

A Note on Peak Force QNM

Correct probe stiffness must be used based on sample Pittenger ndash Veeco Product Bulletin

BBML - All R

ights Reserve

d

A Note on Peak Force QNM Extremely sensitive to calibration (details rarely given) Absolute radius and spring constant known

bull especially important from probe to probe bull radius uarr with indent depth ndash must determine R at depth

you expect to image bull Depth may vary as a function of sample (or at different

regions within the sample)

Relative reference material of ldquoknownrdquo modulus used to determine a ratio of 119896119896 119877119877 bull ldquoknownrdquo modulus may come from tension compression

and not indentation bull R is related to depth indentation depth on sample must

be same as on reference BBML - All R

ights Reserve

d

A Note on Peak Force QNM bull Modulus fit on the fly ndash no ability to monitor fit (or range) bull Local sample topography and sample tilt play a role in

measured parameters bull Assuming all modulus values in an image are correct how to

quantify

512 x 512 pixels = 262144 measurements

n=1 n=262144

n from selected ROIs

BBML - All R

ights Reserve

d

BBML - All R

ights Reserve

d

bull Imaging in air on mica ndash substrate effect

bull Same imaging force or indent depth for all

bull For each area were all modulus values pooled

Lamprou et al PLoS ONE 2013 BBML - All R

ights Reserve

d

AFM Applications in Bone Research Other Cool Mechanical Techniques

BBML - All R

ights Reserve

d

ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo Bonds within or between collagen fibrils in bone that rupture to dissipate energy as a toughening mechanism

Thompson et al Nature 2001

Fantner et al Nature Mat 2005

Hansma et al JMNI 2005

Fantner et al Nano Letters 2007 BBML - All R

ights Reserve

d

Other Mechanical Techniques

Jimenez-Palomar J Mech Beh Bio Mat 2012

van der Rijt Macro Bio 2004

Yang J Biomed Mater Res 2007

Yang Biophysical Journal 2008 BBML - All R

ights Reserve

d

Other Cool Techniques bull Piezoresponse Force Microscopy (PFM)

bull Electrochemical AFM (EC-AFM)

bull Force Modulation Microscopy (FMM)

bull Lateral Force Microscopy (LFM)

bull Magnetic Force Microscopy (MFM)

bull Conductive AFM (C-AFM)

bull Force Pulling

bull Cantilever Bending

bull Tensile testing of individual fibrils

bull 3 point bending of fibrils

BBML - All R

ights Reserve

d

Summary Things to rememberhellip

bull Imaging mode imaging medium and probe selection

bull Morphology Measurements bull Sample preparation and hydration state bull Measuring fibril diameter bull Site selection and proper sampling bull Appropriate system calibration

bull Mechanical Measurements

bull Probe selection (stiffness and tip radius) bull Probe calibration (radius angle spring constant) bull Indentation force range and mechanical model bull Hydration in biological samples bull PeakForce QNM

BBML - All R

ights Reserve

d

Acknowledgements BBML

Max Hammond Armando Diaz Gonzalez Zachary Bart Silvia Canelon Creasy Clauser

IU School of Medicine Dr David Burr Dr Matt Allen Dr Stuart Warden Dr Lilian Plotkin

Funding Sources NIHNIDCR 1F32DE018840-01 A1 IUPUI BME Departmental Startup Funds IUPUI Research Support Funds Grant IUPUI Biomechanics and Biomaterials Research Center

NIH NICHD Dr Joan Marini

BBML - All R

ights Reserve

d

Bone Biology and Mechanics Lab (BBML) wwwiupuiedu~bbml Facebook BoneBiologyMechanicsLab

BBML - All R

ights Reserve

d

• Slide Number 1
• General Seminar Outline
• Bonersquos Hierarchical Structure
• Critical Gap in Understanding Collagen
• Methods For Analyzing Collagen Structure
• Methods For Imaging Collagen Structure
• Cryo-TEM
• Atomic Force Microscopy (AFM)
• AFM Image of Pentacene (C22H14)
• AFM Image of Hexabenzocoronene
• Basics of AFM Imaging The Probe
• Basics of AFM Imaging System Components
• van der Waals Potential Energy Curve
• Contact Mode ndash Monitor Deflection
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Peak Force Tapping Mode ndash Monitor Force
• AFM Applications in Bone Research Imaging
• AFM Imaging of Collagen
• Imaging of Osteoclast Activity
• Mineralized collagen deposition in vitro
• Imaging of Canilicular Structure
• AFM Imaging in Bone
• AFM Imaging in Bone
• Removing Mineral in Bone to Expose Collagen
• Removing Mineral in Bone to Expose Collagen
• Demineralization with EDTA
• Demineralization with EDTA
• Demineralization with EDTA
• Issues to Consider Effects of Acid
• Slide Number 32
• Slide Number 33
• Analysis of Collagen Structure in Bone
• Analysis of Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril D-Spacing
• Slide Number 39
• Collagen in Bone Fibril D-Spacing
• Collagen in Bone Fibril D-Spacing
• 2D Fast Fourier Transform Analysis
• 2D Fast Fourier Transform Analysis
• D-Spacing Distribution
• D-Spacing Distribution
• Cumulative Distribution Function
• CDF vs Histogram
• Changes in D-Spacing with Disease
• Changes in D-Spacing with Disease
• Slide Number 50
• Issues to Consider Site Selection and Sampling
• Issues to Consider Site Selection and Sampling
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• AFM calibration ndash Take Away Message
• AFM Applications in Bone ResearchIndentation Mechanics
• AFM-Based Indentation
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Parameters
• Hertz Contact Equation
• Hertz Contact Equation
• Hertz Contact Equation
• Proper Indentation Model is Essential
• Indentation in OI Tendon Study
• OI Tendon Differential Effects of Dehydration
• OI Tendon Differential Effects of Dehydration
• Indentation in Diabetic Tendon (Poster 0495)
• Multiscale Diabetes Study (Poster 0495)
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• A Note on Peak Force QNM for Bruker AFMs
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• Slide Number 83
• Slide Number 84
• AFM Applications in Bone ResearchOther Cool Mechanical Techniques
• ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo
• Other Mechanical Techniques
• Other Cool Techniques
• Summary Things to rememberhellip
• Acknowledgements
• Bone Biology and Mechanics Lab (BBML)

Issues to Consider Effects of Hydration

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT WetBrtl Wet

KS Test plt0001

Brtl+ distribution shifted downward vs WT when wet

0

10

20

30

40

50

60

70

80

90

100

62 63 64 65 66 67 68 69 70 71 72 73 74

Cum

ulat

ive

Gro

up T

otal

()

D-Periodic Spacing (nm)

WT DryBrtl Dry

KS Test plt0001

Brtl+ distribution shifted upward vs WT when dry

Kemp et al J StructBio 2012 180 (3) 428-438 BBML - All R

ights Reserve

d

Issues to Consider AFM calibration

bull Absolute distance measurements require accurate piezo calibration

bull AFM systems have large scan ranges (150 microm to lt 1 microm)

bull Scanner non-linearities can introduce substantial error over that range

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

ights Reserve

d

Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

BBML - All R

ights Reserve

d

Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

100 nm 10 80 602 1198 100 nm 7 60 827 1439 100 nm 5 40 814 1427 100 nm 35 20 917 1724 100 nm 2 10 937 1774

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

ights Reserve

d

AFM calibration ndash Take Away Message

Calibration using a scan size and feature size comparable to features of interest is highly recommended

Calibration addresses absolute accuracy not precision (differential sensitivity between measurements)

bull Does not limit ability to differentiate between groups using the same AFM with the same calibration parameters

BBML - All R

ights Reserve

d

AFM Applications in Bone Research Indentation Mechanics

BBML - All R

ights Reserve

d

AFM-Based Indentation

In addition to high resolution imaging AFM can be used to extract nanoscale mechanical data

ApproachWithdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Can

tilev

er D

efle

ctio

n (V

olts

)

1 Ramp into a rigid sample ndash measure the slope

Deflection sensitivity x 119899119899119899119899119881119881

2 Calculate cantilever spring constant

3 Hookersquos law takes deflection (volts) and converts to units of force F = kx

Spring constant k 119873119873119899119899

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Forc

e (n

N)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration Fo

rce

(nN

)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

Tip-Sample Separation (nm)

BBML - All R

ights Reserve

d

AFM-Based Indentation Parameters

Approach Withdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

Peak Force

Area Between Curves Energy Dissipation

Contact Mechanics Fit for Elastic Modulus (E)

Adhesion Force

Indentation Depth (δ)

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

z

r

δ

R

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119877119877 ∙ 12057512057532

Es Sample modulus νs Poissonrsquos ratio R tip radius of curvature δ load point displacement

Relevant Hertz Assumptions bull Strains are small and within the elastic limit bull Indenter is infinitely stiff (only sample deforms) bull Indentation depth ltlt radius of curvature of indenter bull Surfaces are continuous non-conforming (must be flat) bull Load is normal to surface

BBML - All R

ights Reserve

d

Proper Indentation Model is Essential

δ

r

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119903119903 ∙ 12057512057532

δ

α

119865119865 =2120587120587∙

1198641198641199041199041 minus 1205841205841199041199042

∙ tan120572120572 ∙ 1205751205752

Es Sample modulus νs Poissonrsquos ratio R tip radius α opening angle

Hertz Model Spherical Indenter

depth ltlt radius of curvature

Sneddon Model Conical Indenter

depth ge radius of curvature

BBML - All R

ights Reserve

d

Indentation in OI Tendon Study

x

z

yBrtl+ and WT mice 6 month old males

4 tails per group

2-3 fascicles per tail

25-30 fibrils per tail

4-5 locations per fibril

Indent force 20 nN wet (Sneddon) 50 nN dry (Hertz) BBML - A

ll Rights R

eserved

OI Tendon Differential Effects of Dehydration

Dry 1268 plusmn 718 MPa

Wet 305 plusmn 243 MPa

Dry 1737 plusmn 973 MPa

Wet 301 plusmn 202 MPa

Kemp et al J StructBio 2012 180 (3) 428-438

bull Significant stiffening of fibrils with dehydration

bull More pronounced modulus modulus as a function of disease

BBML - All R

ights Reserve

d

OI Tendon Differential Effects of Dehydration

Kemp et al J StructBio 2012 180 (3) 428-438

Diseased phenotype not present when wet phenotype observed when dried

0

10

20

30

40

50

60

0 20 40 60 80 100 120 140 160 180 200

Gro

up S

ampl

es (

)

Indent Modulus (MPa)

WT WetBrtl Wet

0

2

4

6

8

10

12

14

16

18

20

025

050

075

010

0012

5015

0017

5020

0022

5025

0027

5030

0032

5035

0037

5040

0042

5045

0047

5050

00

Gro

up S

ampl

es (

)Indent Modulus (MPa)

WT DryBrtl Dry

555 WT Indents 305 plusmn 243 MPa

614 Brtl+ Indents 301 plusmn 202 MPa

KS Test p=0129 t Test p=0464 464 WT Indents

1268 plusmn 718 MPa 479 Brtl+ Indents 1737 plusmn 973 MPa

KS Test plt0001 T Test plt0001

BBML - All R

ights Reserve

d

Indentation in Diabetic Tendon (Poster 0495)

x

z

y

Control and Diabetic Rat Tail Tendon

4-5 rats per group

2-3 fascicles per rat

~70 fibrilstail

4-5 locationsfibril

20 nN (Sneddon)

BBML - All R

ights Reserve

d

Multiscale Diabetes Study (Poster 0495)

0

5

10

15

20

25

300 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 gt25

Perc

enta

ge o

f Gro

up S

ampl

es

Elastic Modulus (MPa)

CD (1530 indents)ZDSD (913 indents)

CD (n=5) 381 plusmn 149 MPa

ZDSD (n=3) 706 plusmn 263 MPa

0

100

200

300

400

500

600

700

800

900

0 2 4 6 8 10

Mic

rosc

ale

Mod

ulus

(MPa

)

Nanoscale Modulus (MPa)

plt0001

r2=0531 p=0040

bull Increased indentation modulus in diabetic tendon

bull Strong relationship between nanoscale indent modulus and microscale tensile modulus

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

Proper probe selection for intended application bull Cantilever must be stiff relative to sample bull Sharp probes may damage sample easily dull

R or α must be determined for each probe bull Properties provided by manufacturer are unreliable bull Vary from probe to probe can dull during indent process

Spring constant must be determined for each probe

bull Value provided by manufacturer is inaccurate bull Thermal tuning OK for ldquosoftrdquo probes poor performance

for stiffer probes bull Sader method (geometry) added mass reference

cantilevers

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

Veeco Application Technical note Ben Ohler Practical Advice on the Determination of Cantilever Spring Constants

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

bull Sample processing

bull Sample hydration

bull Proper force range

bull Substrate effects for thin samples

bull Proper modulus model and range of data goodness of fit

Bearing in mind these assumptions and limitations AFM can provide true ldquonanoscalerdquo mechanical properties both in terms of forces and deformations

BBML - All R

ights Reserve

d

A Note on Peak Force QNM for Bruker AFMs bull Mechanical properties mapped pixel-by-pixel at same

resolution as the height image (PeakForce QNM)

Height Modulus BBML - All R

ights Reserve

d

A Note on Peak Force QNM

Correct probe stiffness must be used based on sample Pittenger ndash Veeco Product Bulletin

BBML - All R

ights Reserve

d

A Note on Peak Force QNM Extremely sensitive to calibration (details rarely given) Absolute radius and spring constant known

bull especially important from probe to probe bull radius uarr with indent depth ndash must determine R at depth

you expect to image bull Depth may vary as a function of sample (or at different

regions within the sample)

Relative reference material of ldquoknownrdquo modulus used to determine a ratio of 119896119896 119877119877 bull ldquoknownrdquo modulus may come from tension compression

and not indentation bull R is related to depth indentation depth on sample must

be same as on reference BBML - All R

ights Reserve

d

A Note on Peak Force QNM bull Modulus fit on the fly ndash no ability to monitor fit (or range) bull Local sample topography and sample tilt play a role in

measured parameters bull Assuming all modulus values in an image are correct how to

quantify

512 x 512 pixels = 262144 measurements

n=1 n=262144

n from selected ROIs

BBML - All R

ights Reserve

d

BBML - All R

ights Reserve

d

bull Imaging in air on mica ndash substrate effect

bull Same imaging force or indent depth for all

bull For each area were all modulus values pooled

Lamprou et al PLoS ONE 2013 BBML - All R

ights Reserve

d

AFM Applications in Bone Research Other Cool Mechanical Techniques

BBML - All R

ights Reserve

d

ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo Bonds within or between collagen fibrils in bone that rupture to dissipate energy as a toughening mechanism

Thompson et al Nature 2001

Fantner et al Nature Mat 2005

Hansma et al JMNI 2005

Fantner et al Nano Letters 2007 BBML - All R

ights Reserve

d

Other Mechanical Techniques

Jimenez-Palomar J Mech Beh Bio Mat 2012

van der Rijt Macro Bio 2004

Yang J Biomed Mater Res 2007

Yang Biophysical Journal 2008 BBML - All R

ights Reserve

d

Other Cool Techniques bull Piezoresponse Force Microscopy (PFM)

bull Electrochemical AFM (EC-AFM)

bull Force Modulation Microscopy (FMM)

bull Lateral Force Microscopy (LFM)

bull Magnetic Force Microscopy (MFM)

bull Conductive AFM (C-AFM)

bull Force Pulling

bull Cantilever Bending

bull Tensile testing of individual fibrils

bull 3 point bending of fibrils

BBML - All R

ights Reserve

d

Summary Things to rememberhellip

bull Imaging mode imaging medium and probe selection

bull Morphology Measurements bull Sample preparation and hydration state bull Measuring fibril diameter bull Site selection and proper sampling bull Appropriate system calibration

bull Mechanical Measurements

bull Probe selection (stiffness and tip radius) bull Probe calibration (radius angle spring constant) bull Indentation force range and mechanical model bull Hydration in biological samples bull PeakForce QNM

BBML - All R

ights Reserve

d

Acknowledgements BBML

Max Hammond Armando Diaz Gonzalez Zachary Bart Silvia Canelon Creasy Clauser

IU School of Medicine Dr David Burr Dr Matt Allen Dr Stuart Warden Dr Lilian Plotkin

Funding Sources NIHNIDCR 1F32DE018840-01 A1 IUPUI BME Departmental Startup Funds IUPUI Research Support Funds Grant IUPUI Biomechanics and Biomaterials Research Center

NIH NICHD Dr Joan Marini

BBML - All R

ights Reserve

d

Bone Biology and Mechanics Lab (BBML) wwwiupuiedu~bbml Facebook BoneBiologyMechanicsLab

BBML - All R

ights Reserve

d

• Slide Number 1
• General Seminar Outline
• Bonersquos Hierarchical Structure
• Critical Gap in Understanding Collagen
• Methods For Analyzing Collagen Structure
• Methods For Imaging Collagen Structure
• Cryo-TEM
• Atomic Force Microscopy (AFM)
• AFM Image of Pentacene (C22H14)
• AFM Image of Hexabenzocoronene
• Basics of AFM Imaging The Probe
• Basics of AFM Imaging System Components
• van der Waals Potential Energy Curve
• Contact Mode ndash Monitor Deflection
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Peak Force Tapping Mode ndash Monitor Force
• AFM Applications in Bone Research Imaging
• AFM Imaging of Collagen
• Imaging of Osteoclast Activity
• Mineralized collagen deposition in vitro
• Imaging of Canilicular Structure
• AFM Imaging in Bone
• AFM Imaging in Bone
• Removing Mineral in Bone to Expose Collagen
• Removing Mineral in Bone to Expose Collagen
• Demineralization with EDTA
• Demineralization with EDTA
• Demineralization with EDTA
• Issues to Consider Effects of Acid
• Slide Number 32
• Slide Number 33
• Analysis of Collagen Structure in Bone
• Analysis of Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril D-Spacing
• Slide Number 39
• Collagen in Bone Fibril D-Spacing
• Collagen in Bone Fibril D-Spacing
• 2D Fast Fourier Transform Analysis
• 2D Fast Fourier Transform Analysis
• D-Spacing Distribution
• D-Spacing Distribution
• Cumulative Distribution Function
• CDF vs Histogram
• Changes in D-Spacing with Disease
• Changes in D-Spacing with Disease
• Slide Number 50
• Issues to Consider Site Selection and Sampling
• Issues to Consider Site Selection and Sampling
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• AFM calibration ndash Take Away Message
• AFM Applications in Bone ResearchIndentation Mechanics
• AFM-Based Indentation
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Parameters
• Hertz Contact Equation
• Hertz Contact Equation
• Hertz Contact Equation
• Proper Indentation Model is Essential
• Indentation in OI Tendon Study
• OI Tendon Differential Effects of Dehydration
• OI Tendon Differential Effects of Dehydration
• Indentation in Diabetic Tendon (Poster 0495)
• Multiscale Diabetes Study (Poster 0495)
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• A Note on Peak Force QNM for Bruker AFMs
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• Slide Number 83
• Slide Number 84
• AFM Applications in Bone ResearchOther Cool Mechanical Techniques
• ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo
• Other Mechanical Techniques
• Other Cool Techniques
• Summary Things to rememberhellip
• Acknowledgements
• Bone Biology and Mechanics Lab (BBML)

Issues to Consider AFM calibration

bull Absolute distance measurements require accurate piezo calibration

bull AFM systems have large scan ranges (150 microm to lt 1 microm)

bull Scanner non-linearities can introduce substantial error over that range

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

ights Reserve

d

Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

BBML - All R

ights Reserve

d

Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

100 nm 10 80 602 1198 100 nm 7 60 827 1439 100 nm 5 40 814 1427 100 nm 35 20 917 1724 100 nm 2 10 937 1774

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

ights Reserve

d

AFM calibration ndash Take Away Message

Calibration using a scan size and feature size comparable to features of interest is highly recommended

Calibration addresses absolute accuracy not precision (differential sensitivity between measurements)

bull Does not limit ability to differentiate between groups using the same AFM with the same calibration parameters

BBML - All R

ights Reserve

d

AFM Applications in Bone Research Indentation Mechanics

BBML - All R

ights Reserve

d

AFM-Based Indentation

In addition to high resolution imaging AFM can be used to extract nanoscale mechanical data

ApproachWithdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Can

tilev

er D

efle

ctio

n (V

olts

)

1 Ramp into a rigid sample ndash measure the slope

Deflection sensitivity x 119899119899119899119899119881119881

2 Calculate cantilever spring constant

3 Hookersquos law takes deflection (volts) and converts to units of force F = kx

Spring constant k 119873119873119899119899

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Forc

e (n

N)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration Fo

rce

(nN

)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

Tip-Sample Separation (nm)

BBML - All R

ights Reserve

d

AFM-Based Indentation Parameters

Approach Withdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

Peak Force

Area Between Curves Energy Dissipation

Contact Mechanics Fit for Elastic Modulus (E)

Adhesion Force

Indentation Depth (δ)

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

z

r

δ

R

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119877119877 ∙ 12057512057532

Es Sample modulus νs Poissonrsquos ratio R tip radius of curvature δ load point displacement

Relevant Hertz Assumptions bull Strains are small and within the elastic limit bull Indenter is infinitely stiff (only sample deforms) bull Indentation depth ltlt radius of curvature of indenter bull Surfaces are continuous non-conforming (must be flat) bull Load is normal to surface

BBML - All R

ights Reserve

d

Proper Indentation Model is Essential

δ

r

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119903119903 ∙ 12057512057532

δ

α

119865119865 =2120587120587∙

1198641198641199041199041 minus 1205841205841199041199042

∙ tan120572120572 ∙ 1205751205752

Es Sample modulus νs Poissonrsquos ratio R tip radius α opening angle

Hertz Model Spherical Indenter

depth ltlt radius of curvature

Sneddon Model Conical Indenter

depth ge radius of curvature

BBML - All R

ights Reserve

d

Indentation in OI Tendon Study

x

z

yBrtl+ and WT mice 6 month old males

4 tails per group

2-3 fascicles per tail

25-30 fibrils per tail

4-5 locations per fibril

Indent force 20 nN wet (Sneddon) 50 nN dry (Hertz) BBML - A

ll Rights R

eserved

OI Tendon Differential Effects of Dehydration

Dry 1268 plusmn 718 MPa

Wet 305 plusmn 243 MPa

Dry 1737 plusmn 973 MPa

Wet 301 plusmn 202 MPa

Kemp et al J StructBio 2012 180 (3) 428-438

bull Significant stiffening of fibrils with dehydration

bull More pronounced modulus modulus as a function of disease

BBML - All R

ights Reserve

d

OI Tendon Differential Effects of Dehydration

Kemp et al J StructBio 2012 180 (3) 428-438

Diseased phenotype not present when wet phenotype observed when dried

0

10

20

30

40

50

60

0 20 40 60 80 100 120 140 160 180 200

Gro

up S

ampl

es (

)

Indent Modulus (MPa)

WT WetBrtl Wet

0

2

4

6

8

10

12

14

16

18

20

025

050

075

010

0012

5015

0017

5020

0022

5025

0027

5030

0032

5035

0037

5040

0042

5045

0047

5050

00

Gro

up S

ampl

es (

)Indent Modulus (MPa)

WT DryBrtl Dry

555 WT Indents 305 plusmn 243 MPa

614 Brtl+ Indents 301 plusmn 202 MPa

KS Test p=0129 t Test p=0464 464 WT Indents

1268 plusmn 718 MPa 479 Brtl+ Indents 1737 plusmn 973 MPa

KS Test plt0001 T Test plt0001

BBML - All R

ights Reserve

d

Indentation in Diabetic Tendon (Poster 0495)

x

z

y

Control and Diabetic Rat Tail Tendon

4-5 rats per group

2-3 fascicles per rat

~70 fibrilstail

4-5 locationsfibril

20 nN (Sneddon)

BBML - All R

ights Reserve

d

Multiscale Diabetes Study (Poster 0495)

0

5

10

15

20

25

300 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 gt25

Perc

enta

ge o

f Gro

up S

ampl

es

Elastic Modulus (MPa)

CD (1530 indents)ZDSD (913 indents)

CD (n=5) 381 plusmn 149 MPa

ZDSD (n=3) 706 plusmn 263 MPa

0

100

200

300

400

500

600

700

800

900

0 2 4 6 8 10

Mic

rosc

ale

Mod

ulus

(MPa

)

Nanoscale Modulus (MPa)

plt0001

r2=0531 p=0040

bull Increased indentation modulus in diabetic tendon

bull Strong relationship between nanoscale indent modulus and microscale tensile modulus

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

Proper probe selection for intended application bull Cantilever must be stiff relative to sample bull Sharp probes may damage sample easily dull

R or α must be determined for each probe bull Properties provided by manufacturer are unreliable bull Vary from probe to probe can dull during indent process

Spring constant must be determined for each probe

bull Value provided by manufacturer is inaccurate bull Thermal tuning OK for ldquosoftrdquo probes poor performance

for stiffer probes bull Sader method (geometry) added mass reference

cantilevers

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

Veeco Application Technical note Ben Ohler Practical Advice on the Determination of Cantilever Spring Constants

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

bull Sample processing

bull Sample hydration

bull Proper force range

bull Substrate effects for thin samples

bull Proper modulus model and range of data goodness of fit

Bearing in mind these assumptions and limitations AFM can provide true ldquonanoscalerdquo mechanical properties both in terms of forces and deformations

BBML - All R

ights Reserve

d

A Note on Peak Force QNM for Bruker AFMs bull Mechanical properties mapped pixel-by-pixel at same

resolution as the height image (PeakForce QNM)

Height Modulus BBML - All R

ights Reserve

d

A Note on Peak Force QNM

Correct probe stiffness must be used based on sample Pittenger ndash Veeco Product Bulletin

BBML - All R

ights Reserve

d

A Note on Peak Force QNM Extremely sensitive to calibration (details rarely given) Absolute radius and spring constant known

bull especially important from probe to probe bull radius uarr with indent depth ndash must determine R at depth

you expect to image bull Depth may vary as a function of sample (or at different

regions within the sample)

Relative reference material of ldquoknownrdquo modulus used to determine a ratio of 119896119896 119877119877 bull ldquoknownrdquo modulus may come from tension compression

and not indentation bull R is related to depth indentation depth on sample must

be same as on reference BBML - All R

ights Reserve

d

A Note on Peak Force QNM bull Modulus fit on the fly ndash no ability to monitor fit (or range) bull Local sample topography and sample tilt play a role in

measured parameters bull Assuming all modulus values in an image are correct how to

quantify

512 x 512 pixels = 262144 measurements

n=1 n=262144

n from selected ROIs

BBML - All R

ights Reserve

d

BBML - All R

ights Reserve

d

bull Imaging in air on mica ndash substrate effect

bull Same imaging force or indent depth for all

bull For each area were all modulus values pooled

Lamprou et al PLoS ONE 2013 BBML - All R

ights Reserve

d

AFM Applications in Bone Research Other Cool Mechanical Techniques

BBML - All R

ights Reserve

d

ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo Bonds within or between collagen fibrils in bone that rupture to dissipate energy as a toughening mechanism

Thompson et al Nature 2001

Fantner et al Nature Mat 2005

Hansma et al JMNI 2005

Fantner et al Nano Letters 2007 BBML - All R

ights Reserve

d

Other Mechanical Techniques

Jimenez-Palomar J Mech Beh Bio Mat 2012

van der Rijt Macro Bio 2004

Yang J Biomed Mater Res 2007

Yang Biophysical Journal 2008 BBML - All R

ights Reserve

d

Other Cool Techniques bull Piezoresponse Force Microscopy (PFM)

bull Electrochemical AFM (EC-AFM)

bull Force Modulation Microscopy (FMM)

bull Lateral Force Microscopy (LFM)

bull Magnetic Force Microscopy (MFM)

bull Conductive AFM (C-AFM)

bull Force Pulling

bull Cantilever Bending

bull Tensile testing of individual fibrils

bull 3 point bending of fibrils

BBML - All R

ights Reserve

d

Summary Things to rememberhellip

bull Imaging mode imaging medium and probe selection

bull Morphology Measurements bull Sample preparation and hydration state bull Measuring fibril diameter bull Site selection and proper sampling bull Appropriate system calibration

bull Mechanical Measurements

bull Probe selection (stiffness and tip radius) bull Probe calibration (radius angle spring constant) bull Indentation force range and mechanical model bull Hydration in biological samples bull PeakForce QNM

BBML - All R

ights Reserve

d

Acknowledgements BBML

Max Hammond Armando Diaz Gonzalez Zachary Bart Silvia Canelon Creasy Clauser

IU School of Medicine Dr David Burr Dr Matt Allen Dr Stuart Warden Dr Lilian Plotkin

Funding Sources NIHNIDCR 1F32DE018840-01 A1 IUPUI BME Departmental Startup Funds IUPUI Research Support Funds Grant IUPUI Biomechanics and Biomaterials Research Center

NIH NICHD Dr Joan Marini

BBML - All R

ights Reserve

d

Bone Biology and Mechanics Lab (BBML) wwwiupuiedu~bbml Facebook BoneBiologyMechanicsLab

BBML - All R

ights Reserve

d

• Slide Number 1
• General Seminar Outline
• Bonersquos Hierarchical Structure
• Critical Gap in Understanding Collagen
• Methods For Analyzing Collagen Structure
• Methods For Imaging Collagen Structure
• Cryo-TEM
• Atomic Force Microscopy (AFM)
• AFM Image of Pentacene (C22H14)
• AFM Image of Hexabenzocoronene
• Basics of AFM Imaging The Probe
• Basics of AFM Imaging System Components
• van der Waals Potential Energy Curve
• Contact Mode ndash Monitor Deflection
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Peak Force Tapping Mode ndash Monitor Force
• AFM Applications in Bone Research Imaging
• AFM Imaging of Collagen
• Imaging of Osteoclast Activity
• Mineralized collagen deposition in vitro
• Imaging of Canilicular Structure
• AFM Imaging in Bone
• AFM Imaging in Bone
• Removing Mineral in Bone to Expose Collagen
• Removing Mineral in Bone to Expose Collagen
• Demineralization with EDTA
• Demineralization with EDTA
• Demineralization with EDTA
• Issues to Consider Effects of Acid
• Slide Number 32
• Slide Number 33
• Analysis of Collagen Structure in Bone
• Analysis of Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril D-Spacing
• Slide Number 39
• Collagen in Bone Fibril D-Spacing
• Collagen in Bone Fibril D-Spacing
• 2D Fast Fourier Transform Analysis
• 2D Fast Fourier Transform Analysis
• D-Spacing Distribution
• D-Spacing Distribution
• Cumulative Distribution Function
• CDF vs Histogram
• Changes in D-Spacing with Disease
• Changes in D-Spacing with Disease
• Slide Number 50
• Issues to Consider Site Selection and Sampling
• Issues to Consider Site Selection and Sampling
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• AFM calibration ndash Take Away Message
• AFM Applications in Bone ResearchIndentation Mechanics
• AFM-Based Indentation
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Parameters
• Hertz Contact Equation
• Hertz Contact Equation
• Hertz Contact Equation
• Proper Indentation Model is Essential
• Indentation in OI Tendon Study
• OI Tendon Differential Effects of Dehydration
• OI Tendon Differential Effects of Dehydration
• Indentation in Diabetic Tendon (Poster 0495)
• Multiscale Diabetes Study (Poster 0495)
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• A Note on Peak Force QNM for Bruker AFMs
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• Slide Number 83
• Slide Number 84
• AFM Applications in Bone ResearchOther Cool Mechanical Techniques
• ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo
• Other Mechanical Techniques
• Other Cool Techniques
• Summary Things to rememberhellip
• Acknowledgements
• Bone Biology and Mechanics Lab (BBML)

Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

BBML - All R

ights Reserve

d

Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

100 nm 10 80 602 1198 100 nm 7 60 827 1439 100 nm 5 40 814 1427 100 nm 35 20 917 1724 100 nm 2 10 937 1774

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

ights Reserve

d

AFM calibration ndash Take Away Message

Calibration using a scan size and feature size comparable to features of interest is highly recommended

Calibration addresses absolute accuracy not precision (differential sensitivity between measurements)

bull Does not limit ability to differentiate between groups using the same AFM with the same calibration parameters

BBML - All R

ights Reserve

d

AFM Applications in Bone Research Indentation Mechanics

BBML - All R

ights Reserve

d

AFM-Based Indentation

In addition to high resolution imaging AFM can be used to extract nanoscale mechanical data

ApproachWithdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Can

tilev

er D

efle

ctio

n (V

olts

)

1 Ramp into a rigid sample ndash measure the slope

Deflection sensitivity x 119899119899119899119899119881119881

2 Calculate cantilever spring constant

3 Hookersquos law takes deflection (volts) and converts to units of force F = kx

Spring constant k 119873119873119899119899

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Forc

e (n

N)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration Fo

rce

(nN

)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

Tip-Sample Separation (nm)

BBML - All R

ights Reserve

d

AFM-Based Indentation Parameters

Approach Withdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

Peak Force

Area Between Curves Energy Dissipation

Contact Mechanics Fit for Elastic Modulus (E)

Adhesion Force

Indentation Depth (δ)

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

z

r

δ

R

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119877119877 ∙ 12057512057532

Es Sample modulus νs Poissonrsquos ratio R tip radius of curvature δ load point displacement

Relevant Hertz Assumptions bull Strains are small and within the elastic limit bull Indenter is infinitely stiff (only sample deforms) bull Indentation depth ltlt radius of curvature of indenter bull Surfaces are continuous non-conforming (must be flat) bull Load is normal to surface

BBML - All R

ights Reserve

d

Proper Indentation Model is Essential

δ

r

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119903119903 ∙ 12057512057532

δ

α

119865119865 =2120587120587∙

1198641198641199041199041 minus 1205841205841199041199042

∙ tan120572120572 ∙ 1205751205752

Es Sample modulus νs Poissonrsquos ratio R tip radius α opening angle

Hertz Model Spherical Indenter

depth ltlt radius of curvature

Sneddon Model Conical Indenter

depth ge radius of curvature

BBML - All R

ights Reserve

d

Indentation in OI Tendon Study

x

z

yBrtl+ and WT mice 6 month old males

4 tails per group

2-3 fascicles per tail

25-30 fibrils per tail

4-5 locations per fibril

Indent force 20 nN wet (Sneddon) 50 nN dry (Hertz) BBML - A

ll Rights R

eserved

OI Tendon Differential Effects of Dehydration

Dry 1268 plusmn 718 MPa

Wet 305 plusmn 243 MPa

Dry 1737 plusmn 973 MPa

Wet 301 plusmn 202 MPa

Kemp et al J StructBio 2012 180 (3) 428-438

bull Significant stiffening of fibrils with dehydration

bull More pronounced modulus modulus as a function of disease

BBML - All R

ights Reserve

d

OI Tendon Differential Effects of Dehydration

Kemp et al J StructBio 2012 180 (3) 428-438

Diseased phenotype not present when wet phenotype observed when dried

0

10

20

30

40

50

60

0 20 40 60 80 100 120 140 160 180 200

Gro

up S

ampl

es (

)

Indent Modulus (MPa)

WT WetBrtl Wet

0

2

4

6

8

10

12

14

16

18

20

025

050

075

010

0012

5015

0017

5020

0022

5025

0027

5030

0032

5035

0037

5040

0042

5045

0047

5050

00

Gro

up S

ampl

es (

)Indent Modulus (MPa)

WT DryBrtl Dry

555 WT Indents 305 plusmn 243 MPa

614 Brtl+ Indents 301 plusmn 202 MPa

KS Test p=0129 t Test p=0464 464 WT Indents

1268 plusmn 718 MPa 479 Brtl+ Indents 1737 plusmn 973 MPa

KS Test plt0001 T Test plt0001

BBML - All R

ights Reserve

d

Indentation in Diabetic Tendon (Poster 0495)

x

z

y

Control and Diabetic Rat Tail Tendon

4-5 rats per group

2-3 fascicles per rat

~70 fibrilstail

4-5 locationsfibril

20 nN (Sneddon)

BBML - All R

ights Reserve

d

Multiscale Diabetes Study (Poster 0495)

0

5

10

15

20

25

300 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 gt25

Perc

enta

ge o

f Gro

up S

ampl

es

Elastic Modulus (MPa)

CD (1530 indents)ZDSD (913 indents)

CD (n=5) 381 plusmn 149 MPa

ZDSD (n=3) 706 plusmn 263 MPa

0

100

200

300

400

500

600

700

800

900

0 2 4 6 8 10

Mic

rosc

ale

Mod

ulus

(MPa

)

Nanoscale Modulus (MPa)

plt0001

r2=0531 p=0040

bull Increased indentation modulus in diabetic tendon

bull Strong relationship between nanoscale indent modulus and microscale tensile modulus

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

Proper probe selection for intended application bull Cantilever must be stiff relative to sample bull Sharp probes may damage sample easily dull

R or α must be determined for each probe bull Properties provided by manufacturer are unreliable bull Vary from probe to probe can dull during indent process

Spring constant must be determined for each probe

bull Value provided by manufacturer is inaccurate bull Thermal tuning OK for ldquosoftrdquo probes poor performance

for stiffer probes bull Sader method (geometry) added mass reference

cantilevers

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

Veeco Application Technical note Ben Ohler Practical Advice on the Determination of Cantilever Spring Constants

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

bull Sample processing

bull Sample hydration

bull Proper force range

bull Substrate effects for thin samples

bull Proper modulus model and range of data goodness of fit

Bearing in mind these assumptions and limitations AFM can provide true ldquonanoscalerdquo mechanical properties both in terms of forces and deformations

BBML - All R

ights Reserve

d

A Note on Peak Force QNM for Bruker AFMs bull Mechanical properties mapped pixel-by-pixel at same

resolution as the height image (PeakForce QNM)

Height Modulus BBML - All R

ights Reserve

d

A Note on Peak Force QNM

Correct probe stiffness must be used based on sample Pittenger ndash Veeco Product Bulletin

BBML - All R

ights Reserve

d

A Note on Peak Force QNM Extremely sensitive to calibration (details rarely given) Absolute radius and spring constant known

bull especially important from probe to probe bull radius uarr with indent depth ndash must determine R at depth

you expect to image bull Depth may vary as a function of sample (or at different

regions within the sample)

Relative reference material of ldquoknownrdquo modulus used to determine a ratio of 119896119896 119877119877 bull ldquoknownrdquo modulus may come from tension compression

and not indentation bull R is related to depth indentation depth on sample must

be same as on reference BBML - All R

ights Reserve

d

A Note on Peak Force QNM bull Modulus fit on the fly ndash no ability to monitor fit (or range) bull Local sample topography and sample tilt play a role in

measured parameters bull Assuming all modulus values in an image are correct how to

quantify

512 x 512 pixels = 262144 measurements

n=1 n=262144

n from selected ROIs

BBML - All R

ights Reserve

d

BBML - All R

ights Reserve

d

bull Imaging in air on mica ndash substrate effect

bull Same imaging force or indent depth for all

bull For each area were all modulus values pooled

Lamprou et al PLoS ONE 2013 BBML - All R

ights Reserve

d

AFM Applications in Bone Research Other Cool Mechanical Techniques

BBML - All R

ights Reserve

d

ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo Bonds within or between collagen fibrils in bone that rupture to dissipate energy as a toughening mechanism

Thompson et al Nature 2001

Fantner et al Nature Mat 2005

Hansma et al JMNI 2005

Fantner et al Nano Letters 2007 BBML - All R

ights Reserve

d

Other Mechanical Techniques

Jimenez-Palomar J Mech Beh Bio Mat 2012

van der Rijt Macro Bio 2004

Yang J Biomed Mater Res 2007

Yang Biophysical Journal 2008 BBML - All R

ights Reserve

d

Other Cool Techniques bull Piezoresponse Force Microscopy (PFM)

bull Electrochemical AFM (EC-AFM)

bull Force Modulation Microscopy (FMM)

bull Lateral Force Microscopy (LFM)

bull Magnetic Force Microscopy (MFM)

bull Conductive AFM (C-AFM)

bull Force Pulling

bull Cantilever Bending

bull Tensile testing of individual fibrils

bull 3 point bending of fibrils

BBML - All R

ights Reserve

d

Summary Things to rememberhellip

bull Imaging mode imaging medium and probe selection

bull Morphology Measurements bull Sample preparation and hydration state bull Measuring fibril diameter bull Site selection and proper sampling bull Appropriate system calibration

bull Mechanical Measurements

bull Probe selection (stiffness and tip radius) bull Probe calibration (radius angle spring constant) bull Indentation force range and mechanical model bull Hydration in biological samples bull PeakForce QNM

BBML - All R

ights Reserve

d

Acknowledgements BBML

Max Hammond Armando Diaz Gonzalez Zachary Bart Silvia Canelon Creasy Clauser

IU School of Medicine Dr David Burr Dr Matt Allen Dr Stuart Warden Dr Lilian Plotkin

Funding Sources NIHNIDCR 1F32DE018840-01 A1 IUPUI BME Departmental Startup Funds IUPUI Research Support Funds Grant IUPUI Biomechanics and Biomaterials Research Center

NIH NICHD Dr Joan Marini

BBML - All R

ights Reserve

d

Bone Biology and Mechanics Lab (BBML) wwwiupuiedu~bbml Facebook BoneBiologyMechanicsLab

BBML - All R

ights Reserve

d

• Slide Number 1
• General Seminar Outline
• Bonersquos Hierarchical Structure
• Critical Gap in Understanding Collagen
• Methods For Analyzing Collagen Structure
• Methods For Imaging Collagen Structure
• Cryo-TEM
• Atomic Force Microscopy (AFM)
• AFM Image of Pentacene (C22H14)
• AFM Image of Hexabenzocoronene
• Basics of AFM Imaging The Probe
• Basics of AFM Imaging System Components
• van der Waals Potential Energy Curve
• Contact Mode ndash Monitor Deflection
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Peak Force Tapping Mode ndash Monitor Force
• AFM Applications in Bone Research Imaging
• AFM Imaging of Collagen
• Imaging of Osteoclast Activity
• Mineralized collagen deposition in vitro
• Imaging of Canilicular Structure
• AFM Imaging in Bone
• AFM Imaging in Bone
• Removing Mineral in Bone to Expose Collagen
• Removing Mineral in Bone to Expose Collagen
• Demineralization with EDTA
• Demineralization with EDTA
• Demineralization with EDTA
• Issues to Consider Effects of Acid
• Slide Number 32
• Slide Number 33
• Analysis of Collagen Structure in Bone
• Analysis of Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril D-Spacing
• Slide Number 39
• Collagen in Bone Fibril D-Spacing
• Collagen in Bone Fibril D-Spacing
• 2D Fast Fourier Transform Analysis
• 2D Fast Fourier Transform Analysis
• D-Spacing Distribution
• D-Spacing Distribution
• Cumulative Distribution Function
• CDF vs Histogram
• Changes in D-Spacing with Disease
• Changes in D-Spacing with Disease
• Slide Number 50
• Issues to Consider Site Selection and Sampling
• Issues to Consider Site Selection and Sampling
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• AFM calibration ndash Take Away Message
• AFM Applications in Bone ResearchIndentation Mechanics
• AFM-Based Indentation
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Parameters
• Hertz Contact Equation
• Hertz Contact Equation
• Hertz Contact Equation
• Proper Indentation Model is Essential
• Indentation in OI Tendon Study
• OI Tendon Differential Effects of Dehydration
• OI Tendon Differential Effects of Dehydration
• Indentation in Diabetic Tendon (Poster 0495)
• Multiscale Diabetes Study (Poster 0495)
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• A Note on Peak Force QNM for Bruker AFMs
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• Slide Number 83
• Slide Number 84
• AFM Applications in Bone ResearchOther Cool Mechanical Techniques
• ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo
• Other Mechanical Techniques
• Other Cool Techniques
• Summary Things to rememberhellip
• Acknowledgements
• Bone Biology and Mechanics Lab (BBML)

Issues to Consider AFM calibration Manufacturers suggest a 10 microm pitch standard at full range

150 times larger than D-spacing image at 23 of max range

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273

Erickson B Wallace JM et al Biotechnology Journal 2013

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

Standard Scan size (microm)

of pitches

Fast Scan Error ()

Slow Scan Error ()

10 microm 70 6 18 273 10 microm 50 4 301 539 10 microm 40 3 290 460 1 microm 35 28 532 796 1 microm 20 16 646 1132 1 microm 10 8 796 1459

100 nm 10 80 602 1198 100 nm 7 60 827 1439 100 nm 5 40 814 1427 100 nm 35 20 917 1724 100 nm 2 10 937 1774

Erickson B Wallace JM et al Biotechnology Journal 2013 BBML - All R

ights Reserve

d

AFM calibration ndash Take Away Message

Calibration using a scan size and feature size comparable to features of interest is highly recommended

Calibration addresses absolute accuracy not precision (differential sensitivity between measurements)

bull Does not limit ability to differentiate between groups using the same AFM with the same calibration parameters

BBML - All R

ights Reserve

d

AFM Applications in Bone Research Indentation Mechanics

BBML - All R

ights Reserve

d

AFM-Based Indentation

In addition to high resolution imaging AFM can be used to extract nanoscale mechanical data

ApproachWithdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Can

tilev

er D

efle

ctio

n (V

olts

)

1 Ramp into a rigid sample ndash measure the slope

Deflection sensitivity x 119899119899119899119899119881119881

2 Calculate cantilever spring constant

3 Hookersquos law takes deflection (volts) and converts to units of force F = kx

Spring constant k 119873119873119899119899

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Forc

e (n

N)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration Fo

rce

(nN

)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

Tip-Sample Separation (nm)

BBML - All R

ights Reserve

d

AFM-Based Indentation Parameters

Approach Withdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

Peak Force

Area Between Curves Energy Dissipation

Contact Mechanics Fit for Elastic Modulus (E)

Adhesion Force

Indentation Depth (δ)

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

z

r

δ

R

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119877119877 ∙ 12057512057532

Es Sample modulus νs Poissonrsquos ratio R tip radius of curvature δ load point displacement

Relevant Hertz Assumptions bull Strains are small and within the elastic limit bull Indenter is infinitely stiff (only sample deforms) bull Indentation depth ltlt radius of curvature of indenter bull Surfaces are continuous non-conforming (must be flat) bull Load is normal to surface

BBML - All R

ights Reserve

d

Proper Indentation Model is Essential

δ

r

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119903119903 ∙ 12057512057532

δ

α

119865119865 =2120587120587∙

1198641198641199041199041 minus 1205841205841199041199042

∙ tan120572120572 ∙ 1205751205752

Es Sample modulus νs Poissonrsquos ratio R tip radius α opening angle

Hertz Model Spherical Indenter

depth ltlt radius of curvature

Sneddon Model Conical Indenter

depth ge radius of curvature

BBML - All R

ights Reserve

d

Indentation in OI Tendon Study

x

z

yBrtl+ and WT mice 6 month old males

4 tails per group

2-3 fascicles per tail

25-30 fibrils per tail

4-5 locations per fibril

Indent force 20 nN wet (Sneddon) 50 nN dry (Hertz) BBML - A

ll Rights R

eserved

OI Tendon Differential Effects of Dehydration

Dry 1268 plusmn 718 MPa

Wet 305 plusmn 243 MPa

Dry 1737 plusmn 973 MPa

Wet 301 plusmn 202 MPa

Kemp et al J StructBio 2012 180 (3) 428-438

bull Significant stiffening of fibrils with dehydration

bull More pronounced modulus modulus as a function of disease

BBML - All R

ights Reserve

d

OI Tendon Differential Effects of Dehydration

Kemp et al J StructBio 2012 180 (3) 428-438

Diseased phenotype not present when wet phenotype observed when dried

0

10

20

30

40

50

60

0 20 40 60 80 100 120 140 160 180 200

Gro

up S

ampl

es (

)

Indent Modulus (MPa)

WT WetBrtl Wet

0

2

4

6

8

10

12

14

16

18

20

025

050

075

010

0012

5015

0017

5020

0022

5025

0027

5030

0032

5035

0037

5040

0042

5045

0047

5050

00

Gro

up S

ampl

es (

)Indent Modulus (MPa)

WT DryBrtl Dry

555 WT Indents 305 plusmn 243 MPa

614 Brtl+ Indents 301 plusmn 202 MPa

KS Test p=0129 t Test p=0464 464 WT Indents

1268 plusmn 718 MPa 479 Brtl+ Indents 1737 plusmn 973 MPa

KS Test plt0001 T Test plt0001

BBML - All R

ights Reserve

d

Indentation in Diabetic Tendon (Poster 0495)

x

z

y

Control and Diabetic Rat Tail Tendon

4-5 rats per group

2-3 fascicles per rat

~70 fibrilstail

4-5 locationsfibril

20 nN (Sneddon)

BBML - All R

ights Reserve

d

Multiscale Diabetes Study (Poster 0495)

0

5

10

15

20

25

300 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 gt25

Perc

enta

ge o

f Gro

up S

ampl

es

Elastic Modulus (MPa)

CD (1530 indents)ZDSD (913 indents)

CD (n=5) 381 plusmn 149 MPa

ZDSD (n=3) 706 plusmn 263 MPa

0

100

200

300

400

500

600

700

800

900

0 2 4 6 8 10

Mic

rosc

ale

Mod

ulus

(MPa

)

Nanoscale Modulus (MPa)

plt0001

r2=0531 p=0040

bull Increased indentation modulus in diabetic tendon

bull Strong relationship between nanoscale indent modulus and microscale tensile modulus

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

Proper probe selection for intended application bull Cantilever must be stiff relative to sample bull Sharp probes may damage sample easily dull

R or α must be determined for each probe bull Properties provided by manufacturer are unreliable bull Vary from probe to probe can dull during indent process

Spring constant must be determined for each probe

bull Value provided by manufacturer is inaccurate bull Thermal tuning OK for ldquosoftrdquo probes poor performance

for stiffer probes bull Sader method (geometry) added mass reference

cantilevers

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

Veeco Application Technical note Ben Ohler Practical Advice on the Determination of Cantilever Spring Constants

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

bull Sample processing

bull Sample hydration

bull Proper force range

bull Substrate effects for thin samples

bull Proper modulus model and range of data goodness of fit

Bearing in mind these assumptions and limitations AFM can provide true ldquonanoscalerdquo mechanical properties both in terms of forces and deformations

BBML - All R

ights Reserve

d

A Note on Peak Force QNM for Bruker AFMs bull Mechanical properties mapped pixel-by-pixel at same

resolution as the height image (PeakForce QNM)

Height Modulus BBML - All R

ights Reserve

d

A Note on Peak Force QNM

Correct probe stiffness must be used based on sample Pittenger ndash Veeco Product Bulletin

BBML - All R

ights Reserve

d

A Note on Peak Force QNM Extremely sensitive to calibration (details rarely given) Absolute radius and spring constant known

bull especially important from probe to probe bull radius uarr with indent depth ndash must determine R at depth

you expect to image bull Depth may vary as a function of sample (or at different

regions within the sample)

Relative reference material of ldquoknownrdquo modulus used to determine a ratio of 119896119896 119877119877 bull ldquoknownrdquo modulus may come from tension compression

and not indentation bull R is related to depth indentation depth on sample must

be same as on reference BBML - All R

ights Reserve

d

A Note on Peak Force QNM bull Modulus fit on the fly ndash no ability to monitor fit (or range) bull Local sample topography and sample tilt play a role in

measured parameters bull Assuming all modulus values in an image are correct how to

quantify

512 x 512 pixels = 262144 measurements

n=1 n=262144

n from selected ROIs

BBML - All R

ights Reserve

d

BBML - All R

ights Reserve

d

bull Imaging in air on mica ndash substrate effect

bull Same imaging force or indent depth for all

bull For each area were all modulus values pooled

Lamprou et al PLoS ONE 2013 BBML - All R

ights Reserve

d

AFM Applications in Bone Research Other Cool Mechanical Techniques

BBML - All R

ights Reserve

d

ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo Bonds within or between collagen fibrils in bone that rupture to dissipate energy as a toughening mechanism

Thompson et al Nature 2001

Fantner et al Nature Mat 2005

Hansma et al JMNI 2005

Fantner et al Nano Letters 2007 BBML - All R

ights Reserve

d

Other Mechanical Techniques

Jimenez-Palomar J Mech Beh Bio Mat 2012

van der Rijt Macro Bio 2004

Yang J Biomed Mater Res 2007

Yang Biophysical Journal 2008 BBML - All R

ights Reserve

d

Other Cool Techniques bull Piezoresponse Force Microscopy (PFM)

bull Electrochemical AFM (EC-AFM)

bull Force Modulation Microscopy (FMM)

bull Lateral Force Microscopy (LFM)

bull Magnetic Force Microscopy (MFM)

bull Conductive AFM (C-AFM)

bull Force Pulling

bull Cantilever Bending

bull Tensile testing of individual fibrils

bull 3 point bending of fibrils

BBML - All R

ights Reserve

d

Summary Things to rememberhellip

bull Imaging mode imaging medium and probe selection

bull Morphology Measurements bull Sample preparation and hydration state bull Measuring fibril diameter bull Site selection and proper sampling bull Appropriate system calibration

bull Mechanical Measurements

bull Probe selection (stiffness and tip radius) bull Probe calibration (radius angle spring constant) bull Indentation force range and mechanical model bull Hydration in biological samples bull PeakForce QNM

BBML - All R

ights Reserve

d

Acknowledgements BBML

Max Hammond Armando Diaz Gonzalez Zachary Bart Silvia Canelon Creasy Clauser

IU School of Medicine Dr David Burr Dr Matt Allen Dr Stuart Warden Dr Lilian Plotkin

Funding Sources NIHNIDCR 1F32DE018840-01 A1 IUPUI BME Departmental Startup Funds IUPUI Research Support Funds Grant IUPUI Biomechanics and Biomaterials Research Center

NIH NICHD Dr Joan Marini

BBML - All R

ights Reserve

d

Bone Biology and Mechanics Lab (BBML) wwwiupuiedu~bbml Facebook BoneBiologyMechanicsLab

BBML - All R

ights Reserve

d

• Slide Number 1
• General Seminar Outline
• Bonersquos Hierarchical Structure
• Critical Gap in Understanding Collagen
• Methods For Analyzing Collagen Structure
• Methods For Imaging Collagen Structure
• Cryo-TEM
• Atomic Force Microscopy (AFM)
• AFM Image of Pentacene (C22H14)
• AFM Image of Hexabenzocoronene
• Basics of AFM Imaging The Probe
• Basics of AFM Imaging System Components
• van der Waals Potential Energy Curve
• Contact Mode ndash Monitor Deflection
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Peak Force Tapping Mode ndash Monitor Force
• AFM Applications in Bone Research Imaging
• AFM Imaging of Collagen
• Imaging of Osteoclast Activity
• Mineralized collagen deposition in vitro
• Imaging of Canilicular Structure
• AFM Imaging in Bone
• AFM Imaging in Bone
• Removing Mineral in Bone to Expose Collagen
• Removing Mineral in Bone to Expose Collagen
• Demineralization with EDTA
• Demineralization with EDTA
• Demineralization with EDTA
• Issues to Consider Effects of Acid
• Slide Number 32
• Slide Number 33
• Analysis of Collagen Structure in Bone
• Analysis of Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril D-Spacing
• Slide Number 39
• Collagen in Bone Fibril D-Spacing
• Collagen in Bone Fibril D-Spacing
• 2D Fast Fourier Transform Analysis
• 2D Fast Fourier Transform Analysis
• D-Spacing Distribution
• D-Spacing Distribution
• Cumulative Distribution Function
• CDF vs Histogram
• Changes in D-Spacing with Disease
• Changes in D-Spacing with Disease
• Slide Number 50
• Issues to Consider Site Selection and Sampling
• Issues to Consider Site Selection and Sampling
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• AFM calibration ndash Take Away Message
• AFM Applications in Bone ResearchIndentation Mechanics
• AFM-Based Indentation
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Parameters
• Hertz Contact Equation
• Hertz Contact Equation
• Hertz Contact Equation
• Proper Indentation Model is Essential
• Indentation in OI Tendon Study
• OI Tendon Differential Effects of Dehydration
• OI Tendon Differential Effects of Dehydration
• Indentation in Diabetic Tendon (Poster 0495)
• Multiscale Diabetes Study (Poster 0495)
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• A Note on Peak Force QNM for Bruker AFMs
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• Slide Number 83
• Slide Number 84
• AFM Applications in Bone ResearchOther Cool Mechanical Techniques
• ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo
• Other Mechanical Techniques
• Other Cool Techniques
• Summary Things to rememberhellip
• Acknowledgements
• Bone Biology and Mechanics Lab (BBML)

AFM calibration ndash Take Away Message

Calibration using a scan size and feature size comparable to features of interest is highly recommended

Calibration addresses absolute accuracy not precision (differential sensitivity between measurements)

bull Does not limit ability to differentiate between groups using the same AFM with the same calibration parameters

BBML - All R

ights Reserve

d

AFM Applications in Bone Research Indentation Mechanics

BBML - All R

ights Reserve

d

AFM-Based Indentation

In addition to high resolution imaging AFM can be used to extract nanoscale mechanical data

ApproachWithdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Can

tilev

er D

efle

ctio

n (V

olts

)

1 Ramp into a rigid sample ndash measure the slope

Deflection sensitivity x 119899119899119899119899119881119881

2 Calculate cantilever spring constant

3 Hookersquos law takes deflection (volts) and converts to units of force F = kx

Spring constant k 119873119873119899119899

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Forc

e (n

N)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration Fo

rce

(nN

)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

Tip-Sample Separation (nm)

BBML - All R

ights Reserve

d

AFM-Based Indentation Parameters

Approach Withdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

Peak Force

Area Between Curves Energy Dissipation

Contact Mechanics Fit for Elastic Modulus (E)

Adhesion Force

Indentation Depth (δ)

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

z

r

δ

R

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119877119877 ∙ 12057512057532

Es Sample modulus νs Poissonrsquos ratio R tip radius of curvature δ load point displacement

Relevant Hertz Assumptions bull Strains are small and within the elastic limit bull Indenter is infinitely stiff (only sample deforms) bull Indentation depth ltlt radius of curvature of indenter bull Surfaces are continuous non-conforming (must be flat) bull Load is normal to surface

BBML - All R

ights Reserve

d

Proper Indentation Model is Essential

δ

r

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119903119903 ∙ 12057512057532

δ

α

119865119865 =2120587120587∙

1198641198641199041199041 minus 1205841205841199041199042

∙ tan120572120572 ∙ 1205751205752

Es Sample modulus νs Poissonrsquos ratio R tip radius α opening angle

Hertz Model Spherical Indenter

depth ltlt radius of curvature

Sneddon Model Conical Indenter

depth ge radius of curvature

BBML - All R

ights Reserve

d

Indentation in OI Tendon Study

x

z

yBrtl+ and WT mice 6 month old males

4 tails per group

2-3 fascicles per tail

25-30 fibrils per tail

4-5 locations per fibril

Indent force 20 nN wet (Sneddon) 50 nN dry (Hertz) BBML - A

ll Rights R

eserved

OI Tendon Differential Effects of Dehydration

Dry 1268 plusmn 718 MPa

Wet 305 plusmn 243 MPa

Dry 1737 plusmn 973 MPa

Wet 301 plusmn 202 MPa

Kemp et al J StructBio 2012 180 (3) 428-438

bull Significant stiffening of fibrils with dehydration

bull More pronounced modulus modulus as a function of disease

BBML - All R

ights Reserve

d

OI Tendon Differential Effects of Dehydration

Kemp et al J StructBio 2012 180 (3) 428-438

Diseased phenotype not present when wet phenotype observed when dried

0

10

20

30

40

50

60

0 20 40 60 80 100 120 140 160 180 200

Gro

up S

ampl

es (

)

Indent Modulus (MPa)

WT WetBrtl Wet

0

2

4

6

8

10

12

14

16

18

20

025

050

075

010

0012

5015

0017

5020

0022

5025

0027

5030

0032

5035

0037

5040

0042

5045

0047

5050

00

Gro

up S

ampl

es (

)Indent Modulus (MPa)

WT DryBrtl Dry

555 WT Indents 305 plusmn 243 MPa

614 Brtl+ Indents 301 plusmn 202 MPa

KS Test p=0129 t Test p=0464 464 WT Indents

1268 plusmn 718 MPa 479 Brtl+ Indents 1737 plusmn 973 MPa

KS Test plt0001 T Test plt0001

BBML - All R

ights Reserve

d

Indentation in Diabetic Tendon (Poster 0495)

x

z

y

Control and Diabetic Rat Tail Tendon

4-5 rats per group

2-3 fascicles per rat

~70 fibrilstail

4-5 locationsfibril

20 nN (Sneddon)

BBML - All R

ights Reserve

d

Multiscale Diabetes Study (Poster 0495)

0

5

10

15

20

25

300 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 gt25

Perc

enta

ge o

f Gro

up S

ampl

es

Elastic Modulus (MPa)

CD (1530 indents)ZDSD (913 indents)

CD (n=5) 381 plusmn 149 MPa

ZDSD (n=3) 706 plusmn 263 MPa

0

100

200

300

400

500

600

700

800

900

0 2 4 6 8 10

Mic

rosc

ale

Mod

ulus

(MPa

)

Nanoscale Modulus (MPa)

plt0001

r2=0531 p=0040

bull Increased indentation modulus in diabetic tendon

bull Strong relationship between nanoscale indent modulus and microscale tensile modulus

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

Proper probe selection for intended application bull Cantilever must be stiff relative to sample bull Sharp probes may damage sample easily dull

R or α must be determined for each probe bull Properties provided by manufacturer are unreliable bull Vary from probe to probe can dull during indent process

Spring constant must be determined for each probe

bull Value provided by manufacturer is inaccurate bull Thermal tuning OK for ldquosoftrdquo probes poor performance

for stiffer probes bull Sader method (geometry) added mass reference

cantilevers

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

Veeco Application Technical note Ben Ohler Practical Advice on the Determination of Cantilever Spring Constants

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

bull Sample processing

bull Sample hydration

bull Proper force range

bull Substrate effects for thin samples

bull Proper modulus model and range of data goodness of fit

Bearing in mind these assumptions and limitations AFM can provide true ldquonanoscalerdquo mechanical properties both in terms of forces and deformations

BBML - All R

ights Reserve

d

A Note on Peak Force QNM for Bruker AFMs bull Mechanical properties mapped pixel-by-pixel at same

resolution as the height image (PeakForce QNM)

Height Modulus BBML - All R

ights Reserve

d

A Note on Peak Force QNM

Correct probe stiffness must be used based on sample Pittenger ndash Veeco Product Bulletin

BBML - All R

ights Reserve

d

A Note on Peak Force QNM Extremely sensitive to calibration (details rarely given) Absolute radius and spring constant known

bull especially important from probe to probe bull radius uarr with indent depth ndash must determine R at depth

you expect to image bull Depth may vary as a function of sample (or at different

regions within the sample)

Relative reference material of ldquoknownrdquo modulus used to determine a ratio of 119896119896 119877119877 bull ldquoknownrdquo modulus may come from tension compression

and not indentation bull R is related to depth indentation depth on sample must

be same as on reference BBML - All R

ights Reserve

d

A Note on Peak Force QNM bull Modulus fit on the fly ndash no ability to monitor fit (or range) bull Local sample topography and sample tilt play a role in

measured parameters bull Assuming all modulus values in an image are correct how to

quantify

512 x 512 pixels = 262144 measurements

n=1 n=262144

n from selected ROIs

BBML - All R

ights Reserve

d

BBML - All R

ights Reserve

d

bull Imaging in air on mica ndash substrate effect

bull Same imaging force or indent depth for all

bull For each area were all modulus values pooled

Lamprou et al PLoS ONE 2013 BBML - All R

ights Reserve

d

AFM Applications in Bone Research Other Cool Mechanical Techniques

BBML - All R

ights Reserve

d

ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo Bonds within or between collagen fibrils in bone that rupture to dissipate energy as a toughening mechanism

Thompson et al Nature 2001

Fantner et al Nature Mat 2005

Hansma et al JMNI 2005

Fantner et al Nano Letters 2007 BBML - All R

ights Reserve

d

Other Mechanical Techniques

Jimenez-Palomar J Mech Beh Bio Mat 2012

van der Rijt Macro Bio 2004

Yang J Biomed Mater Res 2007

Yang Biophysical Journal 2008 BBML - All R

ights Reserve

d

Other Cool Techniques bull Piezoresponse Force Microscopy (PFM)

bull Electrochemical AFM (EC-AFM)

bull Force Modulation Microscopy (FMM)

bull Lateral Force Microscopy (LFM)

bull Magnetic Force Microscopy (MFM)

bull Conductive AFM (C-AFM)

bull Force Pulling

bull Cantilever Bending

bull Tensile testing of individual fibrils

bull 3 point bending of fibrils

BBML - All R

ights Reserve

d

Summary Things to rememberhellip

bull Imaging mode imaging medium and probe selection

bull Morphology Measurements bull Sample preparation and hydration state bull Measuring fibril diameter bull Site selection and proper sampling bull Appropriate system calibration

bull Mechanical Measurements

bull Probe selection (stiffness and tip radius) bull Probe calibration (radius angle spring constant) bull Indentation force range and mechanical model bull Hydration in biological samples bull PeakForce QNM

BBML - All R

ights Reserve

d

Acknowledgements BBML

Max Hammond Armando Diaz Gonzalez Zachary Bart Silvia Canelon Creasy Clauser

IU School of Medicine Dr David Burr Dr Matt Allen Dr Stuart Warden Dr Lilian Plotkin

Funding Sources NIHNIDCR 1F32DE018840-01 A1 IUPUI BME Departmental Startup Funds IUPUI Research Support Funds Grant IUPUI Biomechanics and Biomaterials Research Center

NIH NICHD Dr Joan Marini

BBML - All R

ights Reserve

d

Bone Biology and Mechanics Lab (BBML) wwwiupuiedu~bbml Facebook BoneBiologyMechanicsLab

BBML - All R

ights Reserve

d

• Slide Number 1
• General Seminar Outline
• Bonersquos Hierarchical Structure
• Critical Gap in Understanding Collagen
• Methods For Analyzing Collagen Structure
• Methods For Imaging Collagen Structure
• Cryo-TEM
• Atomic Force Microscopy (AFM)
• AFM Image of Pentacene (C22H14)
• AFM Image of Hexabenzocoronene
• Basics of AFM Imaging The Probe
• Basics of AFM Imaging System Components
• van der Waals Potential Energy Curve
• Contact Mode ndash Monitor Deflection
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Peak Force Tapping Mode ndash Monitor Force
• AFM Applications in Bone Research Imaging
• AFM Imaging of Collagen
• Imaging of Osteoclast Activity
• Mineralized collagen deposition in vitro
• Imaging of Canilicular Structure
• AFM Imaging in Bone
• AFM Imaging in Bone
• Removing Mineral in Bone to Expose Collagen
• Removing Mineral in Bone to Expose Collagen
• Demineralization with EDTA
• Demineralization with EDTA
• Demineralization with EDTA
• Issues to Consider Effects of Acid
• Slide Number 32
• Slide Number 33
• Analysis of Collagen Structure in Bone
• Analysis of Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril D-Spacing
• Slide Number 39
• Collagen in Bone Fibril D-Spacing
• Collagen in Bone Fibril D-Spacing
• 2D Fast Fourier Transform Analysis
• 2D Fast Fourier Transform Analysis
• D-Spacing Distribution
• D-Spacing Distribution
• Cumulative Distribution Function
• CDF vs Histogram
• Changes in D-Spacing with Disease
• Changes in D-Spacing with Disease
• Slide Number 50
• Issues to Consider Site Selection and Sampling
• Issues to Consider Site Selection and Sampling
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• AFM calibration ndash Take Away Message
• AFM Applications in Bone ResearchIndentation Mechanics
• AFM-Based Indentation
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Parameters
• Hertz Contact Equation
• Hertz Contact Equation
• Hertz Contact Equation
• Proper Indentation Model is Essential
• Indentation in OI Tendon Study
• OI Tendon Differential Effects of Dehydration
• OI Tendon Differential Effects of Dehydration
• Indentation in Diabetic Tendon (Poster 0495)
• Multiscale Diabetes Study (Poster 0495)
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• A Note on Peak Force QNM for Bruker AFMs
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• Slide Number 83
• Slide Number 84
• AFM Applications in Bone ResearchOther Cool Mechanical Techniques
• ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo
• Other Mechanical Techniques
• Other Cool Techniques
• Summary Things to rememberhellip
• Acknowledgements
• Bone Biology and Mechanics Lab (BBML)

AFM Applications in Bone Research Indentation Mechanics

BBML - All R

ights Reserve

d

AFM-Based Indentation

In addition to high resolution imaging AFM can be used to extract nanoscale mechanical data

ApproachWithdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Can

tilev

er D

efle

ctio

n (V

olts

)

1 Ramp into a rigid sample ndash measure the slope

Deflection sensitivity x 119899119899119899119899119881119881

2 Calculate cantilever spring constant

3 Hookersquos law takes deflection (volts) and converts to units of force F = kx

Spring constant k 119873119873119899119899

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Forc

e (n

N)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration Fo

rce

(nN

)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

Tip-Sample Separation (nm)

BBML - All R

ights Reserve

d

AFM-Based Indentation Parameters

Approach Withdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

Peak Force

Area Between Curves Energy Dissipation

Contact Mechanics Fit for Elastic Modulus (E)

Adhesion Force

Indentation Depth (δ)

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

z

r

δ

R

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119877119877 ∙ 12057512057532

Es Sample modulus νs Poissonrsquos ratio R tip radius of curvature δ load point displacement

Relevant Hertz Assumptions bull Strains are small and within the elastic limit bull Indenter is infinitely stiff (only sample deforms) bull Indentation depth ltlt radius of curvature of indenter bull Surfaces are continuous non-conforming (must be flat) bull Load is normal to surface

BBML - All R

ights Reserve

d

Proper Indentation Model is Essential

δ

r

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119903119903 ∙ 12057512057532

δ

α

119865119865 =2120587120587∙

1198641198641199041199041 minus 1205841205841199041199042

∙ tan120572120572 ∙ 1205751205752

Es Sample modulus νs Poissonrsquos ratio R tip radius α opening angle

Hertz Model Spherical Indenter

depth ltlt radius of curvature

Sneddon Model Conical Indenter

depth ge radius of curvature

BBML - All R

ights Reserve

d

Indentation in OI Tendon Study

x

z

yBrtl+ and WT mice 6 month old males

4 tails per group

2-3 fascicles per tail

25-30 fibrils per tail

4-5 locations per fibril

Indent force 20 nN wet (Sneddon) 50 nN dry (Hertz) BBML - A

ll Rights R

eserved

OI Tendon Differential Effects of Dehydration

Dry 1268 plusmn 718 MPa

Wet 305 plusmn 243 MPa

Dry 1737 plusmn 973 MPa

Wet 301 plusmn 202 MPa

Kemp et al J StructBio 2012 180 (3) 428-438

bull Significant stiffening of fibrils with dehydration

bull More pronounced modulus modulus as a function of disease

BBML - All R

ights Reserve

d

OI Tendon Differential Effects of Dehydration

Kemp et al J StructBio 2012 180 (3) 428-438

Diseased phenotype not present when wet phenotype observed when dried

0

10

20

30

40

50

60

0 20 40 60 80 100 120 140 160 180 200

Gro

up S

ampl

es (

)

Indent Modulus (MPa)

WT WetBrtl Wet

0

2

4

6

8

10

12

14

16

18

20

025

050

075

010

0012

5015

0017

5020

0022

5025

0027

5030

0032

5035

0037

5040

0042

5045

0047

5050

00

Gro

up S

ampl

es (

)Indent Modulus (MPa)

WT DryBrtl Dry

555 WT Indents 305 plusmn 243 MPa

614 Brtl+ Indents 301 plusmn 202 MPa

KS Test p=0129 t Test p=0464 464 WT Indents

1268 plusmn 718 MPa 479 Brtl+ Indents 1737 plusmn 973 MPa

KS Test plt0001 T Test plt0001

BBML - All R

ights Reserve

d

Indentation in Diabetic Tendon (Poster 0495)

x

z

y

Control and Diabetic Rat Tail Tendon

4-5 rats per group

2-3 fascicles per rat

~70 fibrilstail

4-5 locationsfibril

20 nN (Sneddon)

BBML - All R

ights Reserve

d

Multiscale Diabetes Study (Poster 0495)

0

5

10

15

20

25

300 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 gt25

Perc

enta

ge o

f Gro

up S

ampl

es

Elastic Modulus (MPa)

CD (1530 indents)ZDSD (913 indents)

CD (n=5) 381 plusmn 149 MPa

ZDSD (n=3) 706 plusmn 263 MPa

0

100

200

300

400

500

600

700

800

900

0 2 4 6 8 10

Mic

rosc

ale

Mod

ulus

(MPa

)

Nanoscale Modulus (MPa)

plt0001

r2=0531 p=0040

bull Increased indentation modulus in diabetic tendon

bull Strong relationship between nanoscale indent modulus and microscale tensile modulus

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

Proper probe selection for intended application bull Cantilever must be stiff relative to sample bull Sharp probes may damage sample easily dull

R or α must be determined for each probe bull Properties provided by manufacturer are unreliable bull Vary from probe to probe can dull during indent process

Spring constant must be determined for each probe

bull Value provided by manufacturer is inaccurate bull Thermal tuning OK for ldquosoftrdquo probes poor performance

for stiffer probes bull Sader method (geometry) added mass reference

cantilevers

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

Veeco Application Technical note Ben Ohler Practical Advice on the Determination of Cantilever Spring Constants

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

bull Sample processing

bull Sample hydration

bull Proper force range

bull Substrate effects for thin samples

bull Proper modulus model and range of data goodness of fit

Bearing in mind these assumptions and limitations AFM can provide true ldquonanoscalerdquo mechanical properties both in terms of forces and deformations

BBML - All R

ights Reserve

d

A Note on Peak Force QNM for Bruker AFMs bull Mechanical properties mapped pixel-by-pixel at same

resolution as the height image (PeakForce QNM)

Height Modulus BBML - All R

ights Reserve

d

A Note on Peak Force QNM

Correct probe stiffness must be used based on sample Pittenger ndash Veeco Product Bulletin

BBML - All R

ights Reserve

d

A Note on Peak Force QNM Extremely sensitive to calibration (details rarely given) Absolute radius and spring constant known

bull especially important from probe to probe bull radius uarr with indent depth ndash must determine R at depth

you expect to image bull Depth may vary as a function of sample (or at different

regions within the sample)

Relative reference material of ldquoknownrdquo modulus used to determine a ratio of 119896119896 119877119877 bull ldquoknownrdquo modulus may come from tension compression

and not indentation bull R is related to depth indentation depth on sample must

be same as on reference BBML - All R

ights Reserve

d

A Note on Peak Force QNM bull Modulus fit on the fly ndash no ability to monitor fit (or range) bull Local sample topography and sample tilt play a role in

measured parameters bull Assuming all modulus values in an image are correct how to

quantify

512 x 512 pixels = 262144 measurements

n=1 n=262144

n from selected ROIs

BBML - All R

ights Reserve

d

BBML - All R

ights Reserve

d

bull Imaging in air on mica ndash substrate effect

bull Same imaging force or indent depth for all

bull For each area were all modulus values pooled

Lamprou et al PLoS ONE 2013 BBML - All R

ights Reserve

d

AFM Applications in Bone Research Other Cool Mechanical Techniques

BBML - All R

ights Reserve

d

ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo Bonds within or between collagen fibrils in bone that rupture to dissipate energy as a toughening mechanism

Thompson et al Nature 2001

Fantner et al Nature Mat 2005

Hansma et al JMNI 2005

Fantner et al Nano Letters 2007 BBML - All R

ights Reserve

d

Other Mechanical Techniques

Jimenez-Palomar J Mech Beh Bio Mat 2012

van der Rijt Macro Bio 2004

Yang J Biomed Mater Res 2007

Yang Biophysical Journal 2008 BBML - All R

ights Reserve

d

Other Cool Techniques bull Piezoresponse Force Microscopy (PFM)

bull Electrochemical AFM (EC-AFM)

bull Force Modulation Microscopy (FMM)

bull Lateral Force Microscopy (LFM)

bull Magnetic Force Microscopy (MFM)

bull Conductive AFM (C-AFM)

bull Force Pulling

bull Cantilever Bending

bull Tensile testing of individual fibrils

bull 3 point bending of fibrils

BBML - All R

ights Reserve

d

Summary Things to rememberhellip

bull Imaging mode imaging medium and probe selection

bull Morphology Measurements bull Sample preparation and hydration state bull Measuring fibril diameter bull Site selection and proper sampling bull Appropriate system calibration

bull Mechanical Measurements

bull Probe selection (stiffness and tip radius) bull Probe calibration (radius angle spring constant) bull Indentation force range and mechanical model bull Hydration in biological samples bull PeakForce QNM

BBML - All R

ights Reserve

d

Acknowledgements BBML

Max Hammond Armando Diaz Gonzalez Zachary Bart Silvia Canelon Creasy Clauser

IU School of Medicine Dr David Burr Dr Matt Allen Dr Stuart Warden Dr Lilian Plotkin

Funding Sources NIHNIDCR 1F32DE018840-01 A1 IUPUI BME Departmental Startup Funds IUPUI Research Support Funds Grant IUPUI Biomechanics and Biomaterials Research Center

NIH NICHD Dr Joan Marini

BBML - All R

ights Reserve

d

Bone Biology and Mechanics Lab (BBML) wwwiupuiedu~bbml Facebook BoneBiologyMechanicsLab

BBML - All R

ights Reserve

d

• Slide Number 1
• General Seminar Outline
• Bonersquos Hierarchical Structure
• Critical Gap in Understanding Collagen
• Methods For Analyzing Collagen Structure
• Methods For Imaging Collagen Structure
• Cryo-TEM
• Atomic Force Microscopy (AFM)
• AFM Image of Pentacene (C22H14)
• AFM Image of Hexabenzocoronene
• Basics of AFM Imaging The Probe
• Basics of AFM Imaging System Components
• van der Waals Potential Energy Curve
• Contact Mode ndash Monitor Deflection
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Peak Force Tapping Mode ndash Monitor Force
• AFM Applications in Bone Research Imaging
• AFM Imaging of Collagen
• Imaging of Osteoclast Activity
• Mineralized collagen deposition in vitro
• Imaging of Canilicular Structure
• AFM Imaging in Bone
• AFM Imaging in Bone
• Removing Mineral in Bone to Expose Collagen
• Removing Mineral in Bone to Expose Collagen
• Demineralization with EDTA
• Demineralization with EDTA
• Demineralization with EDTA
• Issues to Consider Effects of Acid
• Slide Number 32
• Slide Number 33
• Analysis of Collagen Structure in Bone
• Analysis of Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril D-Spacing
• Slide Number 39
• Collagen in Bone Fibril D-Spacing
• Collagen in Bone Fibril D-Spacing
• 2D Fast Fourier Transform Analysis
• 2D Fast Fourier Transform Analysis
• D-Spacing Distribution
• D-Spacing Distribution
• Cumulative Distribution Function
• CDF vs Histogram
• Changes in D-Spacing with Disease
• Changes in D-Spacing with Disease
• Slide Number 50
• Issues to Consider Site Selection and Sampling
• Issues to Consider Site Selection and Sampling
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• AFM calibration ndash Take Away Message
• AFM Applications in Bone ResearchIndentation Mechanics
• AFM-Based Indentation
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Parameters
• Hertz Contact Equation
• Hertz Contact Equation
• Hertz Contact Equation
• Proper Indentation Model is Essential
• Indentation in OI Tendon Study
• OI Tendon Differential Effects of Dehydration
• OI Tendon Differential Effects of Dehydration
• Indentation in Diabetic Tendon (Poster 0495)
• Multiscale Diabetes Study (Poster 0495)
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• A Note on Peak Force QNM for Bruker AFMs
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• Slide Number 83
• Slide Number 84
• AFM Applications in Bone ResearchOther Cool Mechanical Techniques
• ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo
• Other Mechanical Techniques
• Other Cool Techniques
• Summary Things to rememberhellip
• Acknowledgements
• Bone Biology and Mechanics Lab (BBML)

AFM-Based Indentation

In addition to high resolution imaging AFM can be used to extract nanoscale mechanical data

ApproachWithdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Can

tilev

er D

efle

ctio

n (V

olts

)

1 Ramp into a rigid sample ndash measure the slope

Deflection sensitivity x 119899119899119899119899119881119881

2 Calculate cantilever spring constant

3 Hookersquos law takes deflection (volts) and converts to units of force F = kx

Spring constant k 119873119873119899119899

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Forc

e (n

N)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration Fo

rce

(nN

)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

Tip-Sample Separation (nm)

BBML - All R

ights Reserve

d

AFM-Based Indentation Parameters

Approach Withdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

Peak Force

Area Between Curves Energy Dissipation

Contact Mechanics Fit for Elastic Modulus (E)

Adhesion Force

Indentation Depth (δ)

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

z

r

δ

R

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119877119877 ∙ 12057512057532

Es Sample modulus νs Poissonrsquos ratio R tip radius of curvature δ load point displacement

Relevant Hertz Assumptions bull Strains are small and within the elastic limit bull Indenter is infinitely stiff (only sample deforms) bull Indentation depth ltlt radius of curvature of indenter bull Surfaces are continuous non-conforming (must be flat) bull Load is normal to surface

BBML - All R

ights Reserve

d

Proper Indentation Model is Essential

δ

r

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119903119903 ∙ 12057512057532

δ

α

119865119865 =2120587120587∙

1198641198641199041199041 minus 1205841205841199041199042

∙ tan120572120572 ∙ 1205751205752

Es Sample modulus νs Poissonrsquos ratio R tip radius α opening angle

Hertz Model Spherical Indenter

depth ltlt radius of curvature

Sneddon Model Conical Indenter

depth ge radius of curvature

BBML - All R

ights Reserve

d

Indentation in OI Tendon Study

x

z

yBrtl+ and WT mice 6 month old males

4 tails per group

2-3 fascicles per tail

25-30 fibrils per tail

4-5 locations per fibril

Indent force 20 nN wet (Sneddon) 50 nN dry (Hertz) BBML - A

ll Rights R

eserved

OI Tendon Differential Effects of Dehydration

Dry 1268 plusmn 718 MPa

Wet 305 plusmn 243 MPa

Dry 1737 plusmn 973 MPa

Wet 301 plusmn 202 MPa

Kemp et al J StructBio 2012 180 (3) 428-438

bull Significant stiffening of fibrils with dehydration

bull More pronounced modulus modulus as a function of disease

BBML - All R

ights Reserve

d

OI Tendon Differential Effects of Dehydration

Kemp et al J StructBio 2012 180 (3) 428-438

Diseased phenotype not present when wet phenotype observed when dried

0

10

20

30

40

50

60

0 20 40 60 80 100 120 140 160 180 200

Gro

up S

ampl

es (

)

Indent Modulus (MPa)

WT WetBrtl Wet

0

2

4

6

8

10

12

14

16

18

20

025

050

075

010

0012

5015

0017

5020

0022

5025

0027

5030

0032

5035

0037

5040

0042

5045

0047

5050

00

Gro

up S

ampl

es (

)Indent Modulus (MPa)

WT DryBrtl Dry

555 WT Indents 305 plusmn 243 MPa

614 Brtl+ Indents 301 plusmn 202 MPa

KS Test p=0129 t Test p=0464 464 WT Indents

1268 plusmn 718 MPa 479 Brtl+ Indents 1737 plusmn 973 MPa

KS Test plt0001 T Test plt0001

BBML - All R

ights Reserve

d

Indentation in Diabetic Tendon (Poster 0495)

x

z

y

Control and Diabetic Rat Tail Tendon

4-5 rats per group

2-3 fascicles per rat

~70 fibrilstail

4-5 locationsfibril

20 nN (Sneddon)

BBML - All R

ights Reserve

d

Multiscale Diabetes Study (Poster 0495)

0

5

10

15

20

25

300 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 gt25

Perc

enta

ge o

f Gro

up S

ampl

es

Elastic Modulus (MPa)

CD (1530 indents)ZDSD (913 indents)

CD (n=5) 381 plusmn 149 MPa

ZDSD (n=3) 706 plusmn 263 MPa

0

100

200

300

400

500

600

700

800

900

0 2 4 6 8 10

Mic

rosc

ale

Mod

ulus

(MPa

)

Nanoscale Modulus (MPa)

plt0001

r2=0531 p=0040

bull Increased indentation modulus in diabetic tendon

bull Strong relationship between nanoscale indent modulus and microscale tensile modulus

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

Proper probe selection for intended application bull Cantilever must be stiff relative to sample bull Sharp probes may damage sample easily dull

R or α must be determined for each probe bull Properties provided by manufacturer are unreliable bull Vary from probe to probe can dull during indent process

Spring constant must be determined for each probe

bull Value provided by manufacturer is inaccurate bull Thermal tuning OK for ldquosoftrdquo probes poor performance

for stiffer probes bull Sader method (geometry) added mass reference

cantilevers

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

Veeco Application Technical note Ben Ohler Practical Advice on the Determination of Cantilever Spring Constants

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

bull Sample processing

bull Sample hydration

bull Proper force range

bull Substrate effects for thin samples

bull Proper modulus model and range of data goodness of fit

Bearing in mind these assumptions and limitations AFM can provide true ldquonanoscalerdquo mechanical properties both in terms of forces and deformations

BBML - All R

ights Reserve

d

A Note on Peak Force QNM for Bruker AFMs bull Mechanical properties mapped pixel-by-pixel at same

resolution as the height image (PeakForce QNM)

Height Modulus BBML - All R

ights Reserve

d

A Note on Peak Force QNM

Correct probe stiffness must be used based on sample Pittenger ndash Veeco Product Bulletin

BBML - All R

ights Reserve

d

A Note on Peak Force QNM Extremely sensitive to calibration (details rarely given) Absolute radius and spring constant known

bull especially important from probe to probe bull radius uarr with indent depth ndash must determine R at depth

you expect to image bull Depth may vary as a function of sample (or at different

regions within the sample)

Relative reference material of ldquoknownrdquo modulus used to determine a ratio of 119896119896 119877119877 bull ldquoknownrdquo modulus may come from tension compression

and not indentation bull R is related to depth indentation depth on sample must

be same as on reference BBML - All R

ights Reserve

d

A Note on Peak Force QNM bull Modulus fit on the fly ndash no ability to monitor fit (or range) bull Local sample topography and sample tilt play a role in

measured parameters bull Assuming all modulus values in an image are correct how to

quantify

512 x 512 pixels = 262144 measurements

n=1 n=262144

n from selected ROIs

BBML - All R

ights Reserve

d

BBML - All R

ights Reserve

d

bull Imaging in air on mica ndash substrate effect

bull Same imaging force or indent depth for all

bull For each area were all modulus values pooled

Lamprou et al PLoS ONE 2013 BBML - All R

ights Reserve

d

AFM Applications in Bone Research Other Cool Mechanical Techniques

BBML - All R

ights Reserve

d

ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo Bonds within or between collagen fibrils in bone that rupture to dissipate energy as a toughening mechanism

Thompson et al Nature 2001

Fantner et al Nature Mat 2005

Hansma et al JMNI 2005

Fantner et al Nano Letters 2007 BBML - All R

ights Reserve

d

Other Mechanical Techniques

Jimenez-Palomar J Mech Beh Bio Mat 2012

van der Rijt Macro Bio 2004

Yang J Biomed Mater Res 2007

Yang Biophysical Journal 2008 BBML - All R

ights Reserve

d

Other Cool Techniques bull Piezoresponse Force Microscopy (PFM)

bull Electrochemical AFM (EC-AFM)

bull Force Modulation Microscopy (FMM)

bull Lateral Force Microscopy (LFM)

bull Magnetic Force Microscopy (MFM)

bull Conductive AFM (C-AFM)

bull Force Pulling

bull Cantilever Bending

bull Tensile testing of individual fibrils

bull 3 point bending of fibrils

BBML - All R

ights Reserve

d

Summary Things to rememberhellip

bull Imaging mode imaging medium and probe selection

bull Morphology Measurements bull Sample preparation and hydration state bull Measuring fibril diameter bull Site selection and proper sampling bull Appropriate system calibration

bull Mechanical Measurements

bull Probe selection (stiffness and tip radius) bull Probe calibration (radius angle spring constant) bull Indentation force range and mechanical model bull Hydration in biological samples bull PeakForce QNM

BBML - All R

ights Reserve

d

Acknowledgements BBML

Max Hammond Armando Diaz Gonzalez Zachary Bart Silvia Canelon Creasy Clauser

IU School of Medicine Dr David Burr Dr Matt Allen Dr Stuart Warden Dr Lilian Plotkin

Funding Sources NIHNIDCR 1F32DE018840-01 A1 IUPUI BME Departmental Startup Funds IUPUI Research Support Funds Grant IUPUI Biomechanics and Biomaterials Research Center

NIH NICHD Dr Joan Marini

BBML - All R

ights Reserve

d

Bone Biology and Mechanics Lab (BBML) wwwiupuiedu~bbml Facebook BoneBiologyMechanicsLab

BBML - All R

ights Reserve

d

• Slide Number 1
• General Seminar Outline
• Bonersquos Hierarchical Structure
• Critical Gap in Understanding Collagen
• Methods For Analyzing Collagen Structure
• Methods For Imaging Collagen Structure
• Cryo-TEM
• Atomic Force Microscopy (AFM)
• AFM Image of Pentacene (C22H14)
• AFM Image of Hexabenzocoronene
• Basics of AFM Imaging The Probe
• Basics of AFM Imaging System Components
• van der Waals Potential Energy Curve
• Contact Mode ndash Monitor Deflection
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Peak Force Tapping Mode ndash Monitor Force
• AFM Applications in Bone Research Imaging
• AFM Imaging of Collagen
• Imaging of Osteoclast Activity
• Mineralized collagen deposition in vitro
• Imaging of Canilicular Structure
• AFM Imaging in Bone
• AFM Imaging in Bone
• Removing Mineral in Bone to Expose Collagen
• Removing Mineral in Bone to Expose Collagen
• Demineralization with EDTA
• Demineralization with EDTA
• Demineralization with EDTA
• Issues to Consider Effects of Acid
• Slide Number 32
• Slide Number 33
• Analysis of Collagen Structure in Bone
• Analysis of Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril D-Spacing
• Slide Number 39
• Collagen in Bone Fibril D-Spacing
• Collagen in Bone Fibril D-Spacing
• 2D Fast Fourier Transform Analysis
• 2D Fast Fourier Transform Analysis
• D-Spacing Distribution
• D-Spacing Distribution
• Cumulative Distribution Function
• CDF vs Histogram
• Changes in D-Spacing with Disease
• Changes in D-Spacing with Disease
• Slide Number 50
• Issues to Consider Site Selection and Sampling
• Issues to Consider Site Selection and Sampling
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• AFM calibration ndash Take Away Message
• AFM Applications in Bone ResearchIndentation Mechanics
• AFM-Based Indentation
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Parameters
• Hertz Contact Equation
• Hertz Contact Equation
• Hertz Contact Equation
• Proper Indentation Model is Essential
• Indentation in OI Tendon Study
• OI Tendon Differential Effects of Dehydration
• OI Tendon Differential Effects of Dehydration
• Indentation in Diabetic Tendon (Poster 0495)
• Multiscale Diabetes Study (Poster 0495)
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• A Note on Peak Force QNM for Bruker AFMs
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• Slide Number 83
• Slide Number 84
• AFM Applications in Bone ResearchOther Cool Mechanical Techniques
• ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo
• Other Mechanical Techniques
• Other Cool Techniques
• Summary Things to rememberhellip
• Acknowledgements
• Bone Biology and Mechanics Lab (BBML)

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Can

tilev

er D

efle

ctio

n (V

olts

)

1 Ramp into a rigid sample ndash measure the slope

Deflection sensitivity x 119899119899119899119899119881119881

2 Calculate cantilever spring constant

3 Hookersquos law takes deflection (volts) and converts to units of force F = kx

Spring constant k 119873119873119899119899

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Forc

e (n

N)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration Fo

rce

(nN

)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

Tip-Sample Separation (nm)

BBML - All R

ights Reserve

d

AFM-Based Indentation Parameters

Approach Withdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

Peak Force

Area Between Curves Energy Dissipation

Contact Mechanics Fit for Elastic Modulus (E)

Adhesion Force

Indentation Depth (δ)

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

z

r

δ

R

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119877119877 ∙ 12057512057532

Es Sample modulus νs Poissonrsquos ratio R tip radius of curvature δ load point displacement

Relevant Hertz Assumptions bull Strains are small and within the elastic limit bull Indenter is infinitely stiff (only sample deforms) bull Indentation depth ltlt radius of curvature of indenter bull Surfaces are continuous non-conforming (must be flat) bull Load is normal to surface

BBML - All R

ights Reserve

d

Proper Indentation Model is Essential

δ

r

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119903119903 ∙ 12057512057532

δ

α

119865119865 =2120587120587∙

1198641198641199041199041 minus 1205841205841199041199042

∙ tan120572120572 ∙ 1205751205752

Es Sample modulus νs Poissonrsquos ratio R tip radius α opening angle

Hertz Model Spherical Indenter

depth ltlt radius of curvature

Sneddon Model Conical Indenter

depth ge radius of curvature

BBML - All R

ights Reserve

d

Indentation in OI Tendon Study

x

z

yBrtl+ and WT mice 6 month old males

4 tails per group

2-3 fascicles per tail

25-30 fibrils per tail

4-5 locations per fibril

Indent force 20 nN wet (Sneddon) 50 nN dry (Hertz) BBML - A

ll Rights R

eserved

OI Tendon Differential Effects of Dehydration

Dry 1268 plusmn 718 MPa

Wet 305 plusmn 243 MPa

Dry 1737 plusmn 973 MPa

Wet 301 plusmn 202 MPa

Kemp et al J StructBio 2012 180 (3) 428-438

bull Significant stiffening of fibrils with dehydration

bull More pronounced modulus modulus as a function of disease

BBML - All R

ights Reserve

d

OI Tendon Differential Effects of Dehydration

Kemp et al J StructBio 2012 180 (3) 428-438

Diseased phenotype not present when wet phenotype observed when dried

0

10

20

30

40

50

60

0 20 40 60 80 100 120 140 160 180 200

Gro

up S

ampl

es (

)

Indent Modulus (MPa)

WT WetBrtl Wet

0

2

4

6

8

10

12

14

16

18

20

025

050

075

010

0012

5015

0017

5020

0022

5025

0027

5030

0032

5035

0037

5040

0042

5045

0047

5050

00

Gro

up S

ampl

es (

)Indent Modulus (MPa)

WT DryBrtl Dry

555 WT Indents 305 plusmn 243 MPa

614 Brtl+ Indents 301 plusmn 202 MPa

KS Test p=0129 t Test p=0464 464 WT Indents

1268 plusmn 718 MPa 479 Brtl+ Indents 1737 plusmn 973 MPa

KS Test plt0001 T Test plt0001

BBML - All R

ights Reserve

d

Indentation in Diabetic Tendon (Poster 0495)

x

z

y

Control and Diabetic Rat Tail Tendon

4-5 rats per group

2-3 fascicles per rat

~70 fibrilstail

4-5 locationsfibril

20 nN (Sneddon)

BBML - All R

ights Reserve

d

Multiscale Diabetes Study (Poster 0495)

0

5

10

15

20

25

300 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 gt25

Perc

enta

ge o

f Gro

up S

ampl

es

Elastic Modulus (MPa)

CD (1530 indents)ZDSD (913 indents)

CD (n=5) 381 plusmn 149 MPa

ZDSD (n=3) 706 plusmn 263 MPa

0

100

200

300

400

500

600

700

800

900

0 2 4 6 8 10

Mic

rosc

ale

Mod

ulus

(MPa

)

Nanoscale Modulus (MPa)

plt0001

r2=0531 p=0040

bull Increased indentation modulus in diabetic tendon

bull Strong relationship between nanoscale indent modulus and microscale tensile modulus

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

Proper probe selection for intended application bull Cantilever must be stiff relative to sample bull Sharp probes may damage sample easily dull

R or α must be determined for each probe bull Properties provided by manufacturer are unreliable bull Vary from probe to probe can dull during indent process

Spring constant must be determined for each probe

bull Value provided by manufacturer is inaccurate bull Thermal tuning OK for ldquosoftrdquo probes poor performance

for stiffer probes bull Sader method (geometry) added mass reference

cantilevers

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

Veeco Application Technical note Ben Ohler Practical Advice on the Determination of Cantilever Spring Constants

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

bull Sample processing

bull Sample hydration

bull Proper force range

bull Substrate effects for thin samples

bull Proper modulus model and range of data goodness of fit

Bearing in mind these assumptions and limitations AFM can provide true ldquonanoscalerdquo mechanical properties both in terms of forces and deformations

BBML - All R

ights Reserve

d

A Note on Peak Force QNM for Bruker AFMs bull Mechanical properties mapped pixel-by-pixel at same

resolution as the height image (PeakForce QNM)

Height Modulus BBML - All R

ights Reserve

d

A Note on Peak Force QNM

Correct probe stiffness must be used based on sample Pittenger ndash Veeco Product Bulletin

BBML - All R

ights Reserve

d

A Note on Peak Force QNM Extremely sensitive to calibration (details rarely given) Absolute radius and spring constant known

bull especially important from probe to probe bull radius uarr with indent depth ndash must determine R at depth

you expect to image bull Depth may vary as a function of sample (or at different

regions within the sample)

Relative reference material of ldquoknownrdquo modulus used to determine a ratio of 119896119896 119877119877 bull ldquoknownrdquo modulus may come from tension compression

and not indentation bull R is related to depth indentation depth on sample must

be same as on reference BBML - All R

ights Reserve

d

A Note on Peak Force QNM bull Modulus fit on the fly ndash no ability to monitor fit (or range) bull Local sample topography and sample tilt play a role in

measured parameters bull Assuming all modulus values in an image are correct how to

quantify

512 x 512 pixels = 262144 measurements

n=1 n=262144

n from selected ROIs

BBML - All R

ights Reserve

d

BBML - All R

ights Reserve

d

bull Imaging in air on mica ndash substrate effect

bull Same imaging force or indent depth for all

bull For each area were all modulus values pooled

Lamprou et al PLoS ONE 2013 BBML - All R

ights Reserve

d

AFM Applications in Bone Research Other Cool Mechanical Techniques

BBML - All R

ights Reserve

d

ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo Bonds within or between collagen fibrils in bone that rupture to dissipate energy as a toughening mechanism

Thompson et al Nature 2001

Fantner et al Nature Mat 2005

Hansma et al JMNI 2005

Fantner et al Nano Letters 2007 BBML - All R

ights Reserve

d

Other Mechanical Techniques

Jimenez-Palomar J Mech Beh Bio Mat 2012

van der Rijt Macro Bio 2004

Yang J Biomed Mater Res 2007

Yang Biophysical Journal 2008 BBML - All R

ights Reserve

d

Other Cool Techniques bull Piezoresponse Force Microscopy (PFM)

bull Electrochemical AFM (EC-AFM)

bull Force Modulation Microscopy (FMM)

bull Lateral Force Microscopy (LFM)

bull Magnetic Force Microscopy (MFM)

bull Conductive AFM (C-AFM)

bull Force Pulling

bull Cantilever Bending

bull Tensile testing of individual fibrils

bull 3 point bending of fibrils

BBML - All R

ights Reserve

d

Summary Things to rememberhellip

bull Imaging mode imaging medium and probe selection

bull Morphology Measurements bull Sample preparation and hydration state bull Measuring fibril diameter bull Site selection and proper sampling bull Appropriate system calibration

bull Mechanical Measurements

bull Probe selection (stiffness and tip radius) bull Probe calibration (radius angle spring constant) bull Indentation force range and mechanical model bull Hydration in biological samples bull PeakForce QNM

BBML - All R

ights Reserve

d

Acknowledgements BBML

Max Hammond Armando Diaz Gonzalez Zachary Bart Silvia Canelon Creasy Clauser

IU School of Medicine Dr David Burr Dr Matt Allen Dr Stuart Warden Dr Lilian Plotkin

Funding Sources NIHNIDCR 1F32DE018840-01 A1 IUPUI BME Departmental Startup Funds IUPUI Research Support Funds Grant IUPUI Biomechanics and Biomaterials Research Center

NIH NICHD Dr Joan Marini

BBML - All R

ights Reserve

d

Bone Biology and Mechanics Lab (BBML) wwwiupuiedu~bbml Facebook BoneBiologyMechanicsLab

BBML - All R

ights Reserve

d

• Slide Number 1
• General Seminar Outline
• Bonersquos Hierarchical Structure
• Critical Gap in Understanding Collagen
• Methods For Analyzing Collagen Structure
• Methods For Imaging Collagen Structure
• Cryo-TEM
• Atomic Force Microscopy (AFM)
• AFM Image of Pentacene (C22H14)
• AFM Image of Hexabenzocoronene
• Basics of AFM Imaging The Probe
• Basics of AFM Imaging System Components
• van der Waals Potential Energy Curve
• Contact Mode ndash Monitor Deflection
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Peak Force Tapping Mode ndash Monitor Force
• AFM Applications in Bone Research Imaging
• AFM Imaging of Collagen
• Imaging of Osteoclast Activity
• Mineralized collagen deposition in vitro
• Imaging of Canilicular Structure
• AFM Imaging in Bone
• AFM Imaging in Bone
• Removing Mineral in Bone to Expose Collagen
• Removing Mineral in Bone to Expose Collagen
• Demineralization with EDTA
• Demineralization with EDTA
• Demineralization with EDTA
• Issues to Consider Effects of Acid
• Slide Number 32
• Slide Number 33
• Analysis of Collagen Structure in Bone
• Analysis of Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril D-Spacing
• Slide Number 39
• Collagen in Bone Fibril D-Spacing
• Collagen in Bone Fibril D-Spacing
• 2D Fast Fourier Transform Analysis
• 2D Fast Fourier Transform Analysis
• D-Spacing Distribution
• D-Spacing Distribution
• Cumulative Distribution Function
• CDF vs Histogram
• Changes in D-Spacing with Disease
• Changes in D-Spacing with Disease
• Slide Number 50
• Issues to Consider Site Selection and Sampling
• Issues to Consider Site Selection and Sampling
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• AFM calibration ndash Take Away Message
• AFM Applications in Bone ResearchIndentation Mechanics
• AFM-Based Indentation
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Parameters
• Hertz Contact Equation
• Hertz Contact Equation
• Hertz Contact Equation
• Proper Indentation Model is Essential
• Indentation in OI Tendon Study
• OI Tendon Differential Effects of Dehydration
• OI Tendon Differential Effects of Dehydration
• Indentation in Diabetic Tendon (Poster 0495)
• Multiscale Diabetes Study (Poster 0495)
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• A Note on Peak Force QNM for Bruker AFMs
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• Slide Number 83
• Slide Number 84
• AFM Applications in Bone ResearchOther Cool Mechanical Techniques
• ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo
• Other Mechanical Techniques
• Other Cool Techniques
• Summary Things to rememberhellip
• Acknowledgements
• Bone Biology and Mechanics Lab (BBML)

AFM-Based Indentation Cantilever Calibration

Z-Piezo Height (nm)

Forc

e (n

N)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

BBML - All R

ights Reserve

d

AFM-Based Indentation Cantilever Calibration Fo

rce

(nN

)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

Tip-Sample Separation (nm)

BBML - All R

ights Reserve

d

AFM-Based Indentation Parameters

Approach Withdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

Peak Force

Area Between Curves Energy Dissipation

Contact Mechanics Fit for Elastic Modulus (E)

Adhesion Force

Indentation Depth (δ)

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

z

r

δ

R

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119877119877 ∙ 12057512057532

Es Sample modulus νs Poissonrsquos ratio R tip radius of curvature δ load point displacement

Relevant Hertz Assumptions bull Strains are small and within the elastic limit bull Indenter is infinitely stiff (only sample deforms) bull Indentation depth ltlt radius of curvature of indenter bull Surfaces are continuous non-conforming (must be flat) bull Load is normal to surface

BBML - All R

ights Reserve

d

Proper Indentation Model is Essential

δ

r

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119903119903 ∙ 12057512057532

δ

α

119865119865 =2120587120587∙

1198641198641199041199041 minus 1205841205841199041199042

∙ tan120572120572 ∙ 1205751205752

Es Sample modulus νs Poissonrsquos ratio R tip radius α opening angle

Hertz Model Spherical Indenter

depth ltlt radius of curvature

Sneddon Model Conical Indenter

depth ge radius of curvature

BBML - All R

ights Reserve

d

Indentation in OI Tendon Study

x

z

yBrtl+ and WT mice 6 month old males

4 tails per group

2-3 fascicles per tail

25-30 fibrils per tail

4-5 locations per fibril

Indent force 20 nN wet (Sneddon) 50 nN dry (Hertz) BBML - A

ll Rights R

eserved

OI Tendon Differential Effects of Dehydration

Dry 1268 plusmn 718 MPa

Wet 305 plusmn 243 MPa

Dry 1737 plusmn 973 MPa

Wet 301 plusmn 202 MPa

Kemp et al J StructBio 2012 180 (3) 428-438

bull Significant stiffening of fibrils with dehydration

bull More pronounced modulus modulus as a function of disease

BBML - All R

ights Reserve

d

OI Tendon Differential Effects of Dehydration

Kemp et al J StructBio 2012 180 (3) 428-438

Diseased phenotype not present when wet phenotype observed when dried

0

10

20

30

40

50

60

0 20 40 60 80 100 120 140 160 180 200

Gro

up S

ampl

es (

)

Indent Modulus (MPa)

WT WetBrtl Wet

0

2

4

6

8

10

12

14

16

18

20

025

050

075

010

0012

5015

0017

5020

0022

5025

0027

5030

0032

5035

0037

5040

0042

5045

0047

5050

00

Gro

up S

ampl

es (

)Indent Modulus (MPa)

WT DryBrtl Dry

555 WT Indents 305 plusmn 243 MPa

614 Brtl+ Indents 301 plusmn 202 MPa

KS Test p=0129 t Test p=0464 464 WT Indents

1268 plusmn 718 MPa 479 Brtl+ Indents 1737 plusmn 973 MPa

KS Test plt0001 T Test plt0001

BBML - All R

ights Reserve

d

Indentation in Diabetic Tendon (Poster 0495)

x

z

y

Control and Diabetic Rat Tail Tendon

4-5 rats per group

2-3 fascicles per rat

~70 fibrilstail

4-5 locationsfibril

20 nN (Sneddon)

BBML - All R

ights Reserve

d

Multiscale Diabetes Study (Poster 0495)

0

5

10

15

20

25

300 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 gt25

Perc

enta

ge o

f Gro

up S

ampl

es

Elastic Modulus (MPa)

CD (1530 indents)ZDSD (913 indents)

CD (n=5) 381 plusmn 149 MPa

ZDSD (n=3) 706 plusmn 263 MPa

0

100

200

300

400

500

600

700

800

900

0 2 4 6 8 10

Mic

rosc

ale

Mod

ulus

(MPa

)

Nanoscale Modulus (MPa)

plt0001

r2=0531 p=0040

bull Increased indentation modulus in diabetic tendon

bull Strong relationship between nanoscale indent modulus and microscale tensile modulus

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

Proper probe selection for intended application bull Cantilever must be stiff relative to sample bull Sharp probes may damage sample easily dull

R or α must be determined for each probe bull Properties provided by manufacturer are unreliable bull Vary from probe to probe can dull during indent process

Spring constant must be determined for each probe

bull Value provided by manufacturer is inaccurate bull Thermal tuning OK for ldquosoftrdquo probes poor performance

for stiffer probes bull Sader method (geometry) added mass reference

cantilevers

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

Veeco Application Technical note Ben Ohler Practical Advice on the Determination of Cantilever Spring Constants

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

bull Sample processing

bull Sample hydration

bull Proper force range

bull Substrate effects for thin samples

bull Proper modulus model and range of data goodness of fit

Bearing in mind these assumptions and limitations AFM can provide true ldquonanoscalerdquo mechanical properties both in terms of forces and deformations

BBML - All R

ights Reserve

d

A Note on Peak Force QNM for Bruker AFMs bull Mechanical properties mapped pixel-by-pixel at same

resolution as the height image (PeakForce QNM)

Height Modulus BBML - All R

ights Reserve

d

A Note on Peak Force QNM

Correct probe stiffness must be used based on sample Pittenger ndash Veeco Product Bulletin

BBML - All R

ights Reserve

d

A Note on Peak Force QNM Extremely sensitive to calibration (details rarely given) Absolute radius and spring constant known

bull especially important from probe to probe bull radius uarr with indent depth ndash must determine R at depth

you expect to image bull Depth may vary as a function of sample (or at different

regions within the sample)

Relative reference material of ldquoknownrdquo modulus used to determine a ratio of 119896119896 119877119877 bull ldquoknownrdquo modulus may come from tension compression

and not indentation bull R is related to depth indentation depth on sample must

be same as on reference BBML - All R

ights Reserve

d

A Note on Peak Force QNM bull Modulus fit on the fly ndash no ability to monitor fit (or range) bull Local sample topography and sample tilt play a role in

measured parameters bull Assuming all modulus values in an image are correct how to

quantify

512 x 512 pixels = 262144 measurements

n=1 n=262144

n from selected ROIs

BBML - All R

ights Reserve

d

BBML - All R

ights Reserve

d

bull Imaging in air on mica ndash substrate effect

bull Same imaging force or indent depth for all

bull For each area were all modulus values pooled

Lamprou et al PLoS ONE 2013 BBML - All R

ights Reserve

d

AFM Applications in Bone Research Other Cool Mechanical Techniques

BBML - All R

ights Reserve

d

ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo Bonds within or between collagen fibrils in bone that rupture to dissipate energy as a toughening mechanism

Thompson et al Nature 2001

Fantner et al Nature Mat 2005

Hansma et al JMNI 2005

Fantner et al Nano Letters 2007 BBML - All R

ights Reserve

d

Other Mechanical Techniques

Jimenez-Palomar J Mech Beh Bio Mat 2012

van der Rijt Macro Bio 2004

Yang J Biomed Mater Res 2007

Yang Biophysical Journal 2008 BBML - All R

ights Reserve

d

Other Cool Techniques bull Piezoresponse Force Microscopy (PFM)

bull Electrochemical AFM (EC-AFM)

bull Force Modulation Microscopy (FMM)

bull Lateral Force Microscopy (LFM)

bull Magnetic Force Microscopy (MFM)

bull Conductive AFM (C-AFM)

bull Force Pulling

bull Cantilever Bending

bull Tensile testing of individual fibrils

bull 3 point bending of fibrils

BBML - All R

ights Reserve

d

Summary Things to rememberhellip

bull Imaging mode imaging medium and probe selection

bull Morphology Measurements bull Sample preparation and hydration state bull Measuring fibril diameter bull Site selection and proper sampling bull Appropriate system calibration

bull Mechanical Measurements

bull Probe selection (stiffness and tip radius) bull Probe calibration (radius angle spring constant) bull Indentation force range and mechanical model bull Hydration in biological samples bull PeakForce QNM

BBML - All R

ights Reserve

d

Acknowledgements BBML

Max Hammond Armando Diaz Gonzalez Zachary Bart Silvia Canelon Creasy Clauser

IU School of Medicine Dr David Burr Dr Matt Allen Dr Stuart Warden Dr Lilian Plotkin

Funding Sources NIHNIDCR 1F32DE018840-01 A1 IUPUI BME Departmental Startup Funds IUPUI Research Support Funds Grant IUPUI Biomechanics and Biomaterials Research Center

NIH NICHD Dr Joan Marini

BBML - All R

ights Reserve

d

Bone Biology and Mechanics Lab (BBML) wwwiupuiedu~bbml Facebook BoneBiologyMechanicsLab

BBML - All R

ights Reserve

d

• Slide Number 1
• General Seminar Outline
• Bonersquos Hierarchical Structure
• Critical Gap in Understanding Collagen
• Methods For Analyzing Collagen Structure
• Methods For Imaging Collagen Structure
• Cryo-TEM
• Atomic Force Microscopy (AFM)
• AFM Image of Pentacene (C22H14)
• AFM Image of Hexabenzocoronene
• Basics of AFM Imaging The Probe
• Basics of AFM Imaging System Components
• van der Waals Potential Energy Curve
• Contact Mode ndash Monitor Deflection
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Peak Force Tapping Mode ndash Monitor Force
• AFM Applications in Bone Research Imaging
• AFM Imaging of Collagen
• Imaging of Osteoclast Activity
• Mineralized collagen deposition in vitro
• Imaging of Canilicular Structure
• AFM Imaging in Bone
• AFM Imaging in Bone
• Removing Mineral in Bone to Expose Collagen
• Removing Mineral in Bone to Expose Collagen
• Demineralization with EDTA
• Demineralization with EDTA
• Demineralization with EDTA
• Issues to Consider Effects of Acid
• Slide Number 32
• Slide Number 33
• Analysis of Collagen Structure in Bone
• Analysis of Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril D-Spacing
• Slide Number 39
• Collagen in Bone Fibril D-Spacing
• Collagen in Bone Fibril D-Spacing
• 2D Fast Fourier Transform Analysis
• 2D Fast Fourier Transform Analysis
• D-Spacing Distribution
• D-Spacing Distribution
• Cumulative Distribution Function
• CDF vs Histogram
• Changes in D-Spacing with Disease
• Changes in D-Spacing with Disease
• Slide Number 50
• Issues to Consider Site Selection and Sampling
• Issues to Consider Site Selection and Sampling
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• AFM calibration ndash Take Away Message
• AFM Applications in Bone ResearchIndentation Mechanics
• AFM-Based Indentation
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Parameters
• Hertz Contact Equation
• Hertz Contact Equation
• Hertz Contact Equation
• Proper Indentation Model is Essential
• Indentation in OI Tendon Study
• OI Tendon Differential Effects of Dehydration
• OI Tendon Differential Effects of Dehydration
• Indentation in Diabetic Tendon (Poster 0495)
• Multiscale Diabetes Study (Poster 0495)
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• A Note on Peak Force QNM for Bruker AFMs
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• Slide Number 83
• Slide Number 84
• AFM Applications in Bone ResearchOther Cool Mechanical Techniques
• ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo
• Other Mechanical Techniques
• Other Cool Techniques
• Summary Things to rememberhellip
• Acknowledgements
• Bone Biology and Mechanics Lab (BBML)

AFM-Based Indentation Cantilever Calibration Fo

rce

(nN

)

Distance traveled in z-direction measured by z-piezo height

Sample deformation must also consider cantilever deflection

Separation = z height - deflection

Tip-Sample Separation (nm)

BBML - All R

ights Reserve

d

AFM-Based Indentation Parameters

Approach Withdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

Peak Force

Area Between Curves Energy Dissipation

Contact Mechanics Fit for Elastic Modulus (E)

Adhesion Force

Indentation Depth (δ)

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

z

r

δ

R

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119877119877 ∙ 12057512057532

Es Sample modulus νs Poissonrsquos ratio R tip radius of curvature δ load point displacement

Relevant Hertz Assumptions bull Strains are small and within the elastic limit bull Indenter is infinitely stiff (only sample deforms) bull Indentation depth ltlt radius of curvature of indenter bull Surfaces are continuous non-conforming (must be flat) bull Load is normal to surface

BBML - All R

ights Reserve

d

Proper Indentation Model is Essential

δ

r

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119903119903 ∙ 12057512057532

δ

α

119865119865 =2120587120587∙

1198641198641199041199041 minus 1205841205841199041199042

∙ tan120572120572 ∙ 1205751205752

Es Sample modulus νs Poissonrsquos ratio R tip radius α opening angle

Hertz Model Spherical Indenter

depth ltlt radius of curvature

Sneddon Model Conical Indenter

depth ge radius of curvature

BBML - All R

ights Reserve

d

Indentation in OI Tendon Study

x

z

yBrtl+ and WT mice 6 month old males

4 tails per group

2-3 fascicles per tail

25-30 fibrils per tail

4-5 locations per fibril

Indent force 20 nN wet (Sneddon) 50 nN dry (Hertz) BBML - A

ll Rights R

eserved

OI Tendon Differential Effects of Dehydration

Dry 1268 plusmn 718 MPa

Wet 305 plusmn 243 MPa

Dry 1737 plusmn 973 MPa

Wet 301 plusmn 202 MPa

Kemp et al J StructBio 2012 180 (3) 428-438

bull Significant stiffening of fibrils with dehydration

bull More pronounced modulus modulus as a function of disease

BBML - All R

ights Reserve

d

OI Tendon Differential Effects of Dehydration

Kemp et al J StructBio 2012 180 (3) 428-438

Diseased phenotype not present when wet phenotype observed when dried

0

10

20

30

40

50

60

0 20 40 60 80 100 120 140 160 180 200

Gro

up S

ampl

es (

)

Indent Modulus (MPa)

WT WetBrtl Wet

0

2

4

6

8

10

12

14

16

18

20

025

050

075

010

0012

5015

0017

5020

0022

5025

0027

5030

0032

5035

0037

5040

0042

5045

0047

5050

00

Gro

up S

ampl

es (

)Indent Modulus (MPa)

WT DryBrtl Dry

555 WT Indents 305 plusmn 243 MPa

614 Brtl+ Indents 301 plusmn 202 MPa

KS Test p=0129 t Test p=0464 464 WT Indents

1268 plusmn 718 MPa 479 Brtl+ Indents 1737 plusmn 973 MPa

KS Test plt0001 T Test plt0001

BBML - All R

ights Reserve

d

Indentation in Diabetic Tendon (Poster 0495)

x

z

y

Control and Diabetic Rat Tail Tendon

4-5 rats per group

2-3 fascicles per rat

~70 fibrilstail

4-5 locationsfibril

20 nN (Sneddon)

BBML - All R

ights Reserve

d

Multiscale Diabetes Study (Poster 0495)

0

5

10

15

20

25

300 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 gt25

Perc

enta

ge o

f Gro

up S

ampl

es

Elastic Modulus (MPa)

CD (1530 indents)ZDSD (913 indents)

CD (n=5) 381 plusmn 149 MPa

ZDSD (n=3) 706 plusmn 263 MPa

0

100

200

300

400

500

600

700

800

900

0 2 4 6 8 10

Mic

rosc

ale

Mod

ulus

(MPa

)

Nanoscale Modulus (MPa)

plt0001

r2=0531 p=0040

bull Increased indentation modulus in diabetic tendon

bull Strong relationship between nanoscale indent modulus and microscale tensile modulus

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

Proper probe selection for intended application bull Cantilever must be stiff relative to sample bull Sharp probes may damage sample easily dull

R or α must be determined for each probe bull Properties provided by manufacturer are unreliable bull Vary from probe to probe can dull during indent process

Spring constant must be determined for each probe

bull Value provided by manufacturer is inaccurate bull Thermal tuning OK for ldquosoftrdquo probes poor performance

for stiffer probes bull Sader method (geometry) added mass reference

cantilevers

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

Veeco Application Technical note Ben Ohler Practical Advice on the Determination of Cantilever Spring Constants

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

bull Sample processing

bull Sample hydration

bull Proper force range

bull Substrate effects for thin samples

bull Proper modulus model and range of data goodness of fit

Bearing in mind these assumptions and limitations AFM can provide true ldquonanoscalerdquo mechanical properties both in terms of forces and deformations

BBML - All R

ights Reserve

d

A Note on Peak Force QNM for Bruker AFMs bull Mechanical properties mapped pixel-by-pixel at same

resolution as the height image (PeakForce QNM)

Height Modulus BBML - All R

ights Reserve

d

A Note on Peak Force QNM

Correct probe stiffness must be used based on sample Pittenger ndash Veeco Product Bulletin

BBML - All R

ights Reserve

d

A Note on Peak Force QNM Extremely sensitive to calibration (details rarely given) Absolute radius and spring constant known

bull especially important from probe to probe bull radius uarr with indent depth ndash must determine R at depth

you expect to image bull Depth may vary as a function of sample (or at different

regions within the sample)

Relative reference material of ldquoknownrdquo modulus used to determine a ratio of 119896119896 119877119877 bull ldquoknownrdquo modulus may come from tension compression

and not indentation bull R is related to depth indentation depth on sample must

be same as on reference BBML - All R

ights Reserve

d

A Note on Peak Force QNM bull Modulus fit on the fly ndash no ability to monitor fit (or range) bull Local sample topography and sample tilt play a role in

measured parameters bull Assuming all modulus values in an image are correct how to

quantify

512 x 512 pixels = 262144 measurements

n=1 n=262144

n from selected ROIs

BBML - All R

ights Reserve

d

BBML - All R

ights Reserve

d

bull Imaging in air on mica ndash substrate effect

bull Same imaging force or indent depth for all

bull For each area were all modulus values pooled

Lamprou et al PLoS ONE 2013 BBML - All R

ights Reserve

d

AFM Applications in Bone Research Other Cool Mechanical Techniques

BBML - All R

ights Reserve

d

ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo Bonds within or between collagen fibrils in bone that rupture to dissipate energy as a toughening mechanism

Thompson et al Nature 2001

Fantner et al Nature Mat 2005

Hansma et al JMNI 2005

Fantner et al Nano Letters 2007 BBML - All R

ights Reserve

d

Other Mechanical Techniques

Jimenez-Palomar J Mech Beh Bio Mat 2012

van der Rijt Macro Bio 2004

Yang J Biomed Mater Res 2007

Yang Biophysical Journal 2008 BBML - All R

ights Reserve

d

Other Cool Techniques bull Piezoresponse Force Microscopy (PFM)

bull Electrochemical AFM (EC-AFM)

bull Force Modulation Microscopy (FMM)

bull Lateral Force Microscopy (LFM)

bull Magnetic Force Microscopy (MFM)

bull Conductive AFM (C-AFM)

bull Force Pulling

bull Cantilever Bending

bull Tensile testing of individual fibrils

bull 3 point bending of fibrils

BBML - All R

ights Reserve

d

Summary Things to rememberhellip

bull Imaging mode imaging medium and probe selection

bull Morphology Measurements bull Sample preparation and hydration state bull Measuring fibril diameter bull Site selection and proper sampling bull Appropriate system calibration

bull Mechanical Measurements

bull Probe selection (stiffness and tip radius) bull Probe calibration (radius angle spring constant) bull Indentation force range and mechanical model bull Hydration in biological samples bull PeakForce QNM

BBML - All R

ights Reserve

d

Acknowledgements BBML

Max Hammond Armando Diaz Gonzalez Zachary Bart Silvia Canelon Creasy Clauser

IU School of Medicine Dr David Burr Dr Matt Allen Dr Stuart Warden Dr Lilian Plotkin

Funding Sources NIHNIDCR 1F32DE018840-01 A1 IUPUI BME Departmental Startup Funds IUPUI Research Support Funds Grant IUPUI Biomechanics and Biomaterials Research Center

NIH NICHD Dr Joan Marini

BBML - All R

ights Reserve

d

Bone Biology and Mechanics Lab (BBML) wwwiupuiedu~bbml Facebook BoneBiologyMechanicsLab

BBML - All R

ights Reserve

d

• Slide Number 1
• General Seminar Outline
• Bonersquos Hierarchical Structure
• Critical Gap in Understanding Collagen
• Methods For Analyzing Collagen Structure
• Methods For Imaging Collagen Structure
• Cryo-TEM
• Atomic Force Microscopy (AFM)
• AFM Image of Pentacene (C22H14)
• AFM Image of Hexabenzocoronene
• Basics of AFM Imaging The Probe
• Basics of AFM Imaging System Components
• van der Waals Potential Energy Curve
• Contact Mode ndash Monitor Deflection
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Peak Force Tapping Mode ndash Monitor Force
• AFM Applications in Bone Research Imaging
• AFM Imaging of Collagen
• Imaging of Osteoclast Activity
• Mineralized collagen deposition in vitro
• Imaging of Canilicular Structure
• AFM Imaging in Bone
• AFM Imaging in Bone
• Removing Mineral in Bone to Expose Collagen
• Removing Mineral in Bone to Expose Collagen
• Demineralization with EDTA
• Demineralization with EDTA
• Demineralization with EDTA
• Issues to Consider Effects of Acid
• Slide Number 32
• Slide Number 33
• Analysis of Collagen Structure in Bone
• Analysis of Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril D-Spacing
• Slide Number 39
• Collagen in Bone Fibril D-Spacing
• Collagen in Bone Fibril D-Spacing
• 2D Fast Fourier Transform Analysis
• 2D Fast Fourier Transform Analysis
• D-Spacing Distribution
• D-Spacing Distribution
• Cumulative Distribution Function
• CDF vs Histogram
• Changes in D-Spacing with Disease
• Changes in D-Spacing with Disease
• Slide Number 50
• Issues to Consider Site Selection and Sampling
• Issues to Consider Site Selection and Sampling
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• AFM calibration ndash Take Away Message
• AFM Applications in Bone ResearchIndentation Mechanics
• AFM-Based Indentation
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Parameters
• Hertz Contact Equation
• Hertz Contact Equation
• Hertz Contact Equation
• Proper Indentation Model is Essential
• Indentation in OI Tendon Study
• OI Tendon Differential Effects of Dehydration
• OI Tendon Differential Effects of Dehydration
• Indentation in Diabetic Tendon (Poster 0495)
• Multiscale Diabetes Study (Poster 0495)
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• A Note on Peak Force QNM for Bruker AFMs
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• Slide Number 83
• Slide Number 84
• AFM Applications in Bone ResearchOther Cool Mechanical Techniques
• ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo
• Other Mechanical Techniques
• Other Cool Techniques
• Summary Things to rememberhellip
• Acknowledgements
• Bone Biology and Mechanics Lab (BBML)

AFM-Based Indentation Parameters

Approach Withdrawal

Tip-Sample Separation (nm)

Forc

e (n

N)

Peak Force

Area Between Curves Energy Dissipation

Contact Mechanics Fit for Elastic Modulus (E)

Adhesion Force

Indentation Depth (δ)

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

z

r

δ

R

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119877119877 ∙ 12057512057532

Es Sample modulus νs Poissonrsquos ratio R tip radius of curvature δ load point displacement

Relevant Hertz Assumptions bull Strains are small and within the elastic limit bull Indenter is infinitely stiff (only sample deforms) bull Indentation depth ltlt radius of curvature of indenter bull Surfaces are continuous non-conforming (must be flat) bull Load is normal to surface

BBML - All R

ights Reserve

d

Proper Indentation Model is Essential

δ

r

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119903119903 ∙ 12057512057532

δ

α

119865119865 =2120587120587∙

1198641198641199041199041 minus 1205841205841199041199042

∙ tan120572120572 ∙ 1205751205752

Es Sample modulus νs Poissonrsquos ratio R tip radius α opening angle

Hertz Model Spherical Indenter

depth ltlt radius of curvature

Sneddon Model Conical Indenter

depth ge radius of curvature

BBML - All R

ights Reserve

d

Indentation in OI Tendon Study

x

z

yBrtl+ and WT mice 6 month old males

4 tails per group

2-3 fascicles per tail

25-30 fibrils per tail

4-5 locations per fibril

Indent force 20 nN wet (Sneddon) 50 nN dry (Hertz) BBML - A

ll Rights R

eserved

OI Tendon Differential Effects of Dehydration

Dry 1268 plusmn 718 MPa

Wet 305 plusmn 243 MPa

Dry 1737 plusmn 973 MPa

Wet 301 plusmn 202 MPa

Kemp et al J StructBio 2012 180 (3) 428-438

bull Significant stiffening of fibrils with dehydration

bull More pronounced modulus modulus as a function of disease

BBML - All R

ights Reserve

d

OI Tendon Differential Effects of Dehydration

Kemp et al J StructBio 2012 180 (3) 428-438

Diseased phenotype not present when wet phenotype observed when dried

0

10

20

30

40

50

60

0 20 40 60 80 100 120 140 160 180 200

Gro

up S

ampl

es (

)

Indent Modulus (MPa)

WT WetBrtl Wet

0

2

4

6

8

10

12

14

16

18

20

025

050

075

010

0012

5015

0017

5020

0022

5025

0027

5030

0032

5035

0037

5040

0042

5045

0047

5050

00

Gro

up S

ampl

es (

)Indent Modulus (MPa)

WT DryBrtl Dry

555 WT Indents 305 plusmn 243 MPa

614 Brtl+ Indents 301 plusmn 202 MPa

KS Test p=0129 t Test p=0464 464 WT Indents

1268 plusmn 718 MPa 479 Brtl+ Indents 1737 plusmn 973 MPa

KS Test plt0001 T Test plt0001

BBML - All R

ights Reserve

d

Indentation in Diabetic Tendon (Poster 0495)

x

z

y

Control and Diabetic Rat Tail Tendon

4-5 rats per group

2-3 fascicles per rat

~70 fibrilstail

4-5 locationsfibril

20 nN (Sneddon)

BBML - All R

ights Reserve

d

Multiscale Diabetes Study (Poster 0495)

0

5

10

15

20

25

300 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 gt25

Perc

enta

ge o

f Gro

up S

ampl

es

Elastic Modulus (MPa)

CD (1530 indents)ZDSD (913 indents)

CD (n=5) 381 plusmn 149 MPa

ZDSD (n=3) 706 plusmn 263 MPa

0

100

200

300

400

500

600

700

800

900

0 2 4 6 8 10

Mic

rosc

ale

Mod

ulus

(MPa

)

Nanoscale Modulus (MPa)

plt0001

r2=0531 p=0040

bull Increased indentation modulus in diabetic tendon

bull Strong relationship between nanoscale indent modulus and microscale tensile modulus

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

Proper probe selection for intended application bull Cantilever must be stiff relative to sample bull Sharp probes may damage sample easily dull

R or α must be determined for each probe bull Properties provided by manufacturer are unreliable bull Vary from probe to probe can dull during indent process

Spring constant must be determined for each probe

bull Value provided by manufacturer is inaccurate bull Thermal tuning OK for ldquosoftrdquo probes poor performance

for stiffer probes bull Sader method (geometry) added mass reference

cantilevers

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

Veeco Application Technical note Ben Ohler Practical Advice on the Determination of Cantilever Spring Constants

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

bull Sample processing

bull Sample hydration

bull Proper force range

bull Substrate effects for thin samples

bull Proper modulus model and range of data goodness of fit

Bearing in mind these assumptions and limitations AFM can provide true ldquonanoscalerdquo mechanical properties both in terms of forces and deformations

BBML - All R

ights Reserve

d

A Note on Peak Force QNM for Bruker AFMs bull Mechanical properties mapped pixel-by-pixel at same

resolution as the height image (PeakForce QNM)

Height Modulus BBML - All R

ights Reserve

d

A Note on Peak Force QNM

Correct probe stiffness must be used based on sample Pittenger ndash Veeco Product Bulletin

BBML - All R

ights Reserve

d

A Note on Peak Force QNM Extremely sensitive to calibration (details rarely given) Absolute radius and spring constant known

bull especially important from probe to probe bull radius uarr with indent depth ndash must determine R at depth

you expect to image bull Depth may vary as a function of sample (or at different

regions within the sample)

Relative reference material of ldquoknownrdquo modulus used to determine a ratio of 119896119896 119877119877 bull ldquoknownrdquo modulus may come from tension compression

and not indentation bull R is related to depth indentation depth on sample must

be same as on reference BBML - All R

ights Reserve

d

A Note on Peak Force QNM bull Modulus fit on the fly ndash no ability to monitor fit (or range) bull Local sample topography and sample tilt play a role in

measured parameters bull Assuming all modulus values in an image are correct how to

quantify

512 x 512 pixels = 262144 measurements

n=1 n=262144

n from selected ROIs

BBML - All R

ights Reserve

d

BBML - All R

ights Reserve

d

bull Imaging in air on mica ndash substrate effect

bull Same imaging force or indent depth for all

bull For each area were all modulus values pooled

Lamprou et al PLoS ONE 2013 BBML - All R

ights Reserve

d

AFM Applications in Bone Research Other Cool Mechanical Techniques

BBML - All R

ights Reserve

d

ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo Bonds within or between collagen fibrils in bone that rupture to dissipate energy as a toughening mechanism

Thompson et al Nature 2001

Fantner et al Nature Mat 2005

Hansma et al JMNI 2005

Fantner et al Nano Letters 2007 BBML - All R

ights Reserve

d

Other Mechanical Techniques

Jimenez-Palomar J Mech Beh Bio Mat 2012

van der Rijt Macro Bio 2004

Yang J Biomed Mater Res 2007

Yang Biophysical Journal 2008 BBML - All R

ights Reserve

d

Other Cool Techniques bull Piezoresponse Force Microscopy (PFM)

bull Electrochemical AFM (EC-AFM)

bull Force Modulation Microscopy (FMM)

bull Lateral Force Microscopy (LFM)

bull Magnetic Force Microscopy (MFM)

bull Conductive AFM (C-AFM)

bull Force Pulling

bull Cantilever Bending

bull Tensile testing of individual fibrils

bull 3 point bending of fibrils

BBML - All R

ights Reserve

d

Summary Things to rememberhellip

bull Imaging mode imaging medium and probe selection

bull Morphology Measurements bull Sample preparation and hydration state bull Measuring fibril diameter bull Site selection and proper sampling bull Appropriate system calibration

bull Mechanical Measurements

bull Probe selection (stiffness and tip radius) bull Probe calibration (radius angle spring constant) bull Indentation force range and mechanical model bull Hydration in biological samples bull PeakForce QNM

BBML - All R

ights Reserve

d

Acknowledgements BBML

Max Hammond Armando Diaz Gonzalez Zachary Bart Silvia Canelon Creasy Clauser

IU School of Medicine Dr David Burr Dr Matt Allen Dr Stuart Warden Dr Lilian Plotkin

Funding Sources NIHNIDCR 1F32DE018840-01 A1 IUPUI BME Departmental Startup Funds IUPUI Research Support Funds Grant IUPUI Biomechanics and Biomaterials Research Center

NIH NICHD Dr Joan Marini

BBML - All R

ights Reserve

d

Bone Biology and Mechanics Lab (BBML) wwwiupuiedu~bbml Facebook BoneBiologyMechanicsLab

BBML - All R

ights Reserve

d

• Slide Number 1
• General Seminar Outline
• Bonersquos Hierarchical Structure
• Critical Gap in Understanding Collagen
• Methods For Analyzing Collagen Structure
• Methods For Imaging Collagen Structure
• Cryo-TEM
• Atomic Force Microscopy (AFM)
• AFM Image of Pentacene (C22H14)
• AFM Image of Hexabenzocoronene
• Basics of AFM Imaging The Probe
• Basics of AFM Imaging System Components
• van der Waals Potential Energy Curve
• Contact Mode ndash Monitor Deflection
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Peak Force Tapping Mode ndash Monitor Force
• AFM Applications in Bone Research Imaging
• AFM Imaging of Collagen
• Imaging of Osteoclast Activity
• Mineralized collagen deposition in vitro
• Imaging of Canilicular Structure
• AFM Imaging in Bone
• AFM Imaging in Bone
• Removing Mineral in Bone to Expose Collagen
• Removing Mineral in Bone to Expose Collagen
• Demineralization with EDTA
• Demineralization with EDTA
• Demineralization with EDTA
• Issues to Consider Effects of Acid
• Slide Number 32
• Slide Number 33
• Analysis of Collagen Structure in Bone
• Analysis of Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril D-Spacing
• Slide Number 39
• Collagen in Bone Fibril D-Spacing
• Collagen in Bone Fibril D-Spacing
• 2D Fast Fourier Transform Analysis
• 2D Fast Fourier Transform Analysis
• D-Spacing Distribution
• D-Spacing Distribution
• Cumulative Distribution Function
• CDF vs Histogram
• Changes in D-Spacing with Disease
• Changes in D-Spacing with Disease
• Slide Number 50
• Issues to Consider Site Selection and Sampling
• Issues to Consider Site Selection and Sampling
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• AFM calibration ndash Take Away Message
• AFM Applications in Bone ResearchIndentation Mechanics
• AFM-Based Indentation
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Parameters
• Hertz Contact Equation
• Hertz Contact Equation
• Hertz Contact Equation
• Proper Indentation Model is Essential
• Indentation in OI Tendon Study
• OI Tendon Differential Effects of Dehydration
• OI Tendon Differential Effects of Dehydration
• Indentation in Diabetic Tendon (Poster 0495)
• Multiscale Diabetes Study (Poster 0495)
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• A Note on Peak Force QNM for Bruker AFMs
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• Slide Number 83
• Slide Number 84
• AFM Applications in Bone ResearchOther Cool Mechanical Techniques
• ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo
• Other Mechanical Techniques
• Other Cool Techniques
• Summary Things to rememberhellip
• Acknowledgements
• Bone Biology and Mechanics Lab (BBML)

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

z

r

δ

R

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119877119877 ∙ 12057512057532

Es Sample modulus νs Poissonrsquos ratio R tip radius of curvature δ load point displacement

Relevant Hertz Assumptions bull Strains are small and within the elastic limit bull Indenter is infinitely stiff (only sample deforms) bull Indentation depth ltlt radius of curvature of indenter bull Surfaces are continuous non-conforming (must be flat) bull Load is normal to surface

BBML - All R

ights Reserve

d

Proper Indentation Model is Essential

δ

r

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119903119903 ∙ 12057512057532

δ

α

119865119865 =2120587120587∙

1198641198641199041199041 minus 1205841205841199041199042

∙ tan120572120572 ∙ 1205751205752

Es Sample modulus νs Poissonrsquos ratio R tip radius α opening angle

Hertz Model Spherical Indenter

depth ltlt radius of curvature

Sneddon Model Conical Indenter

depth ge radius of curvature

BBML - All R

ights Reserve

d

Indentation in OI Tendon Study

x

z

yBrtl+ and WT mice 6 month old males

4 tails per group

2-3 fascicles per tail

25-30 fibrils per tail

4-5 locations per fibril

Indent force 20 nN wet (Sneddon) 50 nN dry (Hertz) BBML - A

ll Rights R

eserved

OI Tendon Differential Effects of Dehydration

Dry 1268 plusmn 718 MPa

Wet 305 plusmn 243 MPa

Dry 1737 plusmn 973 MPa

Wet 301 plusmn 202 MPa

Kemp et al J StructBio 2012 180 (3) 428-438

bull Significant stiffening of fibrils with dehydration

bull More pronounced modulus modulus as a function of disease

BBML - All R

ights Reserve

d

OI Tendon Differential Effects of Dehydration

Kemp et al J StructBio 2012 180 (3) 428-438

Diseased phenotype not present when wet phenotype observed when dried

0

10

20

30

40

50

60

0 20 40 60 80 100 120 140 160 180 200

Gro

up S

ampl

es (

)

Indent Modulus (MPa)

WT WetBrtl Wet

0

2

4

6

8

10

12

14

16

18

20

025

050

075

010

0012

5015

0017

5020

0022

5025

0027

5030

0032

5035

0037

5040

0042

5045

0047

5050

00

Gro

up S

ampl

es (

)Indent Modulus (MPa)

WT DryBrtl Dry

555 WT Indents 305 plusmn 243 MPa

614 Brtl+ Indents 301 plusmn 202 MPa

KS Test p=0129 t Test p=0464 464 WT Indents

1268 plusmn 718 MPa 479 Brtl+ Indents 1737 plusmn 973 MPa

KS Test plt0001 T Test plt0001

BBML - All R

ights Reserve

d

Indentation in Diabetic Tendon (Poster 0495)

x

z

y

Control and Diabetic Rat Tail Tendon

4-5 rats per group

2-3 fascicles per rat

~70 fibrilstail

4-5 locationsfibril

20 nN (Sneddon)

BBML - All R

ights Reserve

d

Multiscale Diabetes Study (Poster 0495)

0

5

10

15

20

25

300 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 gt25

Perc

enta

ge o

f Gro

up S

ampl

es

Elastic Modulus (MPa)

CD (1530 indents)ZDSD (913 indents)

CD (n=5) 381 plusmn 149 MPa

ZDSD (n=3) 706 plusmn 263 MPa

0

100

200

300

400

500

600

700

800

900

0 2 4 6 8 10

Mic

rosc

ale

Mod

ulus

(MPa

)

Nanoscale Modulus (MPa)

plt0001

r2=0531 p=0040

bull Increased indentation modulus in diabetic tendon

bull Strong relationship between nanoscale indent modulus and microscale tensile modulus

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

Proper probe selection for intended application bull Cantilever must be stiff relative to sample bull Sharp probes may damage sample easily dull

R or α must be determined for each probe bull Properties provided by manufacturer are unreliable bull Vary from probe to probe can dull during indent process

Spring constant must be determined for each probe

bull Value provided by manufacturer is inaccurate bull Thermal tuning OK for ldquosoftrdquo probes poor performance

for stiffer probes bull Sader method (geometry) added mass reference

cantilevers

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

Veeco Application Technical note Ben Ohler Practical Advice on the Determination of Cantilever Spring Constants

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

bull Sample processing

bull Sample hydration

bull Proper force range

bull Substrate effects for thin samples

bull Proper modulus model and range of data goodness of fit

Bearing in mind these assumptions and limitations AFM can provide true ldquonanoscalerdquo mechanical properties both in terms of forces and deformations

BBML - All R

ights Reserve

d

A Note on Peak Force QNM for Bruker AFMs bull Mechanical properties mapped pixel-by-pixel at same

resolution as the height image (PeakForce QNM)

Height Modulus BBML - All R

ights Reserve

d

A Note on Peak Force QNM

Correct probe stiffness must be used based on sample Pittenger ndash Veeco Product Bulletin

BBML - All R

ights Reserve

d

A Note on Peak Force QNM Extremely sensitive to calibration (details rarely given) Absolute radius and spring constant known

bull especially important from probe to probe bull radius uarr with indent depth ndash must determine R at depth

you expect to image bull Depth may vary as a function of sample (or at different

regions within the sample)

Relative reference material of ldquoknownrdquo modulus used to determine a ratio of 119896119896 119877119877 bull ldquoknownrdquo modulus may come from tension compression

and not indentation bull R is related to depth indentation depth on sample must

be same as on reference BBML - All R

ights Reserve

d

A Note on Peak Force QNM bull Modulus fit on the fly ndash no ability to monitor fit (or range) bull Local sample topography and sample tilt play a role in

measured parameters bull Assuming all modulus values in an image are correct how to

quantify

512 x 512 pixels = 262144 measurements

n=1 n=262144

n from selected ROIs

BBML - All R

ights Reserve

d

BBML - All R

ights Reserve

d

bull Imaging in air on mica ndash substrate effect

bull Same imaging force or indent depth for all

bull For each area were all modulus values pooled

Lamprou et al PLoS ONE 2013 BBML - All R

ights Reserve

d

AFM Applications in Bone Research Other Cool Mechanical Techniques

BBML - All R

ights Reserve

d

ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo Bonds within or between collagen fibrils in bone that rupture to dissipate energy as a toughening mechanism

Thompson et al Nature 2001

Fantner et al Nature Mat 2005

Hansma et al JMNI 2005

Fantner et al Nano Letters 2007 BBML - All R

ights Reserve

d

Other Mechanical Techniques

Jimenez-Palomar J Mech Beh Bio Mat 2012

van der Rijt Macro Bio 2004

Yang J Biomed Mater Res 2007

Yang Biophysical Journal 2008 BBML - All R

ights Reserve

d

Other Cool Techniques bull Piezoresponse Force Microscopy (PFM)

bull Electrochemical AFM (EC-AFM)

bull Force Modulation Microscopy (FMM)

bull Lateral Force Microscopy (LFM)

bull Magnetic Force Microscopy (MFM)

bull Conductive AFM (C-AFM)

bull Force Pulling

bull Cantilever Bending

bull Tensile testing of individual fibrils

bull 3 point bending of fibrils

BBML - All R

ights Reserve

d

Summary Things to rememberhellip

bull Imaging mode imaging medium and probe selection

bull Morphology Measurements bull Sample preparation and hydration state bull Measuring fibril diameter bull Site selection and proper sampling bull Appropriate system calibration

bull Mechanical Measurements

bull Probe selection (stiffness and tip radius) bull Probe calibration (radius angle spring constant) bull Indentation force range and mechanical model bull Hydration in biological samples bull PeakForce QNM

BBML - All R

ights Reserve

d

Acknowledgements BBML

Max Hammond Armando Diaz Gonzalez Zachary Bart Silvia Canelon Creasy Clauser

IU School of Medicine Dr David Burr Dr Matt Allen Dr Stuart Warden Dr Lilian Plotkin

Funding Sources NIHNIDCR 1F32DE018840-01 A1 IUPUI BME Departmental Startup Funds IUPUI Research Support Funds Grant IUPUI Biomechanics and Biomaterials Research Center

NIH NICHD Dr Joan Marini

BBML - All R

ights Reserve

d

Bone Biology and Mechanics Lab (BBML) wwwiupuiedu~bbml Facebook BoneBiologyMechanicsLab

BBML - All R

ights Reserve

d

• Slide Number 1
• General Seminar Outline
• Bonersquos Hierarchical Structure
• Critical Gap in Understanding Collagen
• Methods For Analyzing Collagen Structure
• Methods For Imaging Collagen Structure
• Cryo-TEM
• Atomic Force Microscopy (AFM)
• AFM Image of Pentacene (C22H14)
• AFM Image of Hexabenzocoronene
• Basics of AFM Imaging The Probe
• Basics of AFM Imaging System Components
• van der Waals Potential Energy Curve
• Contact Mode ndash Monitor Deflection
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Peak Force Tapping Mode ndash Monitor Force
• AFM Applications in Bone Research Imaging
• AFM Imaging of Collagen
• Imaging of Osteoclast Activity
• Mineralized collagen deposition in vitro
• Imaging of Canilicular Structure
• AFM Imaging in Bone
• AFM Imaging in Bone
• Removing Mineral in Bone to Expose Collagen
• Removing Mineral in Bone to Expose Collagen
• Demineralization with EDTA
• Demineralization with EDTA
• Demineralization with EDTA
• Issues to Consider Effects of Acid
• Slide Number 32
• Slide Number 33
• Analysis of Collagen Structure in Bone
• Analysis of Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril D-Spacing
• Slide Number 39
• Collagen in Bone Fibril D-Spacing
• Collagen in Bone Fibril D-Spacing
• 2D Fast Fourier Transform Analysis
• 2D Fast Fourier Transform Analysis
• D-Spacing Distribution
• D-Spacing Distribution
• Cumulative Distribution Function
• CDF vs Histogram
• Changes in D-Spacing with Disease
• Changes in D-Spacing with Disease
• Slide Number 50
• Issues to Consider Site Selection and Sampling
• Issues to Consider Site Selection and Sampling
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• AFM calibration ndash Take Away Message
• AFM Applications in Bone ResearchIndentation Mechanics
• AFM-Based Indentation
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Parameters
• Hertz Contact Equation
• Hertz Contact Equation
• Hertz Contact Equation
• Proper Indentation Model is Essential
• Indentation in OI Tendon Study
• OI Tendon Differential Effects of Dehydration
• OI Tendon Differential Effects of Dehydration
• Indentation in Diabetic Tendon (Poster 0495)
• Multiscale Diabetes Study (Poster 0495)
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• A Note on Peak Force QNM for Bruker AFMs
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• Slide Number 83
• Slide Number 84
• AFM Applications in Bone ResearchOther Cool Mechanical Techniques
• ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo
• Other Mechanical Techniques
• Other Cool Techniques
• Summary Things to rememberhellip
• Acknowledgements
• Bone Biology and Mechanics Lab (BBML)

Hertz Contact Equation

wwwwikipediacom

The Hertz equation models contact of a spherical indenter with an elastic half space

BBML - All R

ights Reserve

d

Hertz Contact Equation

wwwwikipediacom

z

r

δ

R

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119877119877 ∙ 12057512057532

Es Sample modulus νs Poissonrsquos ratio R tip radius of curvature δ load point displacement

Relevant Hertz Assumptions bull Strains are small and within the elastic limit bull Indenter is infinitely stiff (only sample deforms) bull Indentation depth ltlt radius of curvature of indenter bull Surfaces are continuous non-conforming (must be flat) bull Load is normal to surface

BBML - All R

ights Reserve

d

Proper Indentation Model is Essential

δ

r

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119903119903 ∙ 12057512057532

δ

α

119865119865 =2120587120587∙

1198641198641199041199041 minus 1205841205841199041199042

∙ tan120572120572 ∙ 1205751205752

Es Sample modulus νs Poissonrsquos ratio R tip radius α opening angle

Hertz Model Spherical Indenter

depth ltlt radius of curvature

Sneddon Model Conical Indenter

depth ge radius of curvature

BBML - All R

ights Reserve

d

Indentation in OI Tendon Study

x

z

yBrtl+ and WT mice 6 month old males

4 tails per group

2-3 fascicles per tail

25-30 fibrils per tail

4-5 locations per fibril

Indent force 20 nN wet (Sneddon) 50 nN dry (Hertz) BBML - A

ll Rights R

eserved

OI Tendon Differential Effects of Dehydration

Dry 1268 plusmn 718 MPa

Wet 305 plusmn 243 MPa

Dry 1737 plusmn 973 MPa

Wet 301 plusmn 202 MPa

Kemp et al J StructBio 2012 180 (3) 428-438

bull Significant stiffening of fibrils with dehydration

bull More pronounced modulus modulus as a function of disease

BBML - All R

ights Reserve

d

OI Tendon Differential Effects of Dehydration

Kemp et al J StructBio 2012 180 (3) 428-438

Diseased phenotype not present when wet phenotype observed when dried

0

10

20

30

40

50

60

0 20 40 60 80 100 120 140 160 180 200

Gro

up S

ampl

es (

)

Indent Modulus (MPa)

WT WetBrtl Wet

0

2

4

6

8

10

12

14

16

18

20

025

050

075

010

0012

5015

0017

5020

0022

5025

0027

5030

0032

5035

0037

5040

0042

5045

0047

5050

00

Gro

up S

ampl

es (

)Indent Modulus (MPa)

WT DryBrtl Dry

555 WT Indents 305 plusmn 243 MPa

614 Brtl+ Indents 301 plusmn 202 MPa

KS Test p=0129 t Test p=0464 464 WT Indents

1268 plusmn 718 MPa 479 Brtl+ Indents 1737 plusmn 973 MPa

KS Test plt0001 T Test plt0001

BBML - All R

ights Reserve

d

Indentation in Diabetic Tendon (Poster 0495)

x

z

y

Control and Diabetic Rat Tail Tendon

4-5 rats per group

2-3 fascicles per rat

~70 fibrilstail

4-5 locationsfibril

20 nN (Sneddon)

BBML - All R

ights Reserve

d

Multiscale Diabetes Study (Poster 0495)

0

5

10

15

20

25

300 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 gt25

Perc

enta

ge o

f Gro

up S

ampl

es

Elastic Modulus (MPa)

CD (1530 indents)ZDSD (913 indents)

CD (n=5) 381 plusmn 149 MPa

ZDSD (n=3) 706 plusmn 263 MPa

0

100

200

300

400

500

600

700

800

900

0 2 4 6 8 10

Mic

rosc

ale

Mod

ulus

(MPa

)

Nanoscale Modulus (MPa)

plt0001

r2=0531 p=0040

bull Increased indentation modulus in diabetic tendon

bull Strong relationship between nanoscale indent modulus and microscale tensile modulus

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

Proper probe selection for intended application bull Cantilever must be stiff relative to sample bull Sharp probes may damage sample easily dull

R or α must be determined for each probe bull Properties provided by manufacturer are unreliable bull Vary from probe to probe can dull during indent process

Spring constant must be determined for each probe

bull Value provided by manufacturer is inaccurate bull Thermal tuning OK for ldquosoftrdquo probes poor performance

for stiffer probes bull Sader method (geometry) added mass reference

cantilevers

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

Veeco Application Technical note Ben Ohler Practical Advice on the Determination of Cantilever Spring Constants

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

bull Sample processing

bull Sample hydration

bull Proper force range

bull Substrate effects for thin samples

bull Proper modulus model and range of data goodness of fit

Bearing in mind these assumptions and limitations AFM can provide true ldquonanoscalerdquo mechanical properties both in terms of forces and deformations

BBML - All R

ights Reserve

d

A Note on Peak Force QNM for Bruker AFMs bull Mechanical properties mapped pixel-by-pixel at same

resolution as the height image (PeakForce QNM)

Height Modulus BBML - All R

ights Reserve

d

A Note on Peak Force QNM

Correct probe stiffness must be used based on sample Pittenger ndash Veeco Product Bulletin

BBML - All R

ights Reserve

d

A Note on Peak Force QNM Extremely sensitive to calibration (details rarely given) Absolute radius and spring constant known

bull especially important from probe to probe bull radius uarr with indent depth ndash must determine R at depth

you expect to image bull Depth may vary as a function of sample (or at different

regions within the sample)

Relative reference material of ldquoknownrdquo modulus used to determine a ratio of 119896119896 119877119877 bull ldquoknownrdquo modulus may come from tension compression

and not indentation bull R is related to depth indentation depth on sample must

be same as on reference BBML - All R

ights Reserve

d

A Note on Peak Force QNM bull Modulus fit on the fly ndash no ability to monitor fit (or range) bull Local sample topography and sample tilt play a role in

measured parameters bull Assuming all modulus values in an image are correct how to

quantify

512 x 512 pixels = 262144 measurements

n=1 n=262144

n from selected ROIs

BBML - All R

ights Reserve

d

BBML - All R

ights Reserve

d

bull Imaging in air on mica ndash substrate effect

bull Same imaging force or indent depth for all

bull For each area were all modulus values pooled

Lamprou et al PLoS ONE 2013 BBML - All R

ights Reserve

d

AFM Applications in Bone Research Other Cool Mechanical Techniques

BBML - All R

ights Reserve

d

ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo Bonds within or between collagen fibrils in bone that rupture to dissipate energy as a toughening mechanism

Thompson et al Nature 2001

Fantner et al Nature Mat 2005

Hansma et al JMNI 2005

Fantner et al Nano Letters 2007 BBML - All R

ights Reserve

d

Other Mechanical Techniques

Jimenez-Palomar J Mech Beh Bio Mat 2012

van der Rijt Macro Bio 2004

Yang J Biomed Mater Res 2007

Yang Biophysical Journal 2008 BBML - All R

ights Reserve

d

Other Cool Techniques bull Piezoresponse Force Microscopy (PFM)

bull Electrochemical AFM (EC-AFM)

bull Force Modulation Microscopy (FMM)

bull Lateral Force Microscopy (LFM)

bull Magnetic Force Microscopy (MFM)

bull Conductive AFM (C-AFM)

bull Force Pulling

bull Cantilever Bending

bull Tensile testing of individual fibrils

bull 3 point bending of fibrils

BBML - All R

ights Reserve

d

Summary Things to rememberhellip

bull Imaging mode imaging medium and probe selection

bull Morphology Measurements bull Sample preparation and hydration state bull Measuring fibril diameter bull Site selection and proper sampling bull Appropriate system calibration

bull Mechanical Measurements

bull Probe selection (stiffness and tip radius) bull Probe calibration (radius angle spring constant) bull Indentation force range and mechanical model bull Hydration in biological samples bull PeakForce QNM

BBML - All R

ights Reserve

d

Acknowledgements BBML

Max Hammond Armando Diaz Gonzalez Zachary Bart Silvia Canelon Creasy Clauser

IU School of Medicine Dr David Burr Dr Matt Allen Dr Stuart Warden Dr Lilian Plotkin

Funding Sources NIHNIDCR 1F32DE018840-01 A1 IUPUI BME Departmental Startup Funds IUPUI Research Support Funds Grant IUPUI Biomechanics and Biomaterials Research Center

NIH NICHD Dr Joan Marini

BBML - All R

ights Reserve

d

Bone Biology and Mechanics Lab (BBML) wwwiupuiedu~bbml Facebook BoneBiologyMechanicsLab

BBML - All R

ights Reserve

d

• Slide Number 1
• General Seminar Outline
• Bonersquos Hierarchical Structure
• Critical Gap in Understanding Collagen
• Methods For Analyzing Collagen Structure
• Methods For Imaging Collagen Structure
• Cryo-TEM
• Atomic Force Microscopy (AFM)
• AFM Image of Pentacene (C22H14)
• AFM Image of Hexabenzocoronene
• Basics of AFM Imaging The Probe
• Basics of AFM Imaging System Components
• van der Waals Potential Energy Curve
• Contact Mode ndash Monitor Deflection
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Peak Force Tapping Mode ndash Monitor Force
• AFM Applications in Bone Research Imaging
• AFM Imaging of Collagen
• Imaging of Osteoclast Activity
• Mineralized collagen deposition in vitro
• Imaging of Canilicular Structure
• AFM Imaging in Bone
• AFM Imaging in Bone
• Removing Mineral in Bone to Expose Collagen
• Removing Mineral in Bone to Expose Collagen
• Demineralization with EDTA
• Demineralization with EDTA
• Demineralization with EDTA
• Issues to Consider Effects of Acid
• Slide Number 32
• Slide Number 33
• Analysis of Collagen Structure in Bone
• Analysis of Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril D-Spacing
• Slide Number 39
• Collagen in Bone Fibril D-Spacing
• Collagen in Bone Fibril D-Spacing
• 2D Fast Fourier Transform Analysis
• 2D Fast Fourier Transform Analysis
• D-Spacing Distribution
• D-Spacing Distribution
• Cumulative Distribution Function
• CDF vs Histogram
• Changes in D-Spacing with Disease
• Changes in D-Spacing with Disease
• Slide Number 50
• Issues to Consider Site Selection and Sampling
• Issues to Consider Site Selection and Sampling
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• AFM calibration ndash Take Away Message
• AFM Applications in Bone ResearchIndentation Mechanics
• AFM-Based Indentation
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Parameters
• Hertz Contact Equation
• Hertz Contact Equation
• Hertz Contact Equation
• Proper Indentation Model is Essential
• Indentation in OI Tendon Study
• OI Tendon Differential Effects of Dehydration
• OI Tendon Differential Effects of Dehydration
• Indentation in Diabetic Tendon (Poster 0495)
• Multiscale Diabetes Study (Poster 0495)
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• A Note on Peak Force QNM for Bruker AFMs
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• Slide Number 83
• Slide Number 84
• AFM Applications in Bone ResearchOther Cool Mechanical Techniques
• ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo
• Other Mechanical Techniques
• Other Cool Techniques
• Summary Things to rememberhellip
• Acknowledgements
• Bone Biology and Mechanics Lab (BBML)

Hertz Contact Equation

wwwwikipediacom

z

r

δ

R

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119877119877 ∙ 12057512057532

Es Sample modulus νs Poissonrsquos ratio R tip radius of curvature δ load point displacement

Relevant Hertz Assumptions bull Strains are small and within the elastic limit bull Indenter is infinitely stiff (only sample deforms) bull Indentation depth ltlt radius of curvature of indenter bull Surfaces are continuous non-conforming (must be flat) bull Load is normal to surface

BBML - All R

ights Reserve

d

Proper Indentation Model is Essential

δ

r

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119903119903 ∙ 12057512057532

δ

α

119865119865 =2120587120587∙

1198641198641199041199041 minus 1205841205841199041199042

∙ tan120572120572 ∙ 1205751205752

Es Sample modulus νs Poissonrsquos ratio R tip radius α opening angle

Hertz Model Spherical Indenter

depth ltlt radius of curvature

Sneddon Model Conical Indenter

depth ge radius of curvature

BBML - All R

ights Reserve

d

Indentation in OI Tendon Study

x

z

yBrtl+ and WT mice 6 month old males

4 tails per group

2-3 fascicles per tail

25-30 fibrils per tail

4-5 locations per fibril

Indent force 20 nN wet (Sneddon) 50 nN dry (Hertz) BBML - A

ll Rights R

eserved

OI Tendon Differential Effects of Dehydration

Dry 1268 plusmn 718 MPa

Wet 305 plusmn 243 MPa

Dry 1737 plusmn 973 MPa

Wet 301 plusmn 202 MPa

Kemp et al J StructBio 2012 180 (3) 428-438

bull Significant stiffening of fibrils with dehydration

bull More pronounced modulus modulus as a function of disease

BBML - All R

ights Reserve

d

OI Tendon Differential Effects of Dehydration

Kemp et al J StructBio 2012 180 (3) 428-438

Diseased phenotype not present when wet phenotype observed when dried

0

10

20

30

40

50

60

0 20 40 60 80 100 120 140 160 180 200

Gro

up S

ampl

es (

)

Indent Modulus (MPa)

WT WetBrtl Wet

0

2

4

6

8

10

12

14

16

18

20

025

050

075

010

0012

5015

0017

5020

0022

5025

0027

5030

0032

5035

0037

5040

0042

5045

0047

5050

00

Gro

up S

ampl

es (

)Indent Modulus (MPa)

WT DryBrtl Dry

555 WT Indents 305 plusmn 243 MPa

614 Brtl+ Indents 301 plusmn 202 MPa

KS Test p=0129 t Test p=0464 464 WT Indents

1268 plusmn 718 MPa 479 Brtl+ Indents 1737 plusmn 973 MPa

KS Test plt0001 T Test plt0001

BBML - All R

ights Reserve

d

Indentation in Diabetic Tendon (Poster 0495)

x

z

y

Control and Diabetic Rat Tail Tendon

4-5 rats per group

2-3 fascicles per rat

~70 fibrilstail

4-5 locationsfibril

20 nN (Sneddon)

BBML - All R

ights Reserve

d

Multiscale Diabetes Study (Poster 0495)

0

5

10

15

20

25

300 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 gt25

Perc

enta

ge o

f Gro

up S

ampl

es

Elastic Modulus (MPa)

CD (1530 indents)ZDSD (913 indents)

CD (n=5) 381 plusmn 149 MPa

ZDSD (n=3) 706 plusmn 263 MPa

0

100

200

300

400

500

600

700

800

900

0 2 4 6 8 10

Mic

rosc

ale

Mod

ulus

(MPa

)

Nanoscale Modulus (MPa)

plt0001

r2=0531 p=0040

bull Increased indentation modulus in diabetic tendon

bull Strong relationship between nanoscale indent modulus and microscale tensile modulus

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

Proper probe selection for intended application bull Cantilever must be stiff relative to sample bull Sharp probes may damage sample easily dull

R or α must be determined for each probe bull Properties provided by manufacturer are unreliable bull Vary from probe to probe can dull during indent process

Spring constant must be determined for each probe

bull Value provided by manufacturer is inaccurate bull Thermal tuning OK for ldquosoftrdquo probes poor performance

for stiffer probes bull Sader method (geometry) added mass reference

cantilevers

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

Veeco Application Technical note Ben Ohler Practical Advice on the Determination of Cantilever Spring Constants

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

bull Sample processing

bull Sample hydration

bull Proper force range

bull Substrate effects for thin samples

bull Proper modulus model and range of data goodness of fit

Bearing in mind these assumptions and limitations AFM can provide true ldquonanoscalerdquo mechanical properties both in terms of forces and deformations

BBML - All R

ights Reserve

d

A Note on Peak Force QNM for Bruker AFMs bull Mechanical properties mapped pixel-by-pixel at same

resolution as the height image (PeakForce QNM)

Height Modulus BBML - All R

ights Reserve

d

A Note on Peak Force QNM

Correct probe stiffness must be used based on sample Pittenger ndash Veeco Product Bulletin

BBML - All R

ights Reserve

d

A Note on Peak Force QNM Extremely sensitive to calibration (details rarely given) Absolute radius and spring constant known

bull especially important from probe to probe bull radius uarr with indent depth ndash must determine R at depth

you expect to image bull Depth may vary as a function of sample (or at different

regions within the sample)

Relative reference material of ldquoknownrdquo modulus used to determine a ratio of 119896119896 119877119877 bull ldquoknownrdquo modulus may come from tension compression

and not indentation bull R is related to depth indentation depth on sample must

be same as on reference BBML - All R

ights Reserve

d

A Note on Peak Force QNM bull Modulus fit on the fly ndash no ability to monitor fit (or range) bull Local sample topography and sample tilt play a role in

measured parameters bull Assuming all modulus values in an image are correct how to

quantify

512 x 512 pixels = 262144 measurements

n=1 n=262144

n from selected ROIs

BBML - All R

ights Reserve

d

BBML - All R

ights Reserve

d

bull Imaging in air on mica ndash substrate effect

bull Same imaging force or indent depth for all

bull For each area were all modulus values pooled

Lamprou et al PLoS ONE 2013 BBML - All R

ights Reserve

d

AFM Applications in Bone Research Other Cool Mechanical Techniques

BBML - All R

ights Reserve

d

ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo Bonds within or between collagen fibrils in bone that rupture to dissipate energy as a toughening mechanism

Thompson et al Nature 2001

Fantner et al Nature Mat 2005

Hansma et al JMNI 2005

Fantner et al Nano Letters 2007 BBML - All R

ights Reserve

d

Other Mechanical Techniques

Jimenez-Palomar J Mech Beh Bio Mat 2012

van der Rijt Macro Bio 2004

Yang J Biomed Mater Res 2007

Yang Biophysical Journal 2008 BBML - All R

ights Reserve

d

Other Cool Techniques bull Piezoresponse Force Microscopy (PFM)

bull Electrochemical AFM (EC-AFM)

bull Force Modulation Microscopy (FMM)

bull Lateral Force Microscopy (LFM)

bull Magnetic Force Microscopy (MFM)

bull Conductive AFM (C-AFM)

bull Force Pulling

bull Cantilever Bending

bull Tensile testing of individual fibrils

bull 3 point bending of fibrils

BBML - All R

ights Reserve

d

Summary Things to rememberhellip

bull Imaging mode imaging medium and probe selection

bull Morphology Measurements bull Sample preparation and hydration state bull Measuring fibril diameter bull Site selection and proper sampling bull Appropriate system calibration

bull Mechanical Measurements

bull Probe selection (stiffness and tip radius) bull Probe calibration (radius angle spring constant) bull Indentation force range and mechanical model bull Hydration in biological samples bull PeakForce QNM

BBML - All R

ights Reserve

d

Acknowledgements BBML

Max Hammond Armando Diaz Gonzalez Zachary Bart Silvia Canelon Creasy Clauser

IU School of Medicine Dr David Burr Dr Matt Allen Dr Stuart Warden Dr Lilian Plotkin

Funding Sources NIHNIDCR 1F32DE018840-01 A1 IUPUI BME Departmental Startup Funds IUPUI Research Support Funds Grant IUPUI Biomechanics and Biomaterials Research Center

NIH NICHD Dr Joan Marini

BBML - All R

ights Reserve

d

Bone Biology and Mechanics Lab (BBML) wwwiupuiedu~bbml Facebook BoneBiologyMechanicsLab

BBML - All R

ights Reserve

d

• Slide Number 1
• General Seminar Outline
• Bonersquos Hierarchical Structure
• Critical Gap in Understanding Collagen
• Methods For Analyzing Collagen Structure
• Methods For Imaging Collagen Structure
• Cryo-TEM
• Atomic Force Microscopy (AFM)
• AFM Image of Pentacene (C22H14)
• AFM Image of Hexabenzocoronene
• Basics of AFM Imaging The Probe
• Basics of AFM Imaging System Components
• van der Waals Potential Energy Curve
• Contact Mode ndash Monitor Deflection
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Peak Force Tapping Mode ndash Monitor Force
• AFM Applications in Bone Research Imaging
• AFM Imaging of Collagen
• Imaging of Osteoclast Activity
• Mineralized collagen deposition in vitro
• Imaging of Canilicular Structure
• AFM Imaging in Bone
• AFM Imaging in Bone
• Removing Mineral in Bone to Expose Collagen
• Removing Mineral in Bone to Expose Collagen
• Demineralization with EDTA
• Demineralization with EDTA
• Demineralization with EDTA
• Issues to Consider Effects of Acid
• Slide Number 32
• Slide Number 33
• Analysis of Collagen Structure in Bone
• Analysis of Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril D-Spacing
• Slide Number 39
• Collagen in Bone Fibril D-Spacing
• Collagen in Bone Fibril D-Spacing
• 2D Fast Fourier Transform Analysis
• 2D Fast Fourier Transform Analysis
• D-Spacing Distribution
• D-Spacing Distribution
• Cumulative Distribution Function
• CDF vs Histogram
• Changes in D-Spacing with Disease
• Changes in D-Spacing with Disease
• Slide Number 50
• Issues to Consider Site Selection and Sampling
• Issues to Consider Site Selection and Sampling
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• AFM calibration ndash Take Away Message
• AFM Applications in Bone ResearchIndentation Mechanics
• AFM-Based Indentation
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Parameters
• Hertz Contact Equation
• Hertz Contact Equation
• Hertz Contact Equation
• Proper Indentation Model is Essential
• Indentation in OI Tendon Study
• OI Tendon Differential Effects of Dehydration
• OI Tendon Differential Effects of Dehydration
• Indentation in Diabetic Tendon (Poster 0495)
• Multiscale Diabetes Study (Poster 0495)
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• A Note on Peak Force QNM for Bruker AFMs
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• Slide Number 83
• Slide Number 84
• AFM Applications in Bone ResearchOther Cool Mechanical Techniques
• ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo
• Other Mechanical Techniques
• Other Cool Techniques
• Summary Things to rememberhellip
• Acknowledgements
• Bone Biology and Mechanics Lab (BBML)

Proper Indentation Model is Essential

δ

r

119865119865 =43∙

1198641198641199041199041 minus 1205841205841199041199042

∙ 119903119903 ∙ 12057512057532

δ

α

119865119865 =2120587120587∙

1198641198641199041199041 minus 1205841205841199041199042

∙ tan120572120572 ∙ 1205751205752

Es Sample modulus νs Poissonrsquos ratio R tip radius α opening angle

Hertz Model Spherical Indenter

depth ltlt radius of curvature

Sneddon Model Conical Indenter

depth ge radius of curvature

BBML - All R

ights Reserve

d

Indentation in OI Tendon Study

x

z

yBrtl+ and WT mice 6 month old males

4 tails per group

2-3 fascicles per tail

25-30 fibrils per tail

4-5 locations per fibril

Indent force 20 nN wet (Sneddon) 50 nN dry (Hertz) BBML - A

ll Rights R

eserved

OI Tendon Differential Effects of Dehydration

Dry 1268 plusmn 718 MPa

Wet 305 plusmn 243 MPa

Dry 1737 plusmn 973 MPa

Wet 301 plusmn 202 MPa

Kemp et al J StructBio 2012 180 (3) 428-438

bull Significant stiffening of fibrils with dehydration

bull More pronounced modulus modulus as a function of disease

BBML - All R

ights Reserve

d

OI Tendon Differential Effects of Dehydration

Kemp et al J StructBio 2012 180 (3) 428-438

Diseased phenotype not present when wet phenotype observed when dried

0

10

20

30

40

50

60

0 20 40 60 80 100 120 140 160 180 200

Gro

up S

ampl

es (

)

Indent Modulus (MPa)

WT WetBrtl Wet

0

2

4

6

8

10

12

14

16

18

20

025

050

075

010

0012

5015

0017

5020

0022

5025

0027

5030

0032

5035

0037

5040

0042

5045

0047

5050

00

Gro

up S

ampl

es (

)Indent Modulus (MPa)

WT DryBrtl Dry

555 WT Indents 305 plusmn 243 MPa

614 Brtl+ Indents 301 plusmn 202 MPa

KS Test p=0129 t Test p=0464 464 WT Indents

1268 plusmn 718 MPa 479 Brtl+ Indents 1737 plusmn 973 MPa

KS Test plt0001 T Test plt0001

BBML - All R

ights Reserve

d

Indentation in Diabetic Tendon (Poster 0495)

x

z

y

Control and Diabetic Rat Tail Tendon

4-5 rats per group

2-3 fascicles per rat

~70 fibrilstail

4-5 locationsfibril

20 nN (Sneddon)

BBML - All R

ights Reserve

d

Multiscale Diabetes Study (Poster 0495)

0

5

10

15

20

25

300 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 gt25

Perc

enta

ge o

f Gro

up S

ampl

es

Elastic Modulus (MPa)

CD (1530 indents)ZDSD (913 indents)

CD (n=5) 381 plusmn 149 MPa

ZDSD (n=3) 706 plusmn 263 MPa

0

100

200

300

400

500

600

700

800

900

0 2 4 6 8 10

Mic

rosc

ale

Mod

ulus

(MPa

)

Nanoscale Modulus (MPa)

plt0001

r2=0531 p=0040

bull Increased indentation modulus in diabetic tendon

bull Strong relationship between nanoscale indent modulus and microscale tensile modulus

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

Proper probe selection for intended application bull Cantilever must be stiff relative to sample bull Sharp probes may damage sample easily dull

R or α must be determined for each probe bull Properties provided by manufacturer are unreliable bull Vary from probe to probe can dull during indent process

Spring constant must be determined for each probe

bull Value provided by manufacturer is inaccurate bull Thermal tuning OK for ldquosoftrdquo probes poor performance

for stiffer probes bull Sader method (geometry) added mass reference

cantilevers

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

Veeco Application Technical note Ben Ohler Practical Advice on the Determination of Cantilever Spring Constants

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

bull Sample processing

bull Sample hydration

bull Proper force range

bull Substrate effects for thin samples

bull Proper modulus model and range of data goodness of fit

Bearing in mind these assumptions and limitations AFM can provide true ldquonanoscalerdquo mechanical properties both in terms of forces and deformations

BBML - All R

ights Reserve

d

A Note on Peak Force QNM for Bruker AFMs bull Mechanical properties mapped pixel-by-pixel at same

resolution as the height image (PeakForce QNM)

Height Modulus BBML - All R

ights Reserve

d

A Note on Peak Force QNM

Correct probe stiffness must be used based on sample Pittenger ndash Veeco Product Bulletin

BBML - All R

ights Reserve

d

A Note on Peak Force QNM Extremely sensitive to calibration (details rarely given) Absolute radius and spring constant known

bull especially important from probe to probe bull radius uarr with indent depth ndash must determine R at depth

you expect to image bull Depth may vary as a function of sample (or at different

regions within the sample)

Relative reference material of ldquoknownrdquo modulus used to determine a ratio of 119896119896 119877119877 bull ldquoknownrdquo modulus may come from tension compression

and not indentation bull R is related to depth indentation depth on sample must

be same as on reference BBML - All R

ights Reserve

d

A Note on Peak Force QNM bull Modulus fit on the fly ndash no ability to monitor fit (or range) bull Local sample topography and sample tilt play a role in

measured parameters bull Assuming all modulus values in an image are correct how to

quantify

512 x 512 pixels = 262144 measurements

n=1 n=262144

n from selected ROIs

BBML - All R

ights Reserve

d

BBML - All R

ights Reserve

d

bull Imaging in air on mica ndash substrate effect

bull Same imaging force or indent depth for all

bull For each area were all modulus values pooled

Lamprou et al PLoS ONE 2013 BBML - All R

ights Reserve

d

AFM Applications in Bone Research Other Cool Mechanical Techniques

BBML - All R

ights Reserve

d

ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo Bonds within or between collagen fibrils in bone that rupture to dissipate energy as a toughening mechanism

Thompson et al Nature 2001

Fantner et al Nature Mat 2005

Hansma et al JMNI 2005

Fantner et al Nano Letters 2007 BBML - All R

ights Reserve

d

Other Mechanical Techniques

Jimenez-Palomar J Mech Beh Bio Mat 2012

van der Rijt Macro Bio 2004

Yang J Biomed Mater Res 2007

Yang Biophysical Journal 2008 BBML - All R

ights Reserve

d

Other Cool Techniques bull Piezoresponse Force Microscopy (PFM)

bull Electrochemical AFM (EC-AFM)

bull Force Modulation Microscopy (FMM)

bull Lateral Force Microscopy (LFM)

bull Magnetic Force Microscopy (MFM)

bull Conductive AFM (C-AFM)

bull Force Pulling

bull Cantilever Bending

bull Tensile testing of individual fibrils

bull 3 point bending of fibrils

BBML - All R

ights Reserve

d

Summary Things to rememberhellip

bull Imaging mode imaging medium and probe selection

bull Morphology Measurements bull Sample preparation and hydration state bull Measuring fibril diameter bull Site selection and proper sampling bull Appropriate system calibration

bull Mechanical Measurements

bull Probe selection (stiffness and tip radius) bull Probe calibration (radius angle spring constant) bull Indentation force range and mechanical model bull Hydration in biological samples bull PeakForce QNM

BBML - All R

ights Reserve

d

Acknowledgements BBML

Max Hammond Armando Diaz Gonzalez Zachary Bart Silvia Canelon Creasy Clauser

IU School of Medicine Dr David Burr Dr Matt Allen Dr Stuart Warden Dr Lilian Plotkin

Funding Sources NIHNIDCR 1F32DE018840-01 A1 IUPUI BME Departmental Startup Funds IUPUI Research Support Funds Grant IUPUI Biomechanics and Biomaterials Research Center

NIH NICHD Dr Joan Marini

BBML - All R

ights Reserve

d

Bone Biology and Mechanics Lab (BBML) wwwiupuiedu~bbml Facebook BoneBiologyMechanicsLab

BBML - All R

ights Reserve

d

• Slide Number 1
• General Seminar Outline
• Bonersquos Hierarchical Structure
• Critical Gap in Understanding Collagen
• Methods For Analyzing Collagen Structure
• Methods For Imaging Collagen Structure
• Cryo-TEM
• Atomic Force Microscopy (AFM)
• AFM Image of Pentacene (C22H14)
• AFM Image of Hexabenzocoronene
• Basics of AFM Imaging The Probe
• Basics of AFM Imaging System Components
• van der Waals Potential Energy Curve
• Contact Mode ndash Monitor Deflection
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Peak Force Tapping Mode ndash Monitor Force
• AFM Applications in Bone Research Imaging
• AFM Imaging of Collagen
• Imaging of Osteoclast Activity
• Mineralized collagen deposition in vitro
• Imaging of Canilicular Structure
• AFM Imaging in Bone
• AFM Imaging in Bone
• Removing Mineral in Bone to Expose Collagen
• Removing Mineral in Bone to Expose Collagen
• Demineralization with EDTA
• Demineralization with EDTA
• Demineralization with EDTA
• Issues to Consider Effects of Acid
• Slide Number 32
• Slide Number 33
• Analysis of Collagen Structure in Bone
• Analysis of Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril D-Spacing
• Slide Number 39
• Collagen in Bone Fibril D-Spacing
• Collagen in Bone Fibril D-Spacing
• 2D Fast Fourier Transform Analysis
• 2D Fast Fourier Transform Analysis
• D-Spacing Distribution
• D-Spacing Distribution
• Cumulative Distribution Function
• CDF vs Histogram
• Changes in D-Spacing with Disease
• Changes in D-Spacing with Disease
• Slide Number 50
• Issues to Consider Site Selection and Sampling
• Issues to Consider Site Selection and Sampling
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• AFM calibration ndash Take Away Message
• AFM Applications in Bone ResearchIndentation Mechanics
• AFM-Based Indentation
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Parameters
• Hertz Contact Equation
• Hertz Contact Equation
• Hertz Contact Equation
• Proper Indentation Model is Essential
• Indentation in OI Tendon Study
• OI Tendon Differential Effects of Dehydration
• OI Tendon Differential Effects of Dehydration
• Indentation in Diabetic Tendon (Poster 0495)
• Multiscale Diabetes Study (Poster 0495)
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• A Note on Peak Force QNM for Bruker AFMs
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• Slide Number 83
• Slide Number 84
• AFM Applications in Bone ResearchOther Cool Mechanical Techniques
• ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo
• Other Mechanical Techniques
• Other Cool Techniques
• Summary Things to rememberhellip
• Acknowledgements
• Bone Biology and Mechanics Lab (BBML)

Indentation in OI Tendon Study

x

z

yBrtl+ and WT mice 6 month old males

4 tails per group

2-3 fascicles per tail

25-30 fibrils per tail

4-5 locations per fibril

Indent force 20 nN wet (Sneddon) 50 nN dry (Hertz) BBML - A

ll Rights R

eserved

OI Tendon Differential Effects of Dehydration

Dry 1268 plusmn 718 MPa

Wet 305 plusmn 243 MPa

Dry 1737 plusmn 973 MPa

Wet 301 plusmn 202 MPa

Kemp et al J StructBio 2012 180 (3) 428-438

bull Significant stiffening of fibrils with dehydration

bull More pronounced modulus modulus as a function of disease

BBML - All R

ights Reserve

d

OI Tendon Differential Effects of Dehydration

Kemp et al J StructBio 2012 180 (3) 428-438

Diseased phenotype not present when wet phenotype observed when dried

0

10

20

30

40

50

60

0 20 40 60 80 100 120 140 160 180 200

Gro

up S

ampl

es (

)

Indent Modulus (MPa)

WT WetBrtl Wet

0

2

4

6

8

10

12

14

16

18

20

025

050

075

010

0012

5015

0017

5020

0022

5025

0027

5030

0032

5035

0037

5040

0042

5045

0047

5050

00

Gro

up S

ampl

es (

)Indent Modulus (MPa)

WT DryBrtl Dry

555 WT Indents 305 plusmn 243 MPa

614 Brtl+ Indents 301 plusmn 202 MPa

KS Test p=0129 t Test p=0464 464 WT Indents

1268 plusmn 718 MPa 479 Brtl+ Indents 1737 plusmn 973 MPa

KS Test plt0001 T Test plt0001

BBML - All R

ights Reserve

d

Indentation in Diabetic Tendon (Poster 0495)

x

z

y

Control and Diabetic Rat Tail Tendon

4-5 rats per group

2-3 fascicles per rat

~70 fibrilstail

4-5 locationsfibril

20 nN (Sneddon)

BBML - All R

ights Reserve

d

Multiscale Diabetes Study (Poster 0495)

0

5

10

15

20

25

300 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 gt25

Perc

enta

ge o

f Gro

up S

ampl

es

Elastic Modulus (MPa)

CD (1530 indents)ZDSD (913 indents)

CD (n=5) 381 plusmn 149 MPa

ZDSD (n=3) 706 plusmn 263 MPa

0

100

200

300

400

500

600

700

800

900

0 2 4 6 8 10

Mic

rosc

ale

Mod

ulus

(MPa

)

Nanoscale Modulus (MPa)

plt0001

r2=0531 p=0040

bull Increased indentation modulus in diabetic tendon

bull Strong relationship between nanoscale indent modulus and microscale tensile modulus

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

Proper probe selection for intended application bull Cantilever must be stiff relative to sample bull Sharp probes may damage sample easily dull

R or α must be determined for each probe bull Properties provided by manufacturer are unreliable bull Vary from probe to probe can dull during indent process

Spring constant must be determined for each probe

bull Value provided by manufacturer is inaccurate bull Thermal tuning OK for ldquosoftrdquo probes poor performance

for stiffer probes bull Sader method (geometry) added mass reference

cantilevers

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

Veeco Application Technical note Ben Ohler Practical Advice on the Determination of Cantilever Spring Constants

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

bull Sample processing

bull Sample hydration

bull Proper force range

bull Substrate effects for thin samples

bull Proper modulus model and range of data goodness of fit

Bearing in mind these assumptions and limitations AFM can provide true ldquonanoscalerdquo mechanical properties both in terms of forces and deformations

BBML - All R

ights Reserve

d

A Note on Peak Force QNM for Bruker AFMs bull Mechanical properties mapped pixel-by-pixel at same

resolution as the height image (PeakForce QNM)

Height Modulus BBML - All R

ights Reserve

d

A Note on Peak Force QNM

Correct probe stiffness must be used based on sample Pittenger ndash Veeco Product Bulletin

BBML - All R

ights Reserve

d

A Note on Peak Force QNM Extremely sensitive to calibration (details rarely given) Absolute radius and spring constant known

bull especially important from probe to probe bull radius uarr with indent depth ndash must determine R at depth

you expect to image bull Depth may vary as a function of sample (or at different

regions within the sample)

Relative reference material of ldquoknownrdquo modulus used to determine a ratio of 119896119896 119877119877 bull ldquoknownrdquo modulus may come from tension compression

and not indentation bull R is related to depth indentation depth on sample must

be same as on reference BBML - All R

ights Reserve

d

A Note on Peak Force QNM bull Modulus fit on the fly ndash no ability to monitor fit (or range) bull Local sample topography and sample tilt play a role in

measured parameters bull Assuming all modulus values in an image are correct how to

quantify

512 x 512 pixels = 262144 measurements

n=1 n=262144

n from selected ROIs

BBML - All R

ights Reserve

d

BBML - All R

ights Reserve

d

bull Imaging in air on mica ndash substrate effect

bull Same imaging force or indent depth for all

bull For each area were all modulus values pooled

Lamprou et al PLoS ONE 2013 BBML - All R

ights Reserve

d

AFM Applications in Bone Research Other Cool Mechanical Techniques

BBML - All R

ights Reserve

d

ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo Bonds within or between collagen fibrils in bone that rupture to dissipate energy as a toughening mechanism

Thompson et al Nature 2001

Fantner et al Nature Mat 2005

Hansma et al JMNI 2005

Fantner et al Nano Letters 2007 BBML - All R

ights Reserve

d

Other Mechanical Techniques

Jimenez-Palomar J Mech Beh Bio Mat 2012

van der Rijt Macro Bio 2004

Yang J Biomed Mater Res 2007

Yang Biophysical Journal 2008 BBML - All R

ights Reserve

d

Other Cool Techniques bull Piezoresponse Force Microscopy (PFM)

bull Electrochemical AFM (EC-AFM)

bull Force Modulation Microscopy (FMM)

bull Lateral Force Microscopy (LFM)

bull Magnetic Force Microscopy (MFM)

bull Conductive AFM (C-AFM)

bull Force Pulling

bull Cantilever Bending

bull Tensile testing of individual fibrils

bull 3 point bending of fibrils

BBML - All R

ights Reserve

d

Summary Things to rememberhellip

bull Imaging mode imaging medium and probe selection

bull Morphology Measurements bull Sample preparation and hydration state bull Measuring fibril diameter bull Site selection and proper sampling bull Appropriate system calibration

bull Mechanical Measurements

bull Probe selection (stiffness and tip radius) bull Probe calibration (radius angle spring constant) bull Indentation force range and mechanical model bull Hydration in biological samples bull PeakForce QNM

BBML - All R

ights Reserve

d

Acknowledgements BBML

Max Hammond Armando Diaz Gonzalez Zachary Bart Silvia Canelon Creasy Clauser

IU School of Medicine Dr David Burr Dr Matt Allen Dr Stuart Warden Dr Lilian Plotkin

Funding Sources NIHNIDCR 1F32DE018840-01 A1 IUPUI BME Departmental Startup Funds IUPUI Research Support Funds Grant IUPUI Biomechanics and Biomaterials Research Center

NIH NICHD Dr Joan Marini

BBML - All R

ights Reserve

d

Bone Biology and Mechanics Lab (BBML) wwwiupuiedu~bbml Facebook BoneBiologyMechanicsLab

BBML - All R

ights Reserve

d

• Slide Number 1
• General Seminar Outline
• Bonersquos Hierarchical Structure
• Critical Gap in Understanding Collagen
• Methods For Analyzing Collagen Structure
• Methods For Imaging Collagen Structure
• Cryo-TEM
• Atomic Force Microscopy (AFM)
• AFM Image of Pentacene (C22H14)
• AFM Image of Hexabenzocoronene
• Basics of AFM Imaging The Probe
• Basics of AFM Imaging System Components
• van der Waals Potential Energy Curve
• Contact Mode ndash Monitor Deflection
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Peak Force Tapping Mode ndash Monitor Force
• AFM Applications in Bone Research Imaging
• AFM Imaging of Collagen
• Imaging of Osteoclast Activity
• Mineralized collagen deposition in vitro
• Imaging of Canilicular Structure
• AFM Imaging in Bone
• AFM Imaging in Bone
• Removing Mineral in Bone to Expose Collagen
• Removing Mineral in Bone to Expose Collagen
• Demineralization with EDTA
• Demineralization with EDTA
• Demineralization with EDTA
• Issues to Consider Effects of Acid
• Slide Number 32
• Slide Number 33
• Analysis of Collagen Structure in Bone
• Analysis of Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril D-Spacing
• Slide Number 39
• Collagen in Bone Fibril D-Spacing
• Collagen in Bone Fibril D-Spacing
• 2D Fast Fourier Transform Analysis
• 2D Fast Fourier Transform Analysis
• D-Spacing Distribution
• D-Spacing Distribution
• Cumulative Distribution Function
• CDF vs Histogram
• Changes in D-Spacing with Disease
• Changes in D-Spacing with Disease
• Slide Number 50
• Issues to Consider Site Selection and Sampling
• Issues to Consider Site Selection and Sampling
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• AFM calibration ndash Take Away Message
• AFM Applications in Bone ResearchIndentation Mechanics
• AFM-Based Indentation
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Parameters
• Hertz Contact Equation
• Hertz Contact Equation
• Hertz Contact Equation
• Proper Indentation Model is Essential
• Indentation in OI Tendon Study
• OI Tendon Differential Effects of Dehydration
• OI Tendon Differential Effects of Dehydration
• Indentation in Diabetic Tendon (Poster 0495)
• Multiscale Diabetes Study (Poster 0495)
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• A Note on Peak Force QNM for Bruker AFMs
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• Slide Number 83
• Slide Number 84
• AFM Applications in Bone ResearchOther Cool Mechanical Techniques
• ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo
• Other Mechanical Techniques
• Other Cool Techniques
• Summary Things to rememberhellip
• Acknowledgements
• Bone Biology and Mechanics Lab (BBML)

OI Tendon Differential Effects of Dehydration

Dry 1268 plusmn 718 MPa

Wet 305 plusmn 243 MPa

Dry 1737 plusmn 973 MPa

Wet 301 plusmn 202 MPa

Kemp et al J StructBio 2012 180 (3) 428-438

bull Significant stiffening of fibrils with dehydration

bull More pronounced modulus modulus as a function of disease

BBML - All R

ights Reserve

d

OI Tendon Differential Effects of Dehydration

Kemp et al J StructBio 2012 180 (3) 428-438

Diseased phenotype not present when wet phenotype observed when dried

0

10

20

30

40

50

60

0 20 40 60 80 100 120 140 160 180 200

Gro

up S

ampl

es (

)

Indent Modulus (MPa)

WT WetBrtl Wet

0

2

4

6

8

10

12

14

16

18

20

025

050

075

010

0012

5015

0017

5020

0022

5025

0027

5030

0032

5035

0037

5040

0042

5045

0047

5050

00

Gro

up S

ampl

es (

)Indent Modulus (MPa)

WT DryBrtl Dry

555 WT Indents 305 plusmn 243 MPa

614 Brtl+ Indents 301 plusmn 202 MPa

KS Test p=0129 t Test p=0464 464 WT Indents

1268 plusmn 718 MPa 479 Brtl+ Indents 1737 plusmn 973 MPa

KS Test plt0001 T Test plt0001

BBML - All R

ights Reserve

d

Indentation in Diabetic Tendon (Poster 0495)

x

z

y

Control and Diabetic Rat Tail Tendon

4-5 rats per group

2-3 fascicles per rat

~70 fibrilstail

4-5 locationsfibril

20 nN (Sneddon)

BBML - All R

ights Reserve

d

Multiscale Diabetes Study (Poster 0495)

0

5

10

15

20

25

300 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 gt25

Perc

enta

ge o

f Gro

up S

ampl

es

Elastic Modulus (MPa)

CD (1530 indents)ZDSD (913 indents)

CD (n=5) 381 plusmn 149 MPa

ZDSD (n=3) 706 plusmn 263 MPa

0

100

200

300

400

500

600

700

800

900

0 2 4 6 8 10

Mic

rosc

ale

Mod

ulus

(MPa

)

Nanoscale Modulus (MPa)

plt0001

r2=0531 p=0040

bull Increased indentation modulus in diabetic tendon

bull Strong relationship between nanoscale indent modulus and microscale tensile modulus

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

Proper probe selection for intended application bull Cantilever must be stiff relative to sample bull Sharp probes may damage sample easily dull

R or α must be determined for each probe bull Properties provided by manufacturer are unreliable bull Vary from probe to probe can dull during indent process

Spring constant must be determined for each probe

bull Value provided by manufacturer is inaccurate bull Thermal tuning OK for ldquosoftrdquo probes poor performance

for stiffer probes bull Sader method (geometry) added mass reference

cantilevers

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

Veeco Application Technical note Ben Ohler Practical Advice on the Determination of Cantilever Spring Constants

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

bull Sample processing

bull Sample hydration

bull Proper force range

bull Substrate effects for thin samples

bull Proper modulus model and range of data goodness of fit

Bearing in mind these assumptions and limitations AFM can provide true ldquonanoscalerdquo mechanical properties both in terms of forces and deformations

BBML - All R

ights Reserve

d

A Note on Peak Force QNM for Bruker AFMs bull Mechanical properties mapped pixel-by-pixel at same

resolution as the height image (PeakForce QNM)

Height Modulus BBML - All R

ights Reserve

d

A Note on Peak Force QNM

Correct probe stiffness must be used based on sample Pittenger ndash Veeco Product Bulletin

BBML - All R

ights Reserve

d

A Note on Peak Force QNM Extremely sensitive to calibration (details rarely given) Absolute radius and spring constant known

bull especially important from probe to probe bull radius uarr with indent depth ndash must determine R at depth

you expect to image bull Depth may vary as a function of sample (or at different

regions within the sample)

Relative reference material of ldquoknownrdquo modulus used to determine a ratio of 119896119896 119877119877 bull ldquoknownrdquo modulus may come from tension compression

and not indentation bull R is related to depth indentation depth on sample must

be same as on reference BBML - All R

ights Reserve

d

A Note on Peak Force QNM bull Modulus fit on the fly ndash no ability to monitor fit (or range) bull Local sample topography and sample tilt play a role in

measured parameters bull Assuming all modulus values in an image are correct how to

quantify

512 x 512 pixels = 262144 measurements

n=1 n=262144

n from selected ROIs

BBML - All R

ights Reserve

d

BBML - All R

ights Reserve

d

bull Imaging in air on mica ndash substrate effect

bull Same imaging force or indent depth for all

bull For each area were all modulus values pooled

Lamprou et al PLoS ONE 2013 BBML - All R

ights Reserve

d

AFM Applications in Bone Research Other Cool Mechanical Techniques

BBML - All R

ights Reserve

d

ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo Bonds within or between collagen fibrils in bone that rupture to dissipate energy as a toughening mechanism

Thompson et al Nature 2001

Fantner et al Nature Mat 2005

Hansma et al JMNI 2005

Fantner et al Nano Letters 2007 BBML - All R

ights Reserve

d

Other Mechanical Techniques

Jimenez-Palomar J Mech Beh Bio Mat 2012

van der Rijt Macro Bio 2004

Yang J Biomed Mater Res 2007

Yang Biophysical Journal 2008 BBML - All R

ights Reserve

d

Other Cool Techniques bull Piezoresponse Force Microscopy (PFM)

bull Electrochemical AFM (EC-AFM)

bull Force Modulation Microscopy (FMM)

bull Lateral Force Microscopy (LFM)

bull Magnetic Force Microscopy (MFM)

bull Conductive AFM (C-AFM)

bull Force Pulling

bull Cantilever Bending

bull Tensile testing of individual fibrils

bull 3 point bending of fibrils

BBML - All R

ights Reserve

d

Summary Things to rememberhellip

bull Imaging mode imaging medium and probe selection

bull Morphology Measurements bull Sample preparation and hydration state bull Measuring fibril diameter bull Site selection and proper sampling bull Appropriate system calibration

bull Mechanical Measurements

bull Probe selection (stiffness and tip radius) bull Probe calibration (radius angle spring constant) bull Indentation force range and mechanical model bull Hydration in biological samples bull PeakForce QNM

BBML - All R

ights Reserve

d

Acknowledgements BBML

Max Hammond Armando Diaz Gonzalez Zachary Bart Silvia Canelon Creasy Clauser

IU School of Medicine Dr David Burr Dr Matt Allen Dr Stuart Warden Dr Lilian Plotkin

Funding Sources NIHNIDCR 1F32DE018840-01 A1 IUPUI BME Departmental Startup Funds IUPUI Research Support Funds Grant IUPUI Biomechanics and Biomaterials Research Center

NIH NICHD Dr Joan Marini

BBML - All R

ights Reserve

d

Bone Biology and Mechanics Lab (BBML) wwwiupuiedu~bbml Facebook BoneBiologyMechanicsLab

BBML - All R

ights Reserve

d

• Slide Number 1
• General Seminar Outline
• Bonersquos Hierarchical Structure
• Critical Gap in Understanding Collagen
• Methods For Analyzing Collagen Structure
• Methods For Imaging Collagen Structure
• Cryo-TEM
• Atomic Force Microscopy (AFM)
• AFM Image of Pentacene (C22H14)
• AFM Image of Hexabenzocoronene
• Basics of AFM Imaging The Probe
• Basics of AFM Imaging System Components
• van der Waals Potential Energy Curve
• Contact Mode ndash Monitor Deflection
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Peak Force Tapping Mode ndash Monitor Force
• AFM Applications in Bone Research Imaging
• AFM Imaging of Collagen
• Imaging of Osteoclast Activity
• Mineralized collagen deposition in vitro
• Imaging of Canilicular Structure
• AFM Imaging in Bone
• AFM Imaging in Bone
• Removing Mineral in Bone to Expose Collagen
• Removing Mineral in Bone to Expose Collagen
• Demineralization with EDTA
• Demineralization with EDTA
• Demineralization with EDTA
• Issues to Consider Effects of Acid
• Slide Number 32
• Slide Number 33
• Analysis of Collagen Structure in Bone
• Analysis of Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril D-Spacing
• Slide Number 39
• Collagen in Bone Fibril D-Spacing
• Collagen in Bone Fibril D-Spacing
• 2D Fast Fourier Transform Analysis
• 2D Fast Fourier Transform Analysis
• D-Spacing Distribution
• D-Spacing Distribution
• Cumulative Distribution Function
• CDF vs Histogram
• Changes in D-Spacing with Disease
• Changes in D-Spacing with Disease
• Slide Number 50
• Issues to Consider Site Selection and Sampling
• Issues to Consider Site Selection and Sampling
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• AFM calibration ndash Take Away Message
• AFM Applications in Bone ResearchIndentation Mechanics
• AFM-Based Indentation
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Parameters
• Hertz Contact Equation
• Hertz Contact Equation
• Hertz Contact Equation
• Proper Indentation Model is Essential
• Indentation in OI Tendon Study
• OI Tendon Differential Effects of Dehydration
• OI Tendon Differential Effects of Dehydration
• Indentation in Diabetic Tendon (Poster 0495)
• Multiscale Diabetes Study (Poster 0495)
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• A Note on Peak Force QNM for Bruker AFMs
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• Slide Number 83
• Slide Number 84
• AFM Applications in Bone ResearchOther Cool Mechanical Techniques
• ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo
• Other Mechanical Techniques
• Other Cool Techniques
• Summary Things to rememberhellip
• Acknowledgements
• Bone Biology and Mechanics Lab (BBML)

OI Tendon Differential Effects of Dehydration

Kemp et al J StructBio 2012 180 (3) 428-438

Diseased phenotype not present when wet phenotype observed when dried

0

10

20

30

40

50

60

0 20 40 60 80 100 120 140 160 180 200

Gro

up S

ampl

es (

)

Indent Modulus (MPa)

WT WetBrtl Wet

0

2

4

6

8

10

12

14

16

18

20

025

050

075

010

0012

5015

0017

5020

0022

5025

0027

5030

0032

5035

0037

5040

0042

5045

0047

5050

00

Gro

up S

ampl

es (

)Indent Modulus (MPa)

WT DryBrtl Dry

555 WT Indents 305 plusmn 243 MPa

614 Brtl+ Indents 301 plusmn 202 MPa

KS Test p=0129 t Test p=0464 464 WT Indents

1268 plusmn 718 MPa 479 Brtl+ Indents 1737 plusmn 973 MPa

KS Test plt0001 T Test plt0001

BBML - All R

ights Reserve

d

Indentation in Diabetic Tendon (Poster 0495)

x

z

y

Control and Diabetic Rat Tail Tendon

4-5 rats per group

2-3 fascicles per rat

~70 fibrilstail

4-5 locationsfibril

20 nN (Sneddon)

BBML - All R

ights Reserve

d

Multiscale Diabetes Study (Poster 0495)

0

5

10

15

20

25

300 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 gt25

Perc

enta

ge o

f Gro

up S

ampl

es

Elastic Modulus (MPa)

CD (1530 indents)ZDSD (913 indents)

CD (n=5) 381 plusmn 149 MPa

ZDSD (n=3) 706 plusmn 263 MPa

0

100

200

300

400

500

600

700

800

900

0 2 4 6 8 10

Mic

rosc

ale

Mod

ulus

(MPa

)

Nanoscale Modulus (MPa)

plt0001

r2=0531 p=0040

bull Increased indentation modulus in diabetic tendon

bull Strong relationship between nanoscale indent modulus and microscale tensile modulus

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

Proper probe selection for intended application bull Cantilever must be stiff relative to sample bull Sharp probes may damage sample easily dull

R or α must be determined for each probe bull Properties provided by manufacturer are unreliable bull Vary from probe to probe can dull during indent process

Spring constant must be determined for each probe

bull Value provided by manufacturer is inaccurate bull Thermal tuning OK for ldquosoftrdquo probes poor performance

for stiffer probes bull Sader method (geometry) added mass reference

cantilevers

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

Veeco Application Technical note Ben Ohler Practical Advice on the Determination of Cantilever Spring Constants

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

bull Sample processing

bull Sample hydration

bull Proper force range

bull Substrate effects for thin samples

bull Proper modulus model and range of data goodness of fit

Bearing in mind these assumptions and limitations AFM can provide true ldquonanoscalerdquo mechanical properties both in terms of forces and deformations

BBML - All R

ights Reserve

d

A Note on Peak Force QNM for Bruker AFMs bull Mechanical properties mapped pixel-by-pixel at same

resolution as the height image (PeakForce QNM)

Height Modulus BBML - All R

ights Reserve

d

A Note on Peak Force QNM

Correct probe stiffness must be used based on sample Pittenger ndash Veeco Product Bulletin

BBML - All R

ights Reserve

d

A Note on Peak Force QNM Extremely sensitive to calibration (details rarely given) Absolute radius and spring constant known

bull especially important from probe to probe bull radius uarr with indent depth ndash must determine R at depth

you expect to image bull Depth may vary as a function of sample (or at different

regions within the sample)

Relative reference material of ldquoknownrdquo modulus used to determine a ratio of 119896119896 119877119877 bull ldquoknownrdquo modulus may come from tension compression

and not indentation bull R is related to depth indentation depth on sample must

be same as on reference BBML - All R

ights Reserve

d

A Note on Peak Force QNM bull Modulus fit on the fly ndash no ability to monitor fit (or range) bull Local sample topography and sample tilt play a role in

measured parameters bull Assuming all modulus values in an image are correct how to

quantify

512 x 512 pixels = 262144 measurements

n=1 n=262144

n from selected ROIs

BBML - All R

ights Reserve

d

BBML - All R

ights Reserve

d

bull Imaging in air on mica ndash substrate effect

bull Same imaging force or indent depth for all

bull For each area were all modulus values pooled

Lamprou et al PLoS ONE 2013 BBML - All R

ights Reserve

d

AFM Applications in Bone Research Other Cool Mechanical Techniques

BBML - All R

ights Reserve

d

ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo Bonds within or between collagen fibrils in bone that rupture to dissipate energy as a toughening mechanism

Thompson et al Nature 2001

Fantner et al Nature Mat 2005

Hansma et al JMNI 2005

Fantner et al Nano Letters 2007 BBML - All R

ights Reserve

d

Other Mechanical Techniques

Jimenez-Palomar J Mech Beh Bio Mat 2012

van der Rijt Macro Bio 2004

Yang J Biomed Mater Res 2007

Yang Biophysical Journal 2008 BBML - All R

ights Reserve

d

Other Cool Techniques bull Piezoresponse Force Microscopy (PFM)

bull Electrochemical AFM (EC-AFM)

bull Force Modulation Microscopy (FMM)

bull Lateral Force Microscopy (LFM)

bull Magnetic Force Microscopy (MFM)

bull Conductive AFM (C-AFM)

bull Force Pulling

bull Cantilever Bending

bull Tensile testing of individual fibrils

bull 3 point bending of fibrils

BBML - All R

ights Reserve

d

Summary Things to rememberhellip

bull Imaging mode imaging medium and probe selection

bull Morphology Measurements bull Sample preparation and hydration state bull Measuring fibril diameter bull Site selection and proper sampling bull Appropriate system calibration

bull Mechanical Measurements

bull Probe selection (stiffness and tip radius) bull Probe calibration (radius angle spring constant) bull Indentation force range and mechanical model bull Hydration in biological samples bull PeakForce QNM

BBML - All R

ights Reserve

d

Acknowledgements BBML

Max Hammond Armando Diaz Gonzalez Zachary Bart Silvia Canelon Creasy Clauser

IU School of Medicine Dr David Burr Dr Matt Allen Dr Stuart Warden Dr Lilian Plotkin

Funding Sources NIHNIDCR 1F32DE018840-01 A1 IUPUI BME Departmental Startup Funds IUPUI Research Support Funds Grant IUPUI Biomechanics and Biomaterials Research Center

NIH NICHD Dr Joan Marini

BBML - All R

ights Reserve

d

Bone Biology and Mechanics Lab (BBML) wwwiupuiedu~bbml Facebook BoneBiologyMechanicsLab

BBML - All R

ights Reserve

d

• Slide Number 1
• General Seminar Outline
• Bonersquos Hierarchical Structure
• Critical Gap in Understanding Collagen
• Methods For Analyzing Collagen Structure
• Methods For Imaging Collagen Structure
• Cryo-TEM
• Atomic Force Microscopy (AFM)
• AFM Image of Pentacene (C22H14)
• AFM Image of Hexabenzocoronene
• Basics of AFM Imaging The Probe
• Basics of AFM Imaging System Components
• van der Waals Potential Energy Curve
• Contact Mode ndash Monitor Deflection
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Peak Force Tapping Mode ndash Monitor Force
• AFM Applications in Bone Research Imaging
• AFM Imaging of Collagen
• Imaging of Osteoclast Activity
• Mineralized collagen deposition in vitro
• Imaging of Canilicular Structure
• AFM Imaging in Bone
• AFM Imaging in Bone
• Removing Mineral in Bone to Expose Collagen
• Removing Mineral in Bone to Expose Collagen
• Demineralization with EDTA
• Demineralization with EDTA
• Demineralization with EDTA
• Issues to Consider Effects of Acid
• Slide Number 32
• Slide Number 33
• Analysis of Collagen Structure in Bone
• Analysis of Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril D-Spacing
• Slide Number 39
• Collagen in Bone Fibril D-Spacing
• Collagen in Bone Fibril D-Spacing
• 2D Fast Fourier Transform Analysis
• 2D Fast Fourier Transform Analysis
• D-Spacing Distribution
• D-Spacing Distribution
• Cumulative Distribution Function
• CDF vs Histogram
• Changes in D-Spacing with Disease
• Changes in D-Spacing with Disease
• Slide Number 50
• Issues to Consider Site Selection and Sampling
• Issues to Consider Site Selection and Sampling
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• AFM calibration ndash Take Away Message
• AFM Applications in Bone ResearchIndentation Mechanics
• AFM-Based Indentation
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Parameters
• Hertz Contact Equation
• Hertz Contact Equation
• Hertz Contact Equation
• Proper Indentation Model is Essential
• Indentation in OI Tendon Study
• OI Tendon Differential Effects of Dehydration
• OI Tendon Differential Effects of Dehydration
• Indentation in Diabetic Tendon (Poster 0495)
• Multiscale Diabetes Study (Poster 0495)
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• A Note on Peak Force QNM for Bruker AFMs
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• Slide Number 83
• Slide Number 84
• AFM Applications in Bone ResearchOther Cool Mechanical Techniques
• ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo
• Other Mechanical Techniques
• Other Cool Techniques
• Summary Things to rememberhellip
• Acknowledgements
• Bone Biology and Mechanics Lab (BBML)

Indentation in Diabetic Tendon (Poster 0495)

x

z

y

Control and Diabetic Rat Tail Tendon

4-5 rats per group

2-3 fascicles per rat

~70 fibrilstail

4-5 locationsfibril

20 nN (Sneddon)

BBML - All R

ights Reserve

d

Multiscale Diabetes Study (Poster 0495)

0

5

10

15

20

25

300 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 gt25

Perc

enta

ge o

f Gro

up S

ampl

es

Elastic Modulus (MPa)

CD (1530 indents)ZDSD (913 indents)

CD (n=5) 381 plusmn 149 MPa

ZDSD (n=3) 706 plusmn 263 MPa

0

100

200

300

400

500

600

700

800

900

0 2 4 6 8 10

Mic

rosc

ale

Mod

ulus

(MPa

)

Nanoscale Modulus (MPa)

plt0001

r2=0531 p=0040

bull Increased indentation modulus in diabetic tendon

bull Strong relationship between nanoscale indent modulus and microscale tensile modulus

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

Proper probe selection for intended application bull Cantilever must be stiff relative to sample bull Sharp probes may damage sample easily dull

R or α must be determined for each probe bull Properties provided by manufacturer are unreliable bull Vary from probe to probe can dull during indent process

Spring constant must be determined for each probe

bull Value provided by manufacturer is inaccurate bull Thermal tuning OK for ldquosoftrdquo probes poor performance

for stiffer probes bull Sader method (geometry) added mass reference

cantilevers

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

Veeco Application Technical note Ben Ohler Practical Advice on the Determination of Cantilever Spring Constants

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

bull Sample processing

bull Sample hydration

bull Proper force range

bull Substrate effects for thin samples

bull Proper modulus model and range of data goodness of fit

Bearing in mind these assumptions and limitations AFM can provide true ldquonanoscalerdquo mechanical properties both in terms of forces and deformations

BBML - All R

ights Reserve

d

A Note on Peak Force QNM for Bruker AFMs bull Mechanical properties mapped pixel-by-pixel at same

resolution as the height image (PeakForce QNM)

Height Modulus BBML - All R

ights Reserve

d

A Note on Peak Force QNM

Correct probe stiffness must be used based on sample Pittenger ndash Veeco Product Bulletin

BBML - All R

ights Reserve

d

A Note on Peak Force QNM Extremely sensitive to calibration (details rarely given) Absolute radius and spring constant known

bull especially important from probe to probe bull radius uarr with indent depth ndash must determine R at depth

you expect to image bull Depth may vary as a function of sample (or at different

regions within the sample)

Relative reference material of ldquoknownrdquo modulus used to determine a ratio of 119896119896 119877119877 bull ldquoknownrdquo modulus may come from tension compression

and not indentation bull R is related to depth indentation depth on sample must

be same as on reference BBML - All R

ights Reserve

d

A Note on Peak Force QNM bull Modulus fit on the fly ndash no ability to monitor fit (or range) bull Local sample topography and sample tilt play a role in

measured parameters bull Assuming all modulus values in an image are correct how to

quantify

512 x 512 pixels = 262144 measurements

n=1 n=262144

n from selected ROIs

BBML - All R

ights Reserve

d

BBML - All R

ights Reserve

d

bull Imaging in air on mica ndash substrate effect

bull Same imaging force or indent depth for all

bull For each area were all modulus values pooled

Lamprou et al PLoS ONE 2013 BBML - All R

ights Reserve

d

AFM Applications in Bone Research Other Cool Mechanical Techniques

BBML - All R

ights Reserve

d

ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo Bonds within or between collagen fibrils in bone that rupture to dissipate energy as a toughening mechanism

Thompson et al Nature 2001

Fantner et al Nature Mat 2005

Hansma et al JMNI 2005

Fantner et al Nano Letters 2007 BBML - All R

ights Reserve

d

Other Mechanical Techniques

Jimenez-Palomar J Mech Beh Bio Mat 2012

van der Rijt Macro Bio 2004

Yang J Biomed Mater Res 2007

Yang Biophysical Journal 2008 BBML - All R

ights Reserve

d

Other Cool Techniques bull Piezoresponse Force Microscopy (PFM)

bull Electrochemical AFM (EC-AFM)

bull Force Modulation Microscopy (FMM)

bull Lateral Force Microscopy (LFM)

bull Magnetic Force Microscopy (MFM)

bull Conductive AFM (C-AFM)

bull Force Pulling

bull Cantilever Bending

bull Tensile testing of individual fibrils

bull 3 point bending of fibrils

BBML - All R

ights Reserve

d

Summary Things to rememberhellip

bull Imaging mode imaging medium and probe selection

bull Morphology Measurements bull Sample preparation and hydration state bull Measuring fibril diameter bull Site selection and proper sampling bull Appropriate system calibration

bull Mechanical Measurements

bull Probe selection (stiffness and tip radius) bull Probe calibration (radius angle spring constant) bull Indentation force range and mechanical model bull Hydration in biological samples bull PeakForce QNM

BBML - All R

ights Reserve

d

Acknowledgements BBML

Max Hammond Armando Diaz Gonzalez Zachary Bart Silvia Canelon Creasy Clauser

IU School of Medicine Dr David Burr Dr Matt Allen Dr Stuart Warden Dr Lilian Plotkin

Funding Sources NIHNIDCR 1F32DE018840-01 A1 IUPUI BME Departmental Startup Funds IUPUI Research Support Funds Grant IUPUI Biomechanics and Biomaterials Research Center

NIH NICHD Dr Joan Marini

BBML - All R

ights Reserve

d

Bone Biology and Mechanics Lab (BBML) wwwiupuiedu~bbml Facebook BoneBiologyMechanicsLab

BBML - All R

ights Reserve

d

• Slide Number 1
• General Seminar Outline
• Bonersquos Hierarchical Structure
• Critical Gap in Understanding Collagen
• Methods For Analyzing Collagen Structure
• Methods For Imaging Collagen Structure
• Cryo-TEM
• Atomic Force Microscopy (AFM)
• AFM Image of Pentacene (C22H14)
• AFM Image of Hexabenzocoronene
• Basics of AFM Imaging The Probe
• Basics of AFM Imaging System Components
• van der Waals Potential Energy Curve
• Contact Mode ndash Monitor Deflection
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Peak Force Tapping Mode ndash Monitor Force
• AFM Applications in Bone Research Imaging
• AFM Imaging of Collagen
• Imaging of Osteoclast Activity
• Mineralized collagen deposition in vitro
• Imaging of Canilicular Structure
• AFM Imaging in Bone
• AFM Imaging in Bone
• Removing Mineral in Bone to Expose Collagen
• Removing Mineral in Bone to Expose Collagen
• Demineralization with EDTA
• Demineralization with EDTA
• Demineralization with EDTA
• Issues to Consider Effects of Acid
• Slide Number 32
• Slide Number 33
• Analysis of Collagen Structure in Bone
• Analysis of Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril D-Spacing
• Slide Number 39
• Collagen in Bone Fibril D-Spacing
• Collagen in Bone Fibril D-Spacing
• 2D Fast Fourier Transform Analysis
• 2D Fast Fourier Transform Analysis
• D-Spacing Distribution
• D-Spacing Distribution
• Cumulative Distribution Function
• CDF vs Histogram
• Changes in D-Spacing with Disease
• Changes in D-Spacing with Disease
• Slide Number 50
• Issues to Consider Site Selection and Sampling
• Issues to Consider Site Selection and Sampling
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• AFM calibration ndash Take Away Message
• AFM Applications in Bone ResearchIndentation Mechanics
• AFM-Based Indentation
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Parameters
• Hertz Contact Equation
• Hertz Contact Equation
• Hertz Contact Equation
• Proper Indentation Model is Essential
• Indentation in OI Tendon Study
• OI Tendon Differential Effects of Dehydration
• OI Tendon Differential Effects of Dehydration
• Indentation in Diabetic Tendon (Poster 0495)
• Multiscale Diabetes Study (Poster 0495)
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• A Note on Peak Force QNM for Bruker AFMs
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• Slide Number 83
• Slide Number 84
• AFM Applications in Bone ResearchOther Cool Mechanical Techniques
• ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo
• Other Mechanical Techniques
• Other Cool Techniques
• Summary Things to rememberhellip
• Acknowledgements
• Bone Biology and Mechanics Lab (BBML)

Multiscale Diabetes Study (Poster 0495)

0

5

10

15

20

25

300 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 gt25

Perc

enta

ge o

f Gro

up S

ampl

es

Elastic Modulus (MPa)

CD (1530 indents)ZDSD (913 indents)

CD (n=5) 381 plusmn 149 MPa

ZDSD (n=3) 706 plusmn 263 MPa

0

100

200

300

400

500

600

700

800

900

0 2 4 6 8 10

Mic

rosc

ale

Mod

ulus

(MPa

)

Nanoscale Modulus (MPa)

plt0001

r2=0531 p=0040

bull Increased indentation modulus in diabetic tendon

bull Strong relationship between nanoscale indent modulus and microscale tensile modulus

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

Proper probe selection for intended application bull Cantilever must be stiff relative to sample bull Sharp probes may damage sample easily dull

R or α must be determined for each probe bull Properties provided by manufacturer are unreliable bull Vary from probe to probe can dull during indent process

Spring constant must be determined for each probe

bull Value provided by manufacturer is inaccurate bull Thermal tuning OK for ldquosoftrdquo probes poor performance

for stiffer probes bull Sader method (geometry) added mass reference

cantilevers

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

Veeco Application Technical note Ben Ohler Practical Advice on the Determination of Cantilever Spring Constants

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

bull Sample processing

bull Sample hydration

bull Proper force range

bull Substrate effects for thin samples

bull Proper modulus model and range of data goodness of fit

Bearing in mind these assumptions and limitations AFM can provide true ldquonanoscalerdquo mechanical properties both in terms of forces and deformations

BBML - All R

ights Reserve

d

A Note on Peak Force QNM for Bruker AFMs bull Mechanical properties mapped pixel-by-pixel at same

resolution as the height image (PeakForce QNM)

Height Modulus BBML - All R

ights Reserve

d

A Note on Peak Force QNM

Correct probe stiffness must be used based on sample Pittenger ndash Veeco Product Bulletin

BBML - All R

ights Reserve

d

A Note on Peak Force QNM Extremely sensitive to calibration (details rarely given) Absolute radius and spring constant known

bull especially important from probe to probe bull radius uarr with indent depth ndash must determine R at depth

you expect to image bull Depth may vary as a function of sample (or at different

regions within the sample)

Relative reference material of ldquoknownrdquo modulus used to determine a ratio of 119896119896 119877119877 bull ldquoknownrdquo modulus may come from tension compression

and not indentation bull R is related to depth indentation depth on sample must

be same as on reference BBML - All R

ights Reserve

d

A Note on Peak Force QNM bull Modulus fit on the fly ndash no ability to monitor fit (or range) bull Local sample topography and sample tilt play a role in

measured parameters bull Assuming all modulus values in an image are correct how to

quantify

512 x 512 pixels = 262144 measurements

n=1 n=262144

n from selected ROIs

BBML - All R

ights Reserve

d

BBML - All R

ights Reserve

d

bull Imaging in air on mica ndash substrate effect

bull Same imaging force or indent depth for all

bull For each area were all modulus values pooled

Lamprou et al PLoS ONE 2013 BBML - All R

ights Reserve

d

AFM Applications in Bone Research Other Cool Mechanical Techniques

BBML - All R

ights Reserve

d

ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo Bonds within or between collagen fibrils in bone that rupture to dissipate energy as a toughening mechanism

Thompson et al Nature 2001

Fantner et al Nature Mat 2005

Hansma et al JMNI 2005

Fantner et al Nano Letters 2007 BBML - All R

ights Reserve

d

Other Mechanical Techniques

Jimenez-Palomar J Mech Beh Bio Mat 2012

van der Rijt Macro Bio 2004

Yang J Biomed Mater Res 2007

Yang Biophysical Journal 2008 BBML - All R

ights Reserve

d

Other Cool Techniques bull Piezoresponse Force Microscopy (PFM)

bull Electrochemical AFM (EC-AFM)

bull Force Modulation Microscopy (FMM)

bull Lateral Force Microscopy (LFM)

bull Magnetic Force Microscopy (MFM)

bull Conductive AFM (C-AFM)

bull Force Pulling

bull Cantilever Bending

bull Tensile testing of individual fibrils

bull 3 point bending of fibrils

BBML - All R

ights Reserve

d

Summary Things to rememberhellip

bull Imaging mode imaging medium and probe selection

bull Morphology Measurements bull Sample preparation and hydration state bull Measuring fibril diameter bull Site selection and proper sampling bull Appropriate system calibration

bull Mechanical Measurements

bull Probe selection (stiffness and tip radius) bull Probe calibration (radius angle spring constant) bull Indentation force range and mechanical model bull Hydration in biological samples bull PeakForce QNM

BBML - All R

ights Reserve

d

Acknowledgements BBML

Max Hammond Armando Diaz Gonzalez Zachary Bart Silvia Canelon Creasy Clauser

IU School of Medicine Dr David Burr Dr Matt Allen Dr Stuart Warden Dr Lilian Plotkin

Funding Sources NIHNIDCR 1F32DE018840-01 A1 IUPUI BME Departmental Startup Funds IUPUI Research Support Funds Grant IUPUI Biomechanics and Biomaterials Research Center

NIH NICHD Dr Joan Marini

BBML - All R

ights Reserve

d

Bone Biology and Mechanics Lab (BBML) wwwiupuiedu~bbml Facebook BoneBiologyMechanicsLab

BBML - All R

ights Reserve

d

• Slide Number 1
• General Seminar Outline
• Bonersquos Hierarchical Structure
• Critical Gap in Understanding Collagen
• Methods For Analyzing Collagen Structure
• Methods For Imaging Collagen Structure
• Cryo-TEM
• Atomic Force Microscopy (AFM)
• AFM Image of Pentacene (C22H14)
• AFM Image of Hexabenzocoronene
• Basics of AFM Imaging The Probe
• Basics of AFM Imaging System Components
• van der Waals Potential Energy Curve
• Contact Mode ndash Monitor Deflection
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Peak Force Tapping Mode ndash Monitor Force
• AFM Applications in Bone Research Imaging
• AFM Imaging of Collagen
• Imaging of Osteoclast Activity
• Mineralized collagen deposition in vitro
• Imaging of Canilicular Structure
• AFM Imaging in Bone
• AFM Imaging in Bone
• Removing Mineral in Bone to Expose Collagen
• Removing Mineral in Bone to Expose Collagen
• Demineralization with EDTA
• Demineralization with EDTA
• Demineralization with EDTA
• Issues to Consider Effects of Acid
• Slide Number 32
• Slide Number 33
• Analysis of Collagen Structure in Bone
• Analysis of Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril D-Spacing
• Slide Number 39
• Collagen in Bone Fibril D-Spacing
• Collagen in Bone Fibril D-Spacing
• 2D Fast Fourier Transform Analysis
• 2D Fast Fourier Transform Analysis
• D-Spacing Distribution
• D-Spacing Distribution
• Cumulative Distribution Function
• CDF vs Histogram
• Changes in D-Spacing with Disease
• Changes in D-Spacing with Disease
• Slide Number 50
• Issues to Consider Site Selection and Sampling
• Issues to Consider Site Selection and Sampling
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• AFM calibration ndash Take Away Message
• AFM Applications in Bone ResearchIndentation Mechanics
• AFM-Based Indentation
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Parameters
• Hertz Contact Equation
• Hertz Contact Equation
• Hertz Contact Equation
• Proper Indentation Model is Essential
• Indentation in OI Tendon Study
• OI Tendon Differential Effects of Dehydration
• OI Tendon Differential Effects of Dehydration
• Indentation in Diabetic Tendon (Poster 0495)
• Multiscale Diabetes Study (Poster 0495)
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• A Note on Peak Force QNM for Bruker AFMs
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• Slide Number 83
• Slide Number 84
• AFM Applications in Bone ResearchOther Cool Mechanical Techniques
• ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo
• Other Mechanical Techniques
• Other Cool Techniques
• Summary Things to rememberhellip
• Acknowledgements
• Bone Biology and Mechanics Lab (BBML)

Indentation Limitations and Weaknesses

Proper probe selection for intended application bull Cantilever must be stiff relative to sample bull Sharp probes may damage sample easily dull

R or α must be determined for each probe bull Properties provided by manufacturer are unreliable bull Vary from probe to probe can dull during indent process

Spring constant must be determined for each probe

bull Value provided by manufacturer is inaccurate bull Thermal tuning OK for ldquosoftrdquo probes poor performance

for stiffer probes bull Sader method (geometry) added mass reference

cantilevers

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

Veeco Application Technical note Ben Ohler Practical Advice on the Determination of Cantilever Spring Constants

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

bull Sample processing

bull Sample hydration

bull Proper force range

bull Substrate effects for thin samples

bull Proper modulus model and range of data goodness of fit

Bearing in mind these assumptions and limitations AFM can provide true ldquonanoscalerdquo mechanical properties both in terms of forces and deformations

BBML - All R

ights Reserve

d

A Note on Peak Force QNM for Bruker AFMs bull Mechanical properties mapped pixel-by-pixel at same

resolution as the height image (PeakForce QNM)

Height Modulus BBML - All R

ights Reserve

d

A Note on Peak Force QNM

Correct probe stiffness must be used based on sample Pittenger ndash Veeco Product Bulletin

BBML - All R

ights Reserve

d

A Note on Peak Force QNM Extremely sensitive to calibration (details rarely given) Absolute radius and spring constant known

bull especially important from probe to probe bull radius uarr with indent depth ndash must determine R at depth

you expect to image bull Depth may vary as a function of sample (or at different

regions within the sample)

Relative reference material of ldquoknownrdquo modulus used to determine a ratio of 119896119896 119877119877 bull ldquoknownrdquo modulus may come from tension compression

and not indentation bull R is related to depth indentation depth on sample must

be same as on reference BBML - All R

ights Reserve

d

A Note on Peak Force QNM bull Modulus fit on the fly ndash no ability to monitor fit (or range) bull Local sample topography and sample tilt play a role in

measured parameters bull Assuming all modulus values in an image are correct how to

quantify

512 x 512 pixels = 262144 measurements

n=1 n=262144

n from selected ROIs

BBML - All R

ights Reserve

d

BBML - All R

ights Reserve

d

bull Imaging in air on mica ndash substrate effect

bull Same imaging force or indent depth for all

bull For each area were all modulus values pooled

Lamprou et al PLoS ONE 2013 BBML - All R

ights Reserve

d

AFM Applications in Bone Research Other Cool Mechanical Techniques

BBML - All R

ights Reserve

d

ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo Bonds within or between collagen fibrils in bone that rupture to dissipate energy as a toughening mechanism

Thompson et al Nature 2001

Fantner et al Nature Mat 2005

Hansma et al JMNI 2005

Fantner et al Nano Letters 2007 BBML - All R

ights Reserve

d

Other Mechanical Techniques

Jimenez-Palomar J Mech Beh Bio Mat 2012

van der Rijt Macro Bio 2004

Yang J Biomed Mater Res 2007

Yang Biophysical Journal 2008 BBML - All R

ights Reserve

d

Other Cool Techniques bull Piezoresponse Force Microscopy (PFM)

bull Electrochemical AFM (EC-AFM)

bull Force Modulation Microscopy (FMM)

bull Lateral Force Microscopy (LFM)

bull Magnetic Force Microscopy (MFM)

bull Conductive AFM (C-AFM)

bull Force Pulling

bull Cantilever Bending

bull Tensile testing of individual fibrils

bull 3 point bending of fibrils

BBML - All R

ights Reserve

d

Summary Things to rememberhellip

bull Imaging mode imaging medium and probe selection

bull Morphology Measurements bull Sample preparation and hydration state bull Measuring fibril diameter bull Site selection and proper sampling bull Appropriate system calibration

bull Mechanical Measurements

bull Probe selection (stiffness and tip radius) bull Probe calibration (radius angle spring constant) bull Indentation force range and mechanical model bull Hydration in biological samples bull PeakForce QNM

BBML - All R

ights Reserve

d

Acknowledgements BBML

Max Hammond Armando Diaz Gonzalez Zachary Bart Silvia Canelon Creasy Clauser

IU School of Medicine Dr David Burr Dr Matt Allen Dr Stuart Warden Dr Lilian Plotkin

Funding Sources NIHNIDCR 1F32DE018840-01 A1 IUPUI BME Departmental Startup Funds IUPUI Research Support Funds Grant IUPUI Biomechanics and Biomaterials Research Center

NIH NICHD Dr Joan Marini

BBML - All R

ights Reserve

d

Bone Biology and Mechanics Lab (BBML) wwwiupuiedu~bbml Facebook BoneBiologyMechanicsLab

BBML - All R

ights Reserve

d

• Slide Number 1
• General Seminar Outline
• Bonersquos Hierarchical Structure
• Critical Gap in Understanding Collagen
• Methods For Analyzing Collagen Structure
• Methods For Imaging Collagen Structure
• Cryo-TEM
• Atomic Force Microscopy (AFM)
• AFM Image of Pentacene (C22H14)
• AFM Image of Hexabenzocoronene
• Basics of AFM Imaging The Probe
• Basics of AFM Imaging System Components
• van der Waals Potential Energy Curve
• Contact Mode ndash Monitor Deflection
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Peak Force Tapping Mode ndash Monitor Force
• AFM Applications in Bone Research Imaging
• AFM Imaging of Collagen
• Imaging of Osteoclast Activity
• Mineralized collagen deposition in vitro
• Imaging of Canilicular Structure
• AFM Imaging in Bone
• AFM Imaging in Bone
• Removing Mineral in Bone to Expose Collagen
• Removing Mineral in Bone to Expose Collagen
• Demineralization with EDTA
• Demineralization with EDTA
• Demineralization with EDTA
• Issues to Consider Effects of Acid
• Slide Number 32
• Slide Number 33
• Analysis of Collagen Structure in Bone
• Analysis of Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril D-Spacing
• Slide Number 39
• Collagen in Bone Fibril D-Spacing
• Collagen in Bone Fibril D-Spacing
• 2D Fast Fourier Transform Analysis
• 2D Fast Fourier Transform Analysis
• D-Spacing Distribution
• D-Spacing Distribution
• Cumulative Distribution Function
• CDF vs Histogram
• Changes in D-Spacing with Disease
• Changes in D-Spacing with Disease
• Slide Number 50
• Issues to Consider Site Selection and Sampling
• Issues to Consider Site Selection and Sampling
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• AFM calibration ndash Take Away Message
• AFM Applications in Bone ResearchIndentation Mechanics
• AFM-Based Indentation
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Parameters
• Hertz Contact Equation
• Hertz Contact Equation
• Hertz Contact Equation
• Proper Indentation Model is Essential
• Indentation in OI Tendon Study
• OI Tendon Differential Effects of Dehydration
• OI Tendon Differential Effects of Dehydration
• Indentation in Diabetic Tendon (Poster 0495)
• Multiscale Diabetes Study (Poster 0495)
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• A Note on Peak Force QNM for Bruker AFMs
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• Slide Number 83
• Slide Number 84
• AFM Applications in Bone ResearchOther Cool Mechanical Techniques
• ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo
• Other Mechanical Techniques
• Other Cool Techniques
• Summary Things to rememberhellip
• Acknowledgements
• Bone Biology and Mechanics Lab (BBML)

Indentation Limitations and Weaknesses

Veeco Application Technical note Ben Ohler Practical Advice on the Determination of Cantilever Spring Constants

BBML - All R

ights Reserve

d

Indentation Limitations and Weaknesses

bull Sample processing

bull Sample hydration

bull Proper force range

bull Substrate effects for thin samples

bull Proper modulus model and range of data goodness of fit

Bearing in mind these assumptions and limitations AFM can provide true ldquonanoscalerdquo mechanical properties both in terms of forces and deformations

BBML - All R

ights Reserve

d

A Note on Peak Force QNM for Bruker AFMs bull Mechanical properties mapped pixel-by-pixel at same

resolution as the height image (PeakForce QNM)

Height Modulus BBML - All R

ights Reserve

d

A Note on Peak Force QNM

Correct probe stiffness must be used based on sample Pittenger ndash Veeco Product Bulletin

BBML - All R

ights Reserve

d

A Note on Peak Force QNM Extremely sensitive to calibration (details rarely given) Absolute radius and spring constant known

bull especially important from probe to probe bull radius uarr with indent depth ndash must determine R at depth

you expect to image bull Depth may vary as a function of sample (or at different

regions within the sample)

Relative reference material of ldquoknownrdquo modulus used to determine a ratio of 119896119896 119877119877 bull ldquoknownrdquo modulus may come from tension compression

and not indentation bull R is related to depth indentation depth on sample must

be same as on reference BBML - All R

ights Reserve

d

A Note on Peak Force QNM bull Modulus fit on the fly ndash no ability to monitor fit (or range) bull Local sample topography and sample tilt play a role in

measured parameters bull Assuming all modulus values in an image are correct how to

quantify

512 x 512 pixels = 262144 measurements

n=1 n=262144

n from selected ROIs

BBML - All R

ights Reserve

d

BBML - All R

ights Reserve

d

bull Imaging in air on mica ndash substrate effect

bull Same imaging force or indent depth for all

bull For each area were all modulus values pooled

Lamprou et al PLoS ONE 2013 BBML - All R

ights Reserve

d

AFM Applications in Bone Research Other Cool Mechanical Techniques

BBML - All R

ights Reserve

d

ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo Bonds within or between collagen fibrils in bone that rupture to dissipate energy as a toughening mechanism

Thompson et al Nature 2001

Fantner et al Nature Mat 2005

Hansma et al JMNI 2005

Fantner et al Nano Letters 2007 BBML - All R

ights Reserve

d

Other Mechanical Techniques

Jimenez-Palomar J Mech Beh Bio Mat 2012

van der Rijt Macro Bio 2004

Yang J Biomed Mater Res 2007

Yang Biophysical Journal 2008 BBML - All R

ights Reserve

d

Other Cool Techniques bull Piezoresponse Force Microscopy (PFM)

bull Electrochemical AFM (EC-AFM)

bull Force Modulation Microscopy (FMM)

bull Lateral Force Microscopy (LFM)

bull Magnetic Force Microscopy (MFM)

bull Conductive AFM (C-AFM)

bull Force Pulling

bull Cantilever Bending

bull Tensile testing of individual fibrils

bull 3 point bending of fibrils

BBML - All R

ights Reserve

d

Summary Things to rememberhellip

bull Imaging mode imaging medium and probe selection

bull Morphology Measurements bull Sample preparation and hydration state bull Measuring fibril diameter bull Site selection and proper sampling bull Appropriate system calibration

bull Mechanical Measurements

bull Probe selection (stiffness and tip radius) bull Probe calibration (radius angle spring constant) bull Indentation force range and mechanical model bull Hydration in biological samples bull PeakForce QNM

BBML - All R

ights Reserve

d

Acknowledgements BBML

Max Hammond Armando Diaz Gonzalez Zachary Bart Silvia Canelon Creasy Clauser

IU School of Medicine Dr David Burr Dr Matt Allen Dr Stuart Warden Dr Lilian Plotkin

Funding Sources NIHNIDCR 1F32DE018840-01 A1 IUPUI BME Departmental Startup Funds IUPUI Research Support Funds Grant IUPUI Biomechanics and Biomaterials Research Center

NIH NICHD Dr Joan Marini

BBML - All R

ights Reserve

d

Bone Biology and Mechanics Lab (BBML) wwwiupuiedu~bbml Facebook BoneBiologyMechanicsLab

BBML - All R

ights Reserve

d

• Slide Number 1
• General Seminar Outline
• Bonersquos Hierarchical Structure
• Critical Gap in Understanding Collagen
• Methods For Analyzing Collagen Structure
• Methods For Imaging Collagen Structure
• Cryo-TEM
• Atomic Force Microscopy (AFM)
• AFM Image of Pentacene (C22H14)
• AFM Image of Hexabenzocoronene
• Basics of AFM Imaging The Probe
• Basics of AFM Imaging System Components
• van der Waals Potential Energy Curve
• Contact Mode ndash Monitor Deflection
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Peak Force Tapping Mode ndash Monitor Force
• AFM Applications in Bone Research Imaging
• AFM Imaging of Collagen
• Imaging of Osteoclast Activity
• Mineralized collagen deposition in vitro
• Imaging of Canilicular Structure
• AFM Imaging in Bone
• AFM Imaging in Bone
• Removing Mineral in Bone to Expose Collagen
• Removing Mineral in Bone to Expose Collagen
• Demineralization with EDTA
• Demineralization with EDTA
• Demineralization with EDTA
• Issues to Consider Effects of Acid
• Slide Number 32
• Slide Number 33
• Analysis of Collagen Structure in Bone
• Analysis of Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril D-Spacing
• Slide Number 39
• Collagen in Bone Fibril D-Spacing
• Collagen in Bone Fibril D-Spacing
• 2D Fast Fourier Transform Analysis
• 2D Fast Fourier Transform Analysis
• D-Spacing Distribution
• D-Spacing Distribution
• Cumulative Distribution Function
• CDF vs Histogram
• Changes in D-Spacing with Disease
• Changes in D-Spacing with Disease
• Slide Number 50
• Issues to Consider Site Selection and Sampling
• Issues to Consider Site Selection and Sampling
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• AFM calibration ndash Take Away Message
• AFM Applications in Bone ResearchIndentation Mechanics
• AFM-Based Indentation
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Parameters
• Hertz Contact Equation
• Hertz Contact Equation
• Hertz Contact Equation
• Proper Indentation Model is Essential
• Indentation in OI Tendon Study
• OI Tendon Differential Effects of Dehydration
• OI Tendon Differential Effects of Dehydration
• Indentation in Diabetic Tendon (Poster 0495)
• Multiscale Diabetes Study (Poster 0495)
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• A Note on Peak Force QNM for Bruker AFMs
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• Slide Number 83
• Slide Number 84
• AFM Applications in Bone ResearchOther Cool Mechanical Techniques
• ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo
• Other Mechanical Techniques
• Other Cool Techniques
• Summary Things to rememberhellip
• Acknowledgements
• Bone Biology and Mechanics Lab (BBML)

Indentation Limitations and Weaknesses

bull Sample processing

bull Sample hydration

bull Proper force range

bull Substrate effects for thin samples

bull Proper modulus model and range of data goodness of fit

Bearing in mind these assumptions and limitations AFM can provide true ldquonanoscalerdquo mechanical properties both in terms of forces and deformations

BBML - All R

ights Reserve

d

A Note on Peak Force QNM for Bruker AFMs bull Mechanical properties mapped pixel-by-pixel at same

resolution as the height image (PeakForce QNM)

Height Modulus BBML - All R

ights Reserve

d

A Note on Peak Force QNM

Correct probe stiffness must be used based on sample Pittenger ndash Veeco Product Bulletin

BBML - All R

ights Reserve

d

A Note on Peak Force QNM Extremely sensitive to calibration (details rarely given) Absolute radius and spring constant known

bull especially important from probe to probe bull radius uarr with indent depth ndash must determine R at depth

you expect to image bull Depth may vary as a function of sample (or at different

regions within the sample)

Relative reference material of ldquoknownrdquo modulus used to determine a ratio of 119896119896 119877119877 bull ldquoknownrdquo modulus may come from tension compression

and not indentation bull R is related to depth indentation depth on sample must

be same as on reference BBML - All R

ights Reserve

d

A Note on Peak Force QNM bull Modulus fit on the fly ndash no ability to monitor fit (or range) bull Local sample topography and sample tilt play a role in

measured parameters bull Assuming all modulus values in an image are correct how to

quantify

512 x 512 pixels = 262144 measurements

n=1 n=262144

n from selected ROIs

BBML - All R

ights Reserve

d

BBML - All R

ights Reserve

d

bull Imaging in air on mica ndash substrate effect

bull Same imaging force or indent depth for all

bull For each area were all modulus values pooled

Lamprou et al PLoS ONE 2013 BBML - All R

ights Reserve

d

AFM Applications in Bone Research Other Cool Mechanical Techniques

BBML - All R

ights Reserve

d

ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo Bonds within or between collagen fibrils in bone that rupture to dissipate energy as a toughening mechanism

Thompson et al Nature 2001

Fantner et al Nature Mat 2005

Hansma et al JMNI 2005

Fantner et al Nano Letters 2007 BBML - All R

ights Reserve

d

Other Mechanical Techniques

Jimenez-Palomar J Mech Beh Bio Mat 2012

van der Rijt Macro Bio 2004

Yang J Biomed Mater Res 2007

Yang Biophysical Journal 2008 BBML - All R

ights Reserve

d

Other Cool Techniques bull Piezoresponse Force Microscopy (PFM)

bull Electrochemical AFM (EC-AFM)

bull Force Modulation Microscopy (FMM)

bull Lateral Force Microscopy (LFM)

bull Magnetic Force Microscopy (MFM)

bull Conductive AFM (C-AFM)

bull Force Pulling

bull Cantilever Bending

bull Tensile testing of individual fibrils

bull 3 point bending of fibrils

BBML - All R

ights Reserve

d

Summary Things to rememberhellip

bull Imaging mode imaging medium and probe selection

bull Morphology Measurements bull Sample preparation and hydration state bull Measuring fibril diameter bull Site selection and proper sampling bull Appropriate system calibration

bull Mechanical Measurements

bull Probe selection (stiffness and tip radius) bull Probe calibration (radius angle spring constant) bull Indentation force range and mechanical model bull Hydration in biological samples bull PeakForce QNM

BBML - All R

ights Reserve

d

Acknowledgements BBML

Max Hammond Armando Diaz Gonzalez Zachary Bart Silvia Canelon Creasy Clauser

IU School of Medicine Dr David Burr Dr Matt Allen Dr Stuart Warden Dr Lilian Plotkin

Funding Sources NIHNIDCR 1F32DE018840-01 A1 IUPUI BME Departmental Startup Funds IUPUI Research Support Funds Grant IUPUI Biomechanics and Biomaterials Research Center

NIH NICHD Dr Joan Marini

BBML - All R

ights Reserve

d

Bone Biology and Mechanics Lab (BBML) wwwiupuiedu~bbml Facebook BoneBiologyMechanicsLab

BBML - All R

ights Reserve

d

• Slide Number 1
• General Seminar Outline
• Bonersquos Hierarchical Structure
• Critical Gap in Understanding Collagen
• Methods For Analyzing Collagen Structure
• Methods For Imaging Collagen Structure
• Cryo-TEM
• Atomic Force Microscopy (AFM)
• AFM Image of Pentacene (C22H14)
• AFM Image of Hexabenzocoronene
• Basics of AFM Imaging The Probe
• Basics of AFM Imaging System Components
• van der Waals Potential Energy Curve
• Contact Mode ndash Monitor Deflection
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Peak Force Tapping Mode ndash Monitor Force
• AFM Applications in Bone Research Imaging
• AFM Imaging of Collagen
• Imaging of Osteoclast Activity
• Mineralized collagen deposition in vitro
• Imaging of Canilicular Structure
• AFM Imaging in Bone
• AFM Imaging in Bone
• Removing Mineral in Bone to Expose Collagen
• Removing Mineral in Bone to Expose Collagen
• Demineralization with EDTA
• Demineralization with EDTA
• Demineralization with EDTA
• Issues to Consider Effects of Acid
• Slide Number 32
• Slide Number 33
• Analysis of Collagen Structure in Bone
• Analysis of Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril D-Spacing
• Slide Number 39
• Collagen in Bone Fibril D-Spacing
• Collagen in Bone Fibril D-Spacing
• 2D Fast Fourier Transform Analysis
• 2D Fast Fourier Transform Analysis
• D-Spacing Distribution
• D-Spacing Distribution
• Cumulative Distribution Function
• CDF vs Histogram
• Changes in D-Spacing with Disease
• Changes in D-Spacing with Disease
• Slide Number 50
• Issues to Consider Site Selection and Sampling
• Issues to Consider Site Selection and Sampling
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• AFM calibration ndash Take Away Message
• AFM Applications in Bone ResearchIndentation Mechanics
• AFM-Based Indentation
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Parameters
• Hertz Contact Equation
• Hertz Contact Equation
• Hertz Contact Equation
• Proper Indentation Model is Essential
• Indentation in OI Tendon Study
• OI Tendon Differential Effects of Dehydration
• OI Tendon Differential Effects of Dehydration
• Indentation in Diabetic Tendon (Poster 0495)
• Multiscale Diabetes Study (Poster 0495)
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• A Note on Peak Force QNM for Bruker AFMs
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• Slide Number 83
• Slide Number 84
• AFM Applications in Bone ResearchOther Cool Mechanical Techniques
• ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo
• Other Mechanical Techniques
• Other Cool Techniques
• Summary Things to rememberhellip
• Acknowledgements
• Bone Biology and Mechanics Lab (BBML)

A Note on Peak Force QNM for Bruker AFMs bull Mechanical properties mapped pixel-by-pixel at same

resolution as the height image (PeakForce QNM)

Height Modulus BBML - All R

ights Reserve

d

A Note on Peak Force QNM

Correct probe stiffness must be used based on sample Pittenger ndash Veeco Product Bulletin

BBML - All R

ights Reserve

d

A Note on Peak Force QNM Extremely sensitive to calibration (details rarely given) Absolute radius and spring constant known

bull especially important from probe to probe bull radius uarr with indent depth ndash must determine R at depth

you expect to image bull Depth may vary as a function of sample (or at different

regions within the sample)

Relative reference material of ldquoknownrdquo modulus used to determine a ratio of 119896119896 119877119877 bull ldquoknownrdquo modulus may come from tension compression

and not indentation bull R is related to depth indentation depth on sample must

be same as on reference BBML - All R

ights Reserve

d

A Note on Peak Force QNM bull Modulus fit on the fly ndash no ability to monitor fit (or range) bull Local sample topography and sample tilt play a role in

measured parameters bull Assuming all modulus values in an image are correct how to

quantify

512 x 512 pixels = 262144 measurements

n=1 n=262144

n from selected ROIs

BBML - All R

ights Reserve

d

BBML - All R

ights Reserve

d

bull Imaging in air on mica ndash substrate effect

bull Same imaging force or indent depth for all

bull For each area were all modulus values pooled

Lamprou et al PLoS ONE 2013 BBML - All R

ights Reserve

d

AFM Applications in Bone Research Other Cool Mechanical Techniques

BBML - All R

ights Reserve

d

ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo Bonds within or between collagen fibrils in bone that rupture to dissipate energy as a toughening mechanism

Thompson et al Nature 2001

Fantner et al Nature Mat 2005

Hansma et al JMNI 2005

Fantner et al Nano Letters 2007 BBML - All R

ights Reserve

d

Other Mechanical Techniques

Jimenez-Palomar J Mech Beh Bio Mat 2012

van der Rijt Macro Bio 2004

Yang J Biomed Mater Res 2007

Yang Biophysical Journal 2008 BBML - All R

ights Reserve

d

Other Cool Techniques bull Piezoresponse Force Microscopy (PFM)

bull Electrochemical AFM (EC-AFM)

bull Force Modulation Microscopy (FMM)

bull Lateral Force Microscopy (LFM)

bull Magnetic Force Microscopy (MFM)

bull Conductive AFM (C-AFM)

bull Force Pulling

bull Cantilever Bending

bull Tensile testing of individual fibrils

bull 3 point bending of fibrils

BBML - All R

ights Reserve

d

Summary Things to rememberhellip

bull Imaging mode imaging medium and probe selection

bull Morphology Measurements bull Sample preparation and hydration state bull Measuring fibril diameter bull Site selection and proper sampling bull Appropriate system calibration

bull Mechanical Measurements

bull Probe selection (stiffness and tip radius) bull Probe calibration (radius angle spring constant) bull Indentation force range and mechanical model bull Hydration in biological samples bull PeakForce QNM

BBML - All R

ights Reserve

d

Acknowledgements BBML

Max Hammond Armando Diaz Gonzalez Zachary Bart Silvia Canelon Creasy Clauser

IU School of Medicine Dr David Burr Dr Matt Allen Dr Stuart Warden Dr Lilian Plotkin

Funding Sources NIHNIDCR 1F32DE018840-01 A1 IUPUI BME Departmental Startup Funds IUPUI Research Support Funds Grant IUPUI Biomechanics and Biomaterials Research Center

NIH NICHD Dr Joan Marini

BBML - All R

ights Reserve

d

Bone Biology and Mechanics Lab (BBML) wwwiupuiedu~bbml Facebook BoneBiologyMechanicsLab

BBML - All R

ights Reserve

d

• Slide Number 1
• General Seminar Outline
• Bonersquos Hierarchical Structure
• Critical Gap in Understanding Collagen
• Methods For Analyzing Collagen Structure
• Methods For Imaging Collagen Structure
• Cryo-TEM
• Atomic Force Microscopy (AFM)
• AFM Image of Pentacene (C22H14)
• AFM Image of Hexabenzocoronene
• Basics of AFM Imaging The Probe
• Basics of AFM Imaging System Components
• van der Waals Potential Energy Curve
• Contact Mode ndash Monitor Deflection
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Peak Force Tapping Mode ndash Monitor Force
• AFM Applications in Bone Research Imaging
• AFM Imaging of Collagen
• Imaging of Osteoclast Activity
• Mineralized collagen deposition in vitro
• Imaging of Canilicular Structure
• AFM Imaging in Bone
• AFM Imaging in Bone
• Removing Mineral in Bone to Expose Collagen
• Removing Mineral in Bone to Expose Collagen
• Demineralization with EDTA
• Demineralization with EDTA
• Demineralization with EDTA
• Issues to Consider Effects of Acid
• Slide Number 32
• Slide Number 33
• Analysis of Collagen Structure in Bone
• Analysis of Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril D-Spacing
• Slide Number 39
• Collagen in Bone Fibril D-Spacing
• Collagen in Bone Fibril D-Spacing
• 2D Fast Fourier Transform Analysis
• 2D Fast Fourier Transform Analysis
• D-Spacing Distribution
• D-Spacing Distribution
• Cumulative Distribution Function
• CDF vs Histogram
• Changes in D-Spacing with Disease
• Changes in D-Spacing with Disease
• Slide Number 50
• Issues to Consider Site Selection and Sampling
• Issues to Consider Site Selection and Sampling
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• AFM calibration ndash Take Away Message
• AFM Applications in Bone ResearchIndentation Mechanics
• AFM-Based Indentation
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Parameters
• Hertz Contact Equation
• Hertz Contact Equation
• Hertz Contact Equation
• Proper Indentation Model is Essential
• Indentation in OI Tendon Study
• OI Tendon Differential Effects of Dehydration
• OI Tendon Differential Effects of Dehydration
• Indentation in Diabetic Tendon (Poster 0495)
• Multiscale Diabetes Study (Poster 0495)
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• A Note on Peak Force QNM for Bruker AFMs
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• Slide Number 83
• Slide Number 84
• AFM Applications in Bone ResearchOther Cool Mechanical Techniques
• ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo
• Other Mechanical Techniques
• Other Cool Techniques
• Summary Things to rememberhellip
• Acknowledgements
• Bone Biology and Mechanics Lab (BBML)

A Note on Peak Force QNM

Correct probe stiffness must be used based on sample Pittenger ndash Veeco Product Bulletin

BBML - All R

ights Reserve

d

A Note on Peak Force QNM Extremely sensitive to calibration (details rarely given) Absolute radius and spring constant known

bull especially important from probe to probe bull radius uarr with indent depth ndash must determine R at depth

you expect to image bull Depth may vary as a function of sample (or at different

regions within the sample)

Relative reference material of ldquoknownrdquo modulus used to determine a ratio of 119896119896 119877119877 bull ldquoknownrdquo modulus may come from tension compression

and not indentation bull R is related to depth indentation depth on sample must

be same as on reference BBML - All R

ights Reserve

d

A Note on Peak Force QNM bull Modulus fit on the fly ndash no ability to monitor fit (or range) bull Local sample topography and sample tilt play a role in

measured parameters bull Assuming all modulus values in an image are correct how to

quantify

512 x 512 pixels = 262144 measurements

n=1 n=262144

n from selected ROIs

BBML - All R

ights Reserve

d

BBML - All R

ights Reserve

d

bull Imaging in air on mica ndash substrate effect

bull Same imaging force or indent depth for all

bull For each area were all modulus values pooled

Lamprou et al PLoS ONE 2013 BBML - All R

ights Reserve

d

AFM Applications in Bone Research Other Cool Mechanical Techniques

BBML - All R

ights Reserve

d

ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo Bonds within or between collagen fibrils in bone that rupture to dissipate energy as a toughening mechanism

Thompson et al Nature 2001

Fantner et al Nature Mat 2005

Hansma et al JMNI 2005

Fantner et al Nano Letters 2007 BBML - All R

ights Reserve

d

Other Mechanical Techniques

Jimenez-Palomar J Mech Beh Bio Mat 2012

van der Rijt Macro Bio 2004

Yang J Biomed Mater Res 2007

Yang Biophysical Journal 2008 BBML - All R

ights Reserve

d

Other Cool Techniques bull Piezoresponse Force Microscopy (PFM)

bull Electrochemical AFM (EC-AFM)

bull Force Modulation Microscopy (FMM)

bull Lateral Force Microscopy (LFM)

bull Magnetic Force Microscopy (MFM)

bull Conductive AFM (C-AFM)

bull Force Pulling

bull Cantilever Bending

bull Tensile testing of individual fibrils

bull 3 point bending of fibrils

BBML - All R

ights Reserve

d

Summary Things to rememberhellip

bull Imaging mode imaging medium and probe selection

bull Morphology Measurements bull Sample preparation and hydration state bull Measuring fibril diameter bull Site selection and proper sampling bull Appropriate system calibration

bull Mechanical Measurements

bull Probe selection (stiffness and tip radius) bull Probe calibration (radius angle spring constant) bull Indentation force range and mechanical model bull Hydration in biological samples bull PeakForce QNM

BBML - All R

ights Reserve

d

Acknowledgements BBML

Max Hammond Armando Diaz Gonzalez Zachary Bart Silvia Canelon Creasy Clauser

IU School of Medicine Dr David Burr Dr Matt Allen Dr Stuart Warden Dr Lilian Plotkin

Funding Sources NIHNIDCR 1F32DE018840-01 A1 IUPUI BME Departmental Startup Funds IUPUI Research Support Funds Grant IUPUI Biomechanics and Biomaterials Research Center

NIH NICHD Dr Joan Marini

BBML - All R

ights Reserve

d

Bone Biology and Mechanics Lab (BBML) wwwiupuiedu~bbml Facebook BoneBiologyMechanicsLab

BBML - All R

ights Reserve

d

• Slide Number 1
• General Seminar Outline
• Bonersquos Hierarchical Structure
• Critical Gap in Understanding Collagen
• Methods For Analyzing Collagen Structure
• Methods For Imaging Collagen Structure
• Cryo-TEM
• Atomic Force Microscopy (AFM)
• AFM Image of Pentacene (C22H14)
• AFM Image of Hexabenzocoronene
• Basics of AFM Imaging The Probe
• Basics of AFM Imaging System Components
• van der Waals Potential Energy Curve
• Contact Mode ndash Monitor Deflection
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Peak Force Tapping Mode ndash Monitor Force
• AFM Applications in Bone Research Imaging
• AFM Imaging of Collagen
• Imaging of Osteoclast Activity
• Mineralized collagen deposition in vitro
• Imaging of Canilicular Structure
• AFM Imaging in Bone
• AFM Imaging in Bone
• Removing Mineral in Bone to Expose Collagen
• Removing Mineral in Bone to Expose Collagen
• Demineralization with EDTA
• Demineralization with EDTA
• Demineralization with EDTA
• Issues to Consider Effects of Acid
• Slide Number 32
• Slide Number 33
• Analysis of Collagen Structure in Bone
• Analysis of Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril D-Spacing
• Slide Number 39
• Collagen in Bone Fibril D-Spacing
• Collagen in Bone Fibril D-Spacing
• 2D Fast Fourier Transform Analysis
• 2D Fast Fourier Transform Analysis
• D-Spacing Distribution
• D-Spacing Distribution
• Cumulative Distribution Function
• CDF vs Histogram
• Changes in D-Spacing with Disease
• Changes in D-Spacing with Disease
• Slide Number 50
• Issues to Consider Site Selection and Sampling
• Issues to Consider Site Selection and Sampling
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• AFM calibration ndash Take Away Message
• AFM Applications in Bone ResearchIndentation Mechanics
• AFM-Based Indentation
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Parameters
• Hertz Contact Equation
• Hertz Contact Equation
• Hertz Contact Equation
• Proper Indentation Model is Essential
• Indentation in OI Tendon Study
• OI Tendon Differential Effects of Dehydration
• OI Tendon Differential Effects of Dehydration
• Indentation in Diabetic Tendon (Poster 0495)
• Multiscale Diabetes Study (Poster 0495)
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• A Note on Peak Force QNM for Bruker AFMs
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• Slide Number 83
• Slide Number 84
• AFM Applications in Bone ResearchOther Cool Mechanical Techniques
• ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo
• Other Mechanical Techniques
• Other Cool Techniques
• Summary Things to rememberhellip
• Acknowledgements
• Bone Biology and Mechanics Lab (BBML)

A Note on Peak Force QNM Extremely sensitive to calibration (details rarely given) Absolute radius and spring constant known

bull especially important from probe to probe bull radius uarr with indent depth ndash must determine R at depth

you expect to image bull Depth may vary as a function of sample (or at different

regions within the sample)

Relative reference material of ldquoknownrdquo modulus used to determine a ratio of 119896119896 119877119877 bull ldquoknownrdquo modulus may come from tension compression

and not indentation bull R is related to depth indentation depth on sample must

be same as on reference BBML - All R

ights Reserve

d

A Note on Peak Force QNM bull Modulus fit on the fly ndash no ability to monitor fit (or range) bull Local sample topography and sample tilt play a role in

measured parameters bull Assuming all modulus values in an image are correct how to

quantify

512 x 512 pixels = 262144 measurements

n=1 n=262144

n from selected ROIs

BBML - All R

ights Reserve

d

BBML - All R

ights Reserve

d

bull Imaging in air on mica ndash substrate effect

bull Same imaging force or indent depth for all

bull For each area were all modulus values pooled

Lamprou et al PLoS ONE 2013 BBML - All R

ights Reserve

d

AFM Applications in Bone Research Other Cool Mechanical Techniques

BBML - All R

ights Reserve

d

ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo Bonds within or between collagen fibrils in bone that rupture to dissipate energy as a toughening mechanism

Thompson et al Nature 2001

Fantner et al Nature Mat 2005

Hansma et al JMNI 2005

Fantner et al Nano Letters 2007 BBML - All R

ights Reserve

d

Other Mechanical Techniques

Jimenez-Palomar J Mech Beh Bio Mat 2012

van der Rijt Macro Bio 2004

Yang J Biomed Mater Res 2007

Yang Biophysical Journal 2008 BBML - All R

ights Reserve

d

Other Cool Techniques bull Piezoresponse Force Microscopy (PFM)

bull Electrochemical AFM (EC-AFM)

bull Force Modulation Microscopy (FMM)

bull Lateral Force Microscopy (LFM)

bull Magnetic Force Microscopy (MFM)

bull Conductive AFM (C-AFM)

bull Force Pulling

bull Cantilever Bending

bull Tensile testing of individual fibrils

bull 3 point bending of fibrils

BBML - All R

ights Reserve

d

Summary Things to rememberhellip

bull Imaging mode imaging medium and probe selection

bull Morphology Measurements bull Sample preparation and hydration state bull Measuring fibril diameter bull Site selection and proper sampling bull Appropriate system calibration

bull Mechanical Measurements

bull Probe selection (stiffness and tip radius) bull Probe calibration (radius angle spring constant) bull Indentation force range and mechanical model bull Hydration in biological samples bull PeakForce QNM

BBML - All R

ights Reserve

d

Acknowledgements BBML

Max Hammond Armando Diaz Gonzalez Zachary Bart Silvia Canelon Creasy Clauser

IU School of Medicine Dr David Burr Dr Matt Allen Dr Stuart Warden Dr Lilian Plotkin

Funding Sources NIHNIDCR 1F32DE018840-01 A1 IUPUI BME Departmental Startup Funds IUPUI Research Support Funds Grant IUPUI Biomechanics and Biomaterials Research Center

NIH NICHD Dr Joan Marini

BBML - All R

ights Reserve

d

Bone Biology and Mechanics Lab (BBML) wwwiupuiedu~bbml Facebook BoneBiologyMechanicsLab

BBML - All R

ights Reserve

d

• Slide Number 1
• General Seminar Outline
• Bonersquos Hierarchical Structure
• Critical Gap in Understanding Collagen
• Methods For Analyzing Collagen Structure
• Methods For Imaging Collagen Structure
• Cryo-TEM
• Atomic Force Microscopy (AFM)
• AFM Image of Pentacene (C22H14)
• AFM Image of Hexabenzocoronene
• Basics of AFM Imaging The Probe
• Basics of AFM Imaging System Components
• van der Waals Potential Energy Curve
• Contact Mode ndash Monitor Deflection
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Peak Force Tapping Mode ndash Monitor Force
• AFM Applications in Bone Research Imaging
• AFM Imaging of Collagen
• Imaging of Osteoclast Activity
• Mineralized collagen deposition in vitro
• Imaging of Canilicular Structure
• AFM Imaging in Bone
• AFM Imaging in Bone
• Removing Mineral in Bone to Expose Collagen
• Removing Mineral in Bone to Expose Collagen
• Demineralization with EDTA
• Demineralization with EDTA
• Demineralization with EDTA
• Issues to Consider Effects of Acid
• Slide Number 32
• Slide Number 33
• Analysis of Collagen Structure in Bone
• Analysis of Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril D-Spacing
• Slide Number 39
• Collagen in Bone Fibril D-Spacing
• Collagen in Bone Fibril D-Spacing
• 2D Fast Fourier Transform Analysis
• 2D Fast Fourier Transform Analysis
• D-Spacing Distribution
• D-Spacing Distribution
• Cumulative Distribution Function
• CDF vs Histogram
• Changes in D-Spacing with Disease
• Changes in D-Spacing with Disease
• Slide Number 50
• Issues to Consider Site Selection and Sampling
• Issues to Consider Site Selection and Sampling
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• AFM calibration ndash Take Away Message
• AFM Applications in Bone ResearchIndentation Mechanics
• AFM-Based Indentation
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Parameters
• Hertz Contact Equation
• Hertz Contact Equation
• Hertz Contact Equation
• Proper Indentation Model is Essential
• Indentation in OI Tendon Study
• OI Tendon Differential Effects of Dehydration
• OI Tendon Differential Effects of Dehydration
• Indentation in Diabetic Tendon (Poster 0495)
• Multiscale Diabetes Study (Poster 0495)
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• A Note on Peak Force QNM for Bruker AFMs
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• Slide Number 83
• Slide Number 84
• AFM Applications in Bone ResearchOther Cool Mechanical Techniques
• ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo
• Other Mechanical Techniques
• Other Cool Techniques
• Summary Things to rememberhellip
• Acknowledgements
• Bone Biology and Mechanics Lab (BBML)

A Note on Peak Force QNM bull Modulus fit on the fly ndash no ability to monitor fit (or range) bull Local sample topography and sample tilt play a role in

measured parameters bull Assuming all modulus values in an image are correct how to

quantify

512 x 512 pixels = 262144 measurements

n=1 n=262144

n from selected ROIs

BBML - All R

ights Reserve

d

BBML - All R

ights Reserve

d

bull Imaging in air on mica ndash substrate effect

bull Same imaging force or indent depth for all

bull For each area were all modulus values pooled

Lamprou et al PLoS ONE 2013 BBML - All R

ights Reserve

d

AFM Applications in Bone Research Other Cool Mechanical Techniques

BBML - All R

ights Reserve

d

ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo Bonds within or between collagen fibrils in bone that rupture to dissipate energy as a toughening mechanism

Thompson et al Nature 2001

Fantner et al Nature Mat 2005

Hansma et al JMNI 2005

Fantner et al Nano Letters 2007 BBML - All R

ights Reserve

d

Other Mechanical Techniques

Jimenez-Palomar J Mech Beh Bio Mat 2012

van der Rijt Macro Bio 2004

Yang J Biomed Mater Res 2007

Yang Biophysical Journal 2008 BBML - All R

ights Reserve

d

Other Cool Techniques bull Piezoresponse Force Microscopy (PFM)

bull Electrochemical AFM (EC-AFM)

bull Force Modulation Microscopy (FMM)

bull Lateral Force Microscopy (LFM)

bull Magnetic Force Microscopy (MFM)

bull Conductive AFM (C-AFM)

bull Force Pulling

bull Cantilever Bending

bull Tensile testing of individual fibrils

bull 3 point bending of fibrils

BBML - All R

ights Reserve

d

Summary Things to rememberhellip

bull Imaging mode imaging medium and probe selection

bull Morphology Measurements bull Sample preparation and hydration state bull Measuring fibril diameter bull Site selection and proper sampling bull Appropriate system calibration

bull Mechanical Measurements

bull Probe selection (stiffness and tip radius) bull Probe calibration (radius angle spring constant) bull Indentation force range and mechanical model bull Hydration in biological samples bull PeakForce QNM

BBML - All R

ights Reserve

d

Acknowledgements BBML

Max Hammond Armando Diaz Gonzalez Zachary Bart Silvia Canelon Creasy Clauser

IU School of Medicine Dr David Burr Dr Matt Allen Dr Stuart Warden Dr Lilian Plotkin

Funding Sources NIHNIDCR 1F32DE018840-01 A1 IUPUI BME Departmental Startup Funds IUPUI Research Support Funds Grant IUPUI Biomechanics and Biomaterials Research Center

NIH NICHD Dr Joan Marini

BBML - All R

ights Reserve

d

Bone Biology and Mechanics Lab (BBML) wwwiupuiedu~bbml Facebook BoneBiologyMechanicsLab

BBML - All R

ights Reserve

d

• Slide Number 1
• General Seminar Outline
• Bonersquos Hierarchical Structure
• Critical Gap in Understanding Collagen
• Methods For Analyzing Collagen Structure
• Methods For Imaging Collagen Structure
• Cryo-TEM
• Atomic Force Microscopy (AFM)
• AFM Image of Pentacene (C22H14)
• AFM Image of Hexabenzocoronene
• Basics of AFM Imaging The Probe
• Basics of AFM Imaging System Components
• van der Waals Potential Energy Curve
• Contact Mode ndash Monitor Deflection
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Peak Force Tapping Mode ndash Monitor Force
• AFM Applications in Bone Research Imaging
• AFM Imaging of Collagen
• Imaging of Osteoclast Activity
• Mineralized collagen deposition in vitro
• Imaging of Canilicular Structure
• AFM Imaging in Bone
• AFM Imaging in Bone
• Removing Mineral in Bone to Expose Collagen
• Removing Mineral in Bone to Expose Collagen
• Demineralization with EDTA
• Demineralization with EDTA
• Demineralization with EDTA
• Issues to Consider Effects of Acid
• Slide Number 32
• Slide Number 33
• Analysis of Collagen Structure in Bone
• Analysis of Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril D-Spacing
• Slide Number 39
• Collagen in Bone Fibril D-Spacing
• Collagen in Bone Fibril D-Spacing
• 2D Fast Fourier Transform Analysis
• 2D Fast Fourier Transform Analysis
• D-Spacing Distribution
• D-Spacing Distribution
• Cumulative Distribution Function
• CDF vs Histogram
• Changes in D-Spacing with Disease
• Changes in D-Spacing with Disease
• Slide Number 50
• Issues to Consider Site Selection and Sampling
• Issues to Consider Site Selection and Sampling
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• AFM calibration ndash Take Away Message
• AFM Applications in Bone ResearchIndentation Mechanics
• AFM-Based Indentation
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Parameters
• Hertz Contact Equation
• Hertz Contact Equation
• Hertz Contact Equation
• Proper Indentation Model is Essential
• Indentation in OI Tendon Study
• OI Tendon Differential Effects of Dehydration
• OI Tendon Differential Effects of Dehydration
• Indentation in Diabetic Tendon (Poster 0495)
• Multiscale Diabetes Study (Poster 0495)
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• A Note on Peak Force QNM for Bruker AFMs
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• Slide Number 83
• Slide Number 84
• AFM Applications in Bone ResearchOther Cool Mechanical Techniques
• ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo
• Other Mechanical Techniques
• Other Cool Techniques
• Summary Things to rememberhellip
• Acknowledgements
• Bone Biology and Mechanics Lab (BBML)

BBML - All R

ights Reserve

d

bull Imaging in air on mica ndash substrate effect

bull Same imaging force or indent depth for all

bull For each area were all modulus values pooled

Lamprou et al PLoS ONE 2013 BBML - All R

ights Reserve

d

AFM Applications in Bone Research Other Cool Mechanical Techniques

BBML - All R

ights Reserve

d

ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo Bonds within or between collagen fibrils in bone that rupture to dissipate energy as a toughening mechanism

Thompson et al Nature 2001

Fantner et al Nature Mat 2005

Hansma et al JMNI 2005

Fantner et al Nano Letters 2007 BBML - All R

ights Reserve

d

Other Mechanical Techniques

Jimenez-Palomar J Mech Beh Bio Mat 2012

van der Rijt Macro Bio 2004

Yang J Biomed Mater Res 2007

Yang Biophysical Journal 2008 BBML - All R

ights Reserve

d

Other Cool Techniques bull Piezoresponse Force Microscopy (PFM)

bull Electrochemical AFM (EC-AFM)

bull Force Modulation Microscopy (FMM)

bull Lateral Force Microscopy (LFM)

bull Magnetic Force Microscopy (MFM)

bull Conductive AFM (C-AFM)

bull Force Pulling

bull Cantilever Bending

bull Tensile testing of individual fibrils

bull 3 point bending of fibrils

BBML - All R

ights Reserve

d

Summary Things to rememberhellip

bull Imaging mode imaging medium and probe selection

bull Morphology Measurements bull Sample preparation and hydration state bull Measuring fibril diameter bull Site selection and proper sampling bull Appropriate system calibration

bull Mechanical Measurements

bull Probe selection (stiffness and tip radius) bull Probe calibration (radius angle spring constant) bull Indentation force range and mechanical model bull Hydration in biological samples bull PeakForce QNM

BBML - All R

ights Reserve

d

Acknowledgements BBML

Max Hammond Armando Diaz Gonzalez Zachary Bart Silvia Canelon Creasy Clauser

IU School of Medicine Dr David Burr Dr Matt Allen Dr Stuart Warden Dr Lilian Plotkin

Funding Sources NIHNIDCR 1F32DE018840-01 A1 IUPUI BME Departmental Startup Funds IUPUI Research Support Funds Grant IUPUI Biomechanics and Biomaterials Research Center

NIH NICHD Dr Joan Marini

BBML - All R

ights Reserve

d

Bone Biology and Mechanics Lab (BBML) wwwiupuiedu~bbml Facebook BoneBiologyMechanicsLab

BBML - All R

ights Reserve

d

• Slide Number 1
• General Seminar Outline
• Bonersquos Hierarchical Structure
• Critical Gap in Understanding Collagen
• Methods For Analyzing Collagen Structure
• Methods For Imaging Collagen Structure
• Cryo-TEM
• Atomic Force Microscopy (AFM)
• AFM Image of Pentacene (C22H14)
• AFM Image of Hexabenzocoronene
• Basics of AFM Imaging The Probe
• Basics of AFM Imaging System Components
• van der Waals Potential Energy Curve
• Contact Mode ndash Monitor Deflection
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Peak Force Tapping Mode ndash Monitor Force
• AFM Applications in Bone Research Imaging
• AFM Imaging of Collagen
• Imaging of Osteoclast Activity
• Mineralized collagen deposition in vitro
• Imaging of Canilicular Structure
• AFM Imaging in Bone
• AFM Imaging in Bone
• Removing Mineral in Bone to Expose Collagen
• Removing Mineral in Bone to Expose Collagen
• Demineralization with EDTA
• Demineralization with EDTA
• Demineralization with EDTA
• Issues to Consider Effects of Acid
• Slide Number 32
• Slide Number 33
• Analysis of Collagen Structure in Bone
• Analysis of Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril D-Spacing
• Slide Number 39
• Collagen in Bone Fibril D-Spacing
• Collagen in Bone Fibril D-Spacing
• 2D Fast Fourier Transform Analysis
• 2D Fast Fourier Transform Analysis
• D-Spacing Distribution
• D-Spacing Distribution
• Cumulative Distribution Function
• CDF vs Histogram
• Changes in D-Spacing with Disease
• Changes in D-Spacing with Disease
• Slide Number 50
• Issues to Consider Site Selection and Sampling
• Issues to Consider Site Selection and Sampling
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• AFM calibration ndash Take Away Message
• AFM Applications in Bone ResearchIndentation Mechanics
• AFM-Based Indentation
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Parameters
• Hertz Contact Equation
• Hertz Contact Equation
• Hertz Contact Equation
• Proper Indentation Model is Essential
• Indentation in OI Tendon Study
• OI Tendon Differential Effects of Dehydration
• OI Tendon Differential Effects of Dehydration
• Indentation in Diabetic Tendon (Poster 0495)
• Multiscale Diabetes Study (Poster 0495)
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• A Note on Peak Force QNM for Bruker AFMs
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• Slide Number 83
• Slide Number 84
• AFM Applications in Bone ResearchOther Cool Mechanical Techniques
• ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo
• Other Mechanical Techniques
• Other Cool Techniques
• Summary Things to rememberhellip
• Acknowledgements
• Bone Biology and Mechanics Lab (BBML)

bull Imaging in air on mica ndash substrate effect

bull Same imaging force or indent depth for all

bull For each area were all modulus values pooled

Lamprou et al PLoS ONE 2013 BBML - All R

ights Reserve

d

AFM Applications in Bone Research Other Cool Mechanical Techniques

BBML - All R

ights Reserve

d

ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo Bonds within or between collagen fibrils in bone that rupture to dissipate energy as a toughening mechanism

Thompson et al Nature 2001

Fantner et al Nature Mat 2005

Hansma et al JMNI 2005

Fantner et al Nano Letters 2007 BBML - All R

ights Reserve

d

Other Mechanical Techniques

Jimenez-Palomar J Mech Beh Bio Mat 2012

van der Rijt Macro Bio 2004

Yang J Biomed Mater Res 2007

Yang Biophysical Journal 2008 BBML - All R

ights Reserve

d

Other Cool Techniques bull Piezoresponse Force Microscopy (PFM)

bull Electrochemical AFM (EC-AFM)

bull Force Modulation Microscopy (FMM)

bull Lateral Force Microscopy (LFM)

bull Magnetic Force Microscopy (MFM)

bull Conductive AFM (C-AFM)

bull Force Pulling

bull Cantilever Bending

bull Tensile testing of individual fibrils

bull 3 point bending of fibrils

BBML - All R

ights Reserve

d

Summary Things to rememberhellip

bull Imaging mode imaging medium and probe selection

bull Morphology Measurements bull Sample preparation and hydration state bull Measuring fibril diameter bull Site selection and proper sampling bull Appropriate system calibration

bull Mechanical Measurements

bull Probe selection (stiffness and tip radius) bull Probe calibration (radius angle spring constant) bull Indentation force range and mechanical model bull Hydration in biological samples bull PeakForce QNM

BBML - All R

ights Reserve

d

Acknowledgements BBML

Max Hammond Armando Diaz Gonzalez Zachary Bart Silvia Canelon Creasy Clauser

IU School of Medicine Dr David Burr Dr Matt Allen Dr Stuart Warden Dr Lilian Plotkin

Funding Sources NIHNIDCR 1F32DE018840-01 A1 IUPUI BME Departmental Startup Funds IUPUI Research Support Funds Grant IUPUI Biomechanics and Biomaterials Research Center

NIH NICHD Dr Joan Marini

BBML - All R

ights Reserve

d

Bone Biology and Mechanics Lab (BBML) wwwiupuiedu~bbml Facebook BoneBiologyMechanicsLab

BBML - All R

ights Reserve

d

• Slide Number 1
• General Seminar Outline
• Bonersquos Hierarchical Structure
• Critical Gap in Understanding Collagen
• Methods For Analyzing Collagen Structure
• Methods For Imaging Collagen Structure
• Cryo-TEM
• Atomic Force Microscopy (AFM)
• AFM Image of Pentacene (C22H14)
• AFM Image of Hexabenzocoronene
• Basics of AFM Imaging The Probe
• Basics of AFM Imaging System Components
• van der Waals Potential Energy Curve
• Contact Mode ndash Monitor Deflection
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Peak Force Tapping Mode ndash Monitor Force
• AFM Applications in Bone Research Imaging
• AFM Imaging of Collagen
• Imaging of Osteoclast Activity
• Mineralized collagen deposition in vitro
• Imaging of Canilicular Structure
• AFM Imaging in Bone
• AFM Imaging in Bone
• Removing Mineral in Bone to Expose Collagen
• Removing Mineral in Bone to Expose Collagen
• Demineralization with EDTA
• Demineralization with EDTA
• Demineralization with EDTA
• Issues to Consider Effects of Acid
• Slide Number 32
• Slide Number 33
• Analysis of Collagen Structure in Bone
• Analysis of Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril D-Spacing
• Slide Number 39
• Collagen in Bone Fibril D-Spacing
• Collagen in Bone Fibril D-Spacing
• 2D Fast Fourier Transform Analysis
• 2D Fast Fourier Transform Analysis
• D-Spacing Distribution
• D-Spacing Distribution
• Cumulative Distribution Function
• CDF vs Histogram
• Changes in D-Spacing with Disease
• Changes in D-Spacing with Disease
• Slide Number 50
• Issues to Consider Site Selection and Sampling
• Issues to Consider Site Selection and Sampling
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• AFM calibration ndash Take Away Message
• AFM Applications in Bone ResearchIndentation Mechanics
• AFM-Based Indentation
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Parameters
• Hertz Contact Equation
• Hertz Contact Equation
• Hertz Contact Equation
• Proper Indentation Model is Essential
• Indentation in OI Tendon Study
• OI Tendon Differential Effects of Dehydration
• OI Tendon Differential Effects of Dehydration
• Indentation in Diabetic Tendon (Poster 0495)
• Multiscale Diabetes Study (Poster 0495)
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• A Note on Peak Force QNM for Bruker AFMs
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• Slide Number 83
• Slide Number 84
• AFM Applications in Bone ResearchOther Cool Mechanical Techniques
• ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo
• Other Mechanical Techniques
• Other Cool Techniques
• Summary Things to rememberhellip
• Acknowledgements
• Bone Biology and Mechanics Lab (BBML)

AFM Applications in Bone Research Other Cool Mechanical Techniques

BBML - All R

ights Reserve

d

ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo Bonds within or between collagen fibrils in bone that rupture to dissipate energy as a toughening mechanism

Thompson et al Nature 2001

Fantner et al Nature Mat 2005

Hansma et al JMNI 2005

Fantner et al Nano Letters 2007 BBML - All R

ights Reserve

d

Other Mechanical Techniques

Jimenez-Palomar J Mech Beh Bio Mat 2012

van der Rijt Macro Bio 2004

Yang J Biomed Mater Res 2007

Yang Biophysical Journal 2008 BBML - All R

ights Reserve

d

Other Cool Techniques bull Piezoresponse Force Microscopy (PFM)

bull Electrochemical AFM (EC-AFM)

bull Force Modulation Microscopy (FMM)

bull Lateral Force Microscopy (LFM)

bull Magnetic Force Microscopy (MFM)

bull Conductive AFM (C-AFM)

bull Force Pulling

bull Cantilever Bending

bull Tensile testing of individual fibrils

bull 3 point bending of fibrils

BBML - All R

ights Reserve

d

Summary Things to rememberhellip

bull Imaging mode imaging medium and probe selection

bull Morphology Measurements bull Sample preparation and hydration state bull Measuring fibril diameter bull Site selection and proper sampling bull Appropriate system calibration

bull Mechanical Measurements

bull Probe selection (stiffness and tip radius) bull Probe calibration (radius angle spring constant) bull Indentation force range and mechanical model bull Hydration in biological samples bull PeakForce QNM

BBML - All R

ights Reserve

d

Acknowledgements BBML

Max Hammond Armando Diaz Gonzalez Zachary Bart Silvia Canelon Creasy Clauser

IU School of Medicine Dr David Burr Dr Matt Allen Dr Stuart Warden Dr Lilian Plotkin

Funding Sources NIHNIDCR 1F32DE018840-01 A1 IUPUI BME Departmental Startup Funds IUPUI Research Support Funds Grant IUPUI Biomechanics and Biomaterials Research Center

NIH NICHD Dr Joan Marini

BBML - All R

ights Reserve

d

Bone Biology and Mechanics Lab (BBML) wwwiupuiedu~bbml Facebook BoneBiologyMechanicsLab

BBML - All R

ights Reserve

d

• Slide Number 1
• General Seminar Outline
• Bonersquos Hierarchical Structure
• Critical Gap in Understanding Collagen
• Methods For Analyzing Collagen Structure
• Methods For Imaging Collagen Structure
• Cryo-TEM
• Atomic Force Microscopy (AFM)
• AFM Image of Pentacene (C22H14)
• AFM Image of Hexabenzocoronene
• Basics of AFM Imaging The Probe
• Basics of AFM Imaging System Components
• van der Waals Potential Energy Curve
• Contact Mode ndash Monitor Deflection
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Peak Force Tapping Mode ndash Monitor Force
• AFM Applications in Bone Research Imaging
• AFM Imaging of Collagen
• Imaging of Osteoclast Activity
• Mineralized collagen deposition in vitro
• Imaging of Canilicular Structure
• AFM Imaging in Bone
• AFM Imaging in Bone
• Removing Mineral in Bone to Expose Collagen
• Removing Mineral in Bone to Expose Collagen
• Demineralization with EDTA
• Demineralization with EDTA
• Demineralization with EDTA
• Issues to Consider Effects of Acid
• Slide Number 32
• Slide Number 33
• Analysis of Collagen Structure in Bone
• Analysis of Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril D-Spacing
• Slide Number 39
• Collagen in Bone Fibril D-Spacing
• Collagen in Bone Fibril D-Spacing
• 2D Fast Fourier Transform Analysis
• 2D Fast Fourier Transform Analysis
• D-Spacing Distribution
• D-Spacing Distribution
• Cumulative Distribution Function
• CDF vs Histogram
• Changes in D-Spacing with Disease
• Changes in D-Spacing with Disease
• Slide Number 50
• Issues to Consider Site Selection and Sampling
• Issues to Consider Site Selection and Sampling
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• AFM calibration ndash Take Away Message
• AFM Applications in Bone ResearchIndentation Mechanics
• AFM-Based Indentation
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Parameters
• Hertz Contact Equation
• Hertz Contact Equation
• Hertz Contact Equation
• Proper Indentation Model is Essential
• Indentation in OI Tendon Study
• OI Tendon Differential Effects of Dehydration
• OI Tendon Differential Effects of Dehydration
• Indentation in Diabetic Tendon (Poster 0495)
• Multiscale Diabetes Study (Poster 0495)
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• A Note on Peak Force QNM for Bruker AFMs
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• Slide Number 83
• Slide Number 84
• AFM Applications in Bone ResearchOther Cool Mechanical Techniques
• ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo
• Other Mechanical Techniques
• Other Cool Techniques
• Summary Things to rememberhellip
• Acknowledgements
• Bone Biology and Mechanics Lab (BBML)

ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo Bonds within or between collagen fibrils in bone that rupture to dissipate energy as a toughening mechanism

Thompson et al Nature 2001

Fantner et al Nature Mat 2005

Hansma et al JMNI 2005

Fantner et al Nano Letters 2007 BBML - All R

ights Reserve

d

Other Mechanical Techniques

Jimenez-Palomar J Mech Beh Bio Mat 2012

van der Rijt Macro Bio 2004

Yang J Biomed Mater Res 2007

Yang Biophysical Journal 2008 BBML - All R

ights Reserve

d

Other Cool Techniques bull Piezoresponse Force Microscopy (PFM)

bull Electrochemical AFM (EC-AFM)

bull Force Modulation Microscopy (FMM)

bull Lateral Force Microscopy (LFM)

bull Magnetic Force Microscopy (MFM)

bull Conductive AFM (C-AFM)

bull Force Pulling

bull Cantilever Bending

bull Tensile testing of individual fibrils

bull 3 point bending of fibrils

BBML - All R

ights Reserve

d

Summary Things to rememberhellip

bull Imaging mode imaging medium and probe selection

bull Morphology Measurements bull Sample preparation and hydration state bull Measuring fibril diameter bull Site selection and proper sampling bull Appropriate system calibration

bull Mechanical Measurements

bull Probe selection (stiffness and tip radius) bull Probe calibration (radius angle spring constant) bull Indentation force range and mechanical model bull Hydration in biological samples bull PeakForce QNM

BBML - All R

ights Reserve

d

Acknowledgements BBML

Max Hammond Armando Diaz Gonzalez Zachary Bart Silvia Canelon Creasy Clauser

IU School of Medicine Dr David Burr Dr Matt Allen Dr Stuart Warden Dr Lilian Plotkin

Funding Sources NIHNIDCR 1F32DE018840-01 A1 IUPUI BME Departmental Startup Funds IUPUI Research Support Funds Grant IUPUI Biomechanics and Biomaterials Research Center

NIH NICHD Dr Joan Marini

BBML - All R

ights Reserve

d

Bone Biology and Mechanics Lab (BBML) wwwiupuiedu~bbml Facebook BoneBiologyMechanicsLab

BBML - All R

ights Reserve

d

• Slide Number 1
• General Seminar Outline
• Bonersquos Hierarchical Structure
• Critical Gap in Understanding Collagen
• Methods For Analyzing Collagen Structure
• Methods For Imaging Collagen Structure
• Cryo-TEM
• Atomic Force Microscopy (AFM)
• AFM Image of Pentacene (C22H14)
• AFM Image of Hexabenzocoronene
• Basics of AFM Imaging The Probe
• Basics of AFM Imaging System Components
• van der Waals Potential Energy Curve
• Contact Mode ndash Monitor Deflection
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Peak Force Tapping Mode ndash Monitor Force
• AFM Applications in Bone Research Imaging
• AFM Imaging of Collagen
• Imaging of Osteoclast Activity
• Mineralized collagen deposition in vitro
• Imaging of Canilicular Structure
• AFM Imaging in Bone
• AFM Imaging in Bone
• Removing Mineral in Bone to Expose Collagen
• Removing Mineral in Bone to Expose Collagen
• Demineralization with EDTA
• Demineralization with EDTA
• Demineralization with EDTA
• Issues to Consider Effects of Acid
• Slide Number 32
• Slide Number 33
• Analysis of Collagen Structure in Bone
• Analysis of Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril D-Spacing
• Slide Number 39
• Collagen in Bone Fibril D-Spacing
• Collagen in Bone Fibril D-Spacing
• 2D Fast Fourier Transform Analysis
• 2D Fast Fourier Transform Analysis
• D-Spacing Distribution
• D-Spacing Distribution
• Cumulative Distribution Function
• CDF vs Histogram
• Changes in D-Spacing with Disease
• Changes in D-Spacing with Disease
• Slide Number 50
• Issues to Consider Site Selection and Sampling
• Issues to Consider Site Selection and Sampling
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• AFM calibration ndash Take Away Message
• AFM Applications in Bone ResearchIndentation Mechanics
• AFM-Based Indentation
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Parameters
• Hertz Contact Equation
• Hertz Contact Equation
• Hertz Contact Equation
• Proper Indentation Model is Essential
• Indentation in OI Tendon Study
• OI Tendon Differential Effects of Dehydration
• OI Tendon Differential Effects of Dehydration
• Indentation in Diabetic Tendon (Poster 0495)
• Multiscale Diabetes Study (Poster 0495)
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• A Note on Peak Force QNM for Bruker AFMs
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• Slide Number 83
• Slide Number 84
• AFM Applications in Bone ResearchOther Cool Mechanical Techniques
• ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo
• Other Mechanical Techniques
• Other Cool Techniques
• Summary Things to rememberhellip
• Acknowledgements
• Bone Biology and Mechanics Lab (BBML)

Other Mechanical Techniques

Jimenez-Palomar J Mech Beh Bio Mat 2012

van der Rijt Macro Bio 2004

Yang J Biomed Mater Res 2007

Yang Biophysical Journal 2008 BBML - All R

ights Reserve

d

Other Cool Techniques bull Piezoresponse Force Microscopy (PFM)

bull Electrochemical AFM (EC-AFM)

bull Force Modulation Microscopy (FMM)

bull Lateral Force Microscopy (LFM)

bull Magnetic Force Microscopy (MFM)

bull Conductive AFM (C-AFM)

bull Force Pulling

bull Cantilever Bending

bull Tensile testing of individual fibrils

bull 3 point bending of fibrils

BBML - All R

ights Reserve

d

Summary Things to rememberhellip

bull Imaging mode imaging medium and probe selection

bull Morphology Measurements bull Sample preparation and hydration state bull Measuring fibril diameter bull Site selection and proper sampling bull Appropriate system calibration

bull Mechanical Measurements

bull Probe selection (stiffness and tip radius) bull Probe calibration (radius angle spring constant) bull Indentation force range and mechanical model bull Hydration in biological samples bull PeakForce QNM

BBML - All R

ights Reserve

d

Acknowledgements BBML

Max Hammond Armando Diaz Gonzalez Zachary Bart Silvia Canelon Creasy Clauser

IU School of Medicine Dr David Burr Dr Matt Allen Dr Stuart Warden Dr Lilian Plotkin

Funding Sources NIHNIDCR 1F32DE018840-01 A1 IUPUI BME Departmental Startup Funds IUPUI Research Support Funds Grant IUPUI Biomechanics and Biomaterials Research Center

NIH NICHD Dr Joan Marini

BBML - All R

ights Reserve

d

Bone Biology and Mechanics Lab (BBML) wwwiupuiedu~bbml Facebook BoneBiologyMechanicsLab

BBML - All R

ights Reserve

d

• Slide Number 1
• General Seminar Outline
• Bonersquos Hierarchical Structure
• Critical Gap in Understanding Collagen
• Methods For Analyzing Collagen Structure
• Methods For Imaging Collagen Structure
• Cryo-TEM
• Atomic Force Microscopy (AFM)
• AFM Image of Pentacene (C22H14)
• AFM Image of Hexabenzocoronene
• Basics of AFM Imaging The Probe
• Basics of AFM Imaging System Components
• van der Waals Potential Energy Curve
• Contact Mode ndash Monitor Deflection
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Peak Force Tapping Mode ndash Monitor Force
• AFM Applications in Bone Research Imaging
• AFM Imaging of Collagen
• Imaging of Osteoclast Activity
• Mineralized collagen deposition in vitro
• Imaging of Canilicular Structure
• AFM Imaging in Bone
• AFM Imaging in Bone
• Removing Mineral in Bone to Expose Collagen
• Removing Mineral in Bone to Expose Collagen
• Demineralization with EDTA
• Demineralization with EDTA
• Demineralization with EDTA
• Issues to Consider Effects of Acid
• Slide Number 32
• Slide Number 33
• Analysis of Collagen Structure in Bone
• Analysis of Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril D-Spacing
• Slide Number 39
• Collagen in Bone Fibril D-Spacing
• Collagen in Bone Fibril D-Spacing
• 2D Fast Fourier Transform Analysis
• 2D Fast Fourier Transform Analysis
• D-Spacing Distribution
• D-Spacing Distribution
• Cumulative Distribution Function
• CDF vs Histogram
• Changes in D-Spacing with Disease
• Changes in D-Spacing with Disease
• Slide Number 50
• Issues to Consider Site Selection and Sampling
• Issues to Consider Site Selection and Sampling
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• AFM calibration ndash Take Away Message
• AFM Applications in Bone ResearchIndentation Mechanics
• AFM-Based Indentation
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Parameters
• Hertz Contact Equation
• Hertz Contact Equation
• Hertz Contact Equation
• Proper Indentation Model is Essential
• Indentation in OI Tendon Study
• OI Tendon Differential Effects of Dehydration
• OI Tendon Differential Effects of Dehydration
• Indentation in Diabetic Tendon (Poster 0495)
• Multiscale Diabetes Study (Poster 0495)
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• A Note on Peak Force QNM for Bruker AFMs
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• Slide Number 83
• Slide Number 84
• AFM Applications in Bone ResearchOther Cool Mechanical Techniques
• ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo
• Other Mechanical Techniques
• Other Cool Techniques
• Summary Things to rememberhellip
• Acknowledgements
• Bone Biology and Mechanics Lab (BBML)

Other Cool Techniques bull Piezoresponse Force Microscopy (PFM)

bull Electrochemical AFM (EC-AFM)

bull Force Modulation Microscopy (FMM)

bull Lateral Force Microscopy (LFM)

bull Magnetic Force Microscopy (MFM)

bull Conductive AFM (C-AFM)

bull Force Pulling

bull Cantilever Bending

bull Tensile testing of individual fibrils

bull 3 point bending of fibrils

BBML - All R

ights Reserve

d

Summary Things to rememberhellip

bull Imaging mode imaging medium and probe selection

bull Morphology Measurements bull Sample preparation and hydration state bull Measuring fibril diameter bull Site selection and proper sampling bull Appropriate system calibration

bull Mechanical Measurements

bull Probe selection (stiffness and tip radius) bull Probe calibration (radius angle spring constant) bull Indentation force range and mechanical model bull Hydration in biological samples bull PeakForce QNM

BBML - All R

ights Reserve

d

Acknowledgements BBML

Max Hammond Armando Diaz Gonzalez Zachary Bart Silvia Canelon Creasy Clauser

IU School of Medicine Dr David Burr Dr Matt Allen Dr Stuart Warden Dr Lilian Plotkin

Funding Sources NIHNIDCR 1F32DE018840-01 A1 IUPUI BME Departmental Startup Funds IUPUI Research Support Funds Grant IUPUI Biomechanics and Biomaterials Research Center

NIH NICHD Dr Joan Marini

BBML - All R

ights Reserve

d

Bone Biology and Mechanics Lab (BBML) wwwiupuiedu~bbml Facebook BoneBiologyMechanicsLab

BBML - All R

ights Reserve

d

• Slide Number 1
• General Seminar Outline
• Bonersquos Hierarchical Structure
• Critical Gap in Understanding Collagen
• Methods For Analyzing Collagen Structure
• Methods For Imaging Collagen Structure
• Cryo-TEM
• Atomic Force Microscopy (AFM)
• AFM Image of Pentacene (C22H14)
• AFM Image of Hexabenzocoronene
• Basics of AFM Imaging The Probe
• Basics of AFM Imaging System Components
• van der Waals Potential Energy Curve
• Contact Mode ndash Monitor Deflection
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Peak Force Tapping Mode ndash Monitor Force
• AFM Applications in Bone Research Imaging
• AFM Imaging of Collagen
• Imaging of Osteoclast Activity
• Mineralized collagen deposition in vitro
• Imaging of Canilicular Structure
• AFM Imaging in Bone
• AFM Imaging in Bone
• Removing Mineral in Bone to Expose Collagen
• Removing Mineral in Bone to Expose Collagen
• Demineralization with EDTA
• Demineralization with EDTA
• Demineralization with EDTA
• Issues to Consider Effects of Acid
• Slide Number 32
• Slide Number 33
• Analysis of Collagen Structure in Bone
• Analysis of Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril D-Spacing
• Slide Number 39
• Collagen in Bone Fibril D-Spacing
• Collagen in Bone Fibril D-Spacing
• 2D Fast Fourier Transform Analysis
• 2D Fast Fourier Transform Analysis
• D-Spacing Distribution
• D-Spacing Distribution
• Cumulative Distribution Function
• CDF vs Histogram
• Changes in D-Spacing with Disease
• Changes in D-Spacing with Disease
• Slide Number 50
• Issues to Consider Site Selection and Sampling
• Issues to Consider Site Selection and Sampling
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• AFM calibration ndash Take Away Message
• AFM Applications in Bone ResearchIndentation Mechanics
• AFM-Based Indentation
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Parameters
• Hertz Contact Equation
• Hertz Contact Equation
• Hertz Contact Equation
• Proper Indentation Model is Essential
• Indentation in OI Tendon Study
• OI Tendon Differential Effects of Dehydration
• OI Tendon Differential Effects of Dehydration
• Indentation in Diabetic Tendon (Poster 0495)
• Multiscale Diabetes Study (Poster 0495)
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• A Note on Peak Force QNM for Bruker AFMs
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• Slide Number 83
• Slide Number 84
• AFM Applications in Bone ResearchOther Cool Mechanical Techniques
• ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo
• Other Mechanical Techniques
• Other Cool Techniques
• Summary Things to rememberhellip
• Acknowledgements
• Bone Biology and Mechanics Lab (BBML)

Summary Things to rememberhellip

bull Imaging mode imaging medium and probe selection

bull Morphology Measurements bull Sample preparation and hydration state bull Measuring fibril diameter bull Site selection and proper sampling bull Appropriate system calibration

bull Mechanical Measurements

bull Probe selection (stiffness and tip radius) bull Probe calibration (radius angle spring constant) bull Indentation force range and mechanical model bull Hydration in biological samples bull PeakForce QNM

BBML - All R

ights Reserve

d

Acknowledgements BBML

Max Hammond Armando Diaz Gonzalez Zachary Bart Silvia Canelon Creasy Clauser

IU School of Medicine Dr David Burr Dr Matt Allen Dr Stuart Warden Dr Lilian Plotkin

Funding Sources NIHNIDCR 1F32DE018840-01 A1 IUPUI BME Departmental Startup Funds IUPUI Research Support Funds Grant IUPUI Biomechanics and Biomaterials Research Center

NIH NICHD Dr Joan Marini

BBML - All R

ights Reserve

d

Bone Biology and Mechanics Lab (BBML) wwwiupuiedu~bbml Facebook BoneBiologyMechanicsLab

BBML - All R

ights Reserve

d

• Slide Number 1
• General Seminar Outline
• Bonersquos Hierarchical Structure
• Critical Gap in Understanding Collagen
• Methods For Analyzing Collagen Structure
• Methods For Imaging Collagen Structure
• Cryo-TEM
• Atomic Force Microscopy (AFM)
• AFM Image of Pentacene (C22H14)
• AFM Image of Hexabenzocoronene
• Basics of AFM Imaging The Probe
• Basics of AFM Imaging System Components
• van der Waals Potential Energy Curve
• Contact Mode ndash Monitor Deflection
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Peak Force Tapping Mode ndash Monitor Force
• AFM Applications in Bone Research Imaging
• AFM Imaging of Collagen
• Imaging of Osteoclast Activity
• Mineralized collagen deposition in vitro
• Imaging of Canilicular Structure
• AFM Imaging in Bone
• AFM Imaging in Bone
• Removing Mineral in Bone to Expose Collagen
• Removing Mineral in Bone to Expose Collagen
• Demineralization with EDTA
• Demineralization with EDTA
• Demineralization with EDTA
• Issues to Consider Effects of Acid
• Slide Number 32
• Slide Number 33
• Analysis of Collagen Structure in Bone
• Analysis of Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril D-Spacing
• Slide Number 39
• Collagen in Bone Fibril D-Spacing
• Collagen in Bone Fibril D-Spacing
• 2D Fast Fourier Transform Analysis
• 2D Fast Fourier Transform Analysis
• D-Spacing Distribution
• D-Spacing Distribution
• Cumulative Distribution Function
• CDF vs Histogram
• Changes in D-Spacing with Disease
• Changes in D-Spacing with Disease
• Slide Number 50
• Issues to Consider Site Selection and Sampling
• Issues to Consider Site Selection and Sampling
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• AFM calibration ndash Take Away Message
• AFM Applications in Bone ResearchIndentation Mechanics
• AFM-Based Indentation
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Parameters
• Hertz Contact Equation
• Hertz Contact Equation
• Hertz Contact Equation
• Proper Indentation Model is Essential
• Indentation in OI Tendon Study
• OI Tendon Differential Effects of Dehydration
• OI Tendon Differential Effects of Dehydration
• Indentation in Diabetic Tendon (Poster 0495)
• Multiscale Diabetes Study (Poster 0495)
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• A Note on Peak Force QNM for Bruker AFMs
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• Slide Number 83
• Slide Number 84
• AFM Applications in Bone ResearchOther Cool Mechanical Techniques
• ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo
• Other Mechanical Techniques
• Other Cool Techniques
• Summary Things to rememberhellip
• Acknowledgements
• Bone Biology and Mechanics Lab (BBML)

Acknowledgements BBML

Max Hammond Armando Diaz Gonzalez Zachary Bart Silvia Canelon Creasy Clauser

IU School of Medicine Dr David Burr Dr Matt Allen Dr Stuart Warden Dr Lilian Plotkin

Funding Sources NIHNIDCR 1F32DE018840-01 A1 IUPUI BME Departmental Startup Funds IUPUI Research Support Funds Grant IUPUI Biomechanics and Biomaterials Research Center

NIH NICHD Dr Joan Marini

BBML - All R

ights Reserve

d

Bone Biology and Mechanics Lab (BBML) wwwiupuiedu~bbml Facebook BoneBiologyMechanicsLab

BBML - All R

ights Reserve

d

• Slide Number 1
• General Seminar Outline
• Bonersquos Hierarchical Structure
• Critical Gap in Understanding Collagen
• Methods For Analyzing Collagen Structure
• Methods For Imaging Collagen Structure
• Cryo-TEM
• Atomic Force Microscopy (AFM)
• AFM Image of Pentacene (C22H14)
• AFM Image of Hexabenzocoronene
• Basics of AFM Imaging The Probe
• Basics of AFM Imaging System Components
• van der Waals Potential Energy Curve
• Contact Mode ndash Monitor Deflection
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Peak Force Tapping Mode ndash Monitor Force
• AFM Applications in Bone Research Imaging
• AFM Imaging of Collagen
• Imaging of Osteoclast Activity
• Mineralized collagen deposition in vitro
• Imaging of Canilicular Structure
• AFM Imaging in Bone
• AFM Imaging in Bone
• Removing Mineral in Bone to Expose Collagen
• Removing Mineral in Bone to Expose Collagen
• Demineralization with EDTA
• Demineralization with EDTA
• Demineralization with EDTA
• Issues to Consider Effects of Acid
• Slide Number 32
• Slide Number 33
• Analysis of Collagen Structure in Bone
• Analysis of Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril D-Spacing
• Slide Number 39
• Collagen in Bone Fibril D-Spacing
• Collagen in Bone Fibril D-Spacing
• 2D Fast Fourier Transform Analysis
• 2D Fast Fourier Transform Analysis
• D-Spacing Distribution
• D-Spacing Distribution
• Cumulative Distribution Function
• CDF vs Histogram
• Changes in D-Spacing with Disease
• Changes in D-Spacing with Disease
• Slide Number 50
• Issues to Consider Site Selection and Sampling
• Issues to Consider Site Selection and Sampling
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• AFM calibration ndash Take Away Message
• AFM Applications in Bone ResearchIndentation Mechanics
• AFM-Based Indentation
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Parameters
• Hertz Contact Equation
• Hertz Contact Equation
• Hertz Contact Equation
• Proper Indentation Model is Essential
• Indentation in OI Tendon Study
• OI Tendon Differential Effects of Dehydration
• OI Tendon Differential Effects of Dehydration
• Indentation in Diabetic Tendon (Poster 0495)
• Multiscale Diabetes Study (Poster 0495)
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• A Note on Peak Force QNM for Bruker AFMs
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• Slide Number 83
• Slide Number 84
• AFM Applications in Bone ResearchOther Cool Mechanical Techniques
• ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo
• Other Mechanical Techniques
• Other Cool Techniques
• Summary Things to rememberhellip
• Acknowledgements
• Bone Biology and Mechanics Lab (BBML)

Bone Biology and Mechanics Lab (BBML) wwwiupuiedu~bbml Facebook BoneBiologyMechanicsLab

BBML - All R

ights Reserve

d

• Slide Number 1
• General Seminar Outline
• Bonersquos Hierarchical Structure
• Critical Gap in Understanding Collagen
• Methods For Analyzing Collagen Structure
• Methods For Imaging Collagen Structure
• Cryo-TEM
• Atomic Force Microscopy (AFM)
• AFM Image of Pentacene (C22H14)
• AFM Image of Hexabenzocoronene
• Basics of AFM Imaging The Probe
• Basics of AFM Imaging System Components
• van der Waals Potential Energy Curve
• Contact Mode ndash Monitor Deflection
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Tapping Mode ndash Monitor Amplitude
• Peak Force Tapping Mode ndash Monitor Force
• AFM Applications in Bone Research Imaging
• AFM Imaging of Collagen
• Imaging of Osteoclast Activity
• Mineralized collagen deposition in vitro
• Imaging of Canilicular Structure
• AFM Imaging in Bone
• AFM Imaging in Bone
• Removing Mineral in Bone to Expose Collagen
• Removing Mineral in Bone to Expose Collagen
• Demineralization with EDTA
• Demineralization with EDTA
• Demineralization with EDTA
• Issues to Consider Effects of Acid
• Slide Number 32
• Slide Number 33
• Analysis of Collagen Structure in Bone
• Analysis of Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril Diameter
• Collagen in Bone Fibril D-Spacing
• Slide Number 39
• Collagen in Bone Fibril D-Spacing
• Collagen in Bone Fibril D-Spacing
• 2D Fast Fourier Transform Analysis
• 2D Fast Fourier Transform Analysis
• D-Spacing Distribution
• D-Spacing Distribution
• Cumulative Distribution Function
• CDF vs Histogram
• Changes in D-Spacing with Disease
• Changes in D-Spacing with Disease
• Slide Number 50
• Issues to Consider Site Selection and Sampling
• Issues to Consider Site Selection and Sampling
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider Effects of Hydration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• Issues to Consider AFM calibration
• AFM calibration ndash Take Away Message
• AFM Applications in Bone ResearchIndentation Mechanics
• AFM-Based Indentation
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Cantilever Calibration
• AFM-Based Indentation Parameters
• Hertz Contact Equation
• Hertz Contact Equation
• Hertz Contact Equation
• Proper Indentation Model is Essential
• Indentation in OI Tendon Study
• OI Tendon Differential Effects of Dehydration
• OI Tendon Differential Effects of Dehydration
• Indentation in Diabetic Tendon (Poster 0495)
• Multiscale Diabetes Study (Poster 0495)
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• Indentation Limitations and Weaknesses
• A Note on Peak Force QNM for Bruker AFMs
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• A Note on Peak Force QNM
• Slide Number 83
• Slide Number 84
• AFM Applications in Bone ResearchOther Cool Mechanical Techniques
• ldquoSacrificial Bondsrdquo and ldquoMolecular Gluerdquo
• Other Mechanical Techniques
• Other Cool Techniques
• Summary Things to rememberhellip
• Acknowledgements
• Bone Biology and Mechanics Lab (BBML)