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FTIR Microscopy and Imaging

A world of applications and solutions

Dr. Mustafa Kansiz

FTIR Imaging and Microscopy Product Manager

Agilent Technologies

Email: [email protected]

FTIR Spectroscopy - What is it?

• Fourier Transform InfraRed Spectroscopy is the study of the

interaction of infrared light with matter.

• The vibrations of bonds between atoms in a molecule are

excited by IR light leading to absorbances that are specific

to chemical structure specific

How can FTIR microscopy imaging help me?

An FTIR microscope has two essential purposes:

1. To allow users to visually see small (micron) sized samples

2. Collect accurate FTIR spectra from small samples

FTIR Imaging takes this to another level by providing spatial

and spectral information from an area on your sample

FTIR microscopy can collect data in four modes:

1. Single point

2. Single point mapping

3. Linear array mapping

4. 2-D Focal Plane Array (FPA) imaging

FTIR Imaging gives spatial (WHERE) and spectral (WHAT) information, and FTIR

Chemical Imaging with a IR Focal Plane Array (FPA), gives this simultaneously

FTIR Microscope Measurement Modes

1 : Single Point

Single or multiple spectra of

different zones of a sample

2: Single Point Mapping Automated acquisition of spectra

(one by one) defined by a grid. A

hundred points can take several

hours.

FTIR Microscope Measurement Modes:

4: FPA Imaging

With an FPA detector, up to

16384 spectra can be recorded

simultaneously in a single

measurement.

3: Linear array Mapping Acquisition of spectra by a row

(1x16) of detectors. Faster than

single point mapping, but still

much slower than FPA imaging

Why use FPA chemical imaging?

Two reasons:

1. Provides rapid high spatial resolution chemical distribution – the where (spatial) and the what (spectral)

2. Allows for the measurement of defects as small as a ~2 microns

What do the Coloured Images Mean?

3500 3000 2500 2000 1500 1000

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ATR Chemical Image

Image @

1724 cm-1

PET 7

0 u

m

70 um

C

O

=

O


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bsorb

ance

70

um

70 um

ATR Chemical Image

Image @

1724 cm-1

PET

Image @

3295 cm-1

Nylon

N

H

-

C

=

O

O


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nce

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ance

ATR Chemical Image

Image @

1724 cm-1

PET

Image @

3295 cm-1

Nylon

Image @

2915cm-1

PP

70

um

70 um

C

O

=

O

N

H

-

C

H

-

FTIR Chemical Imaging and its applications Materials/Polymer Polymer laminates (functional

layer & adhesive identification)

Defect analysis

Phase distribution

Composites

Biomedical/Biological Research

Early disease diagnosis (Cancers)

Study of diseases (Alzheimer’s, kidney)

Plant/fungi tissue studies

Live cell chemical imaging (in water)

Microbial identification

Bone, teeth and cartilage

Forensics Car paint layer & structure analysis

Trace evidence analysis

Pharmaceuticals Ingredient distribution

Coating analysis

Defect/particle analysis

Electronics Defect analysis

Art Conservation Painting components & layer identification

Geology Study of inclusions

Food/Cosmetics Study of emulsions, eg cheese, mayonnaise,

cream

Infrared Microscopy Sampling Techniques

Transmission

Sample thickness:

10 – 20 mm

Reflectance

Sample thickness:

NA

Absorption/

Reflectance

Sample thickness:

5 - 10 mm

Objective

Sample

Condenser

Stage

θ θ θ

Micro - ATR

Sample thickness:

NA

“Kevley SlideTM”

θ

ATR - IRE

Large Sample Microscopy Sampling Techniques – Measure small areas on large samples

Large Sample

Reflectance

Sample thickness:

NA Sample thickness:

NA

Sample thickness:

NA

large

sample

External

sample

stage

large

sample

large

sample

Large Sample

Micro ATR

Large Sample

Grazing Angle

Obje

ctive

Obje

ctive

GA

O

External

sample

stage

External

sample

stage

Agilent’s patented1 Large Sample Microscope Objective allows you to measure unlimited sized samples in reflection or ATR single point or imaging mode

It’s as simple as:

1. Taking your sample to the microscope

2. Placing it against the 90 degree objective

3. Press collect and get your results!


Examples:

1. Investigation of new coating materials on helmets (military / aerospace / etc.)


Examples:

1. Investigation of new coating materials on helmets (military / aerospace / etc.)

2. Large vehicle component analysis, ex: car door/panel

Large sample

15x Objective

Mirror assembly

3000 2000 1000

0.4

0.2

0.0 4000

cm-1

Ab

s.

LS

Large Sample Objective

VARIAN/Marketing/Marcom/TVEII/Refined animtions/flexibility/flexibility.exe

Single point analysis (Single-element detector analysis)

Single Point Analysis

1 detector element collects 1 spectrum per scan

Desired spatial resolution is attained by fixing aperture size to desired sampling size. This

eliminates spectral interference from the surrounding area.

Typical best achievable spatial resolution is 10 - 20 µm.

Transmission IR Analysis of Microtomed 3 layer Laminate

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sorb

an

ce

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sorb

an

ce

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sorb

an

ce

Wavenumber (cm-1)

Plastic Tubing – Defect analysis

ATR

ATR

Tube spectrum #2

Tube spectrum #3

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4x glass visible objective

15x reflective vis/IR objective


2.5 mm

2.0

mm

660 mm

50

0 m

m

660 mm

50

0 m

m

4x glass visible objective


ATR

Glue spectrum #1

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ance

2.5 mm

2.0

mm

660 mm

50

0 m

m

Plastic Tubing – Defect analysis

Infrared Mapping

- Single Point

- Linear Arrays

Infrared Single Point Mapping

One data collect at each point gives one interferogram or spectrum

Move the sample with a motorized stage and collect multiple scans

Build up a Map of the sample 1 2

3 4

5 6

Automatic Single Point Mapping

The automated mapping experiment can be used to

obtain IR spectra over a sample.

• Define the field to be mapped.

• Define spacing of the points to be collected (x and y direction).

• Define aperture/spatial resolution (the area you are collecting data on an individual scan).

• You must have visual control of the experiment. You are visually locating areas of interest.

Infrared Linear Array Mapping

• A row of data from each pixel in the array (usually 1 x 16 ) collected

simultaneously

• Move the sample with a motorized stage and collect multiple scans

• Build up a Map of the sample by “stitching” linear array scan together

• Faster than single point mapping, but still much slower than FPA imaging

1 3 4 5 2 6

Layer 1 0.9

0.6

0.3

0.0

Ab

so

rban

ce

4000 3000 2000 1000

Wavenumber (cm-1)

Polyester resin

4000 3000 2000 1000

Wavenumber (cm-1)

1.0

0.5

0.0

Ab

so

rban

ce

1.5

1.0

0.5

0.0

Ab

so

rban

ce

Layer 2

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Wavenumber (cm-1)

0.9

0.6

0.3

0.0

Ab

so

rban

ce Layer 3

4000 3000 2000 1000

Wavenumber (cm-1)

Layer 4 0.9

0.6

0.3

0.0

Ab

so

rban

ce

4000 3000 2000 1000

Wavenumber (cm-1)

Infrared Mapping in Material Analysis:

Multi-layer Paint Sample

Infrared Mapping in Material Analysis:

Multi-layer Paint Sample

Feature image based on absorbance at 3692 cm-1.

Abs. at 3692 cm-1

Infrared 2-D Focal Plane

Array Imaging

FPA-FTIR detectors provide the ability to acquire a grid of spectra in the same

amount of time that it takes single point detectors to acquire one spectrum

One pixel (~5.5µm)

is an entire spectrum

Sample is imaged onto a FPA detector (n n pixel array)

n2 spatially resolved spectra are collected simultaneously

(e.g., 16 16 array 256 spectra)

Principle of Infrared Imaging

Pixel Size (obj mag, NA, mode) Achieved

Spatial

Resolution

3750 cm-1

Achieved

Spatial

Resolution

2500 cm-1

Single FPA tile

FOV (with

128x128FPA)

3.3 um (25x, 0.81NA, std mag) 4.3 um 5.0 um 420x420 um

0.66 um (25x, 0.81NA, high mag) 1.4 um 1.7 um 85x85 um

5.5 um (15x, 0.62NA, std mag) 6.9 um 7.6 um 700x700 um

1.1 um (15x, 0.62NA, high mag) 2.4 um 3.0 um 140x140 um

19 um (4x IR, 0.2NA, std mag) 20.4 um 20.0 um 2400x2400 um

Achieved Spatial Resolution Summary

USAF target imaged (280x280um) at 1.1 um

resolution (high mag mode) with 15x objective in

8 minutes.

2x2 tile mosiac with 128FPA

Entire 2”x2” (50x50mm) USAF target imaged at

19 um pixel resolution with 4xIR objective in 90

minutes

(21x21 tile mosaic with128FPA)

USAF target (700x700um) imaged at 5.5 um pixel

resolution (normal mag. mode) with 15x objective in

2 minutes

Single 128FPA tile

1.4um

0.98um 25x obj

Advancing FTIR Imaging with the Agilent Cary 620

June 12, 2015

27

Highest spatial resolution Largest Field of View

Superior signal-to-noise Fastest data collection

MARKETS AND APPLICATIONS

Polymer Film Laminate FTIR Imaging

Sample Preparation Free FTIR Chemical Imaging of Polymer laminates & Films

Step 1. Cut out small piece Step 2. Place cut-out piece in micro-vice.

Step 3. Cross-section sample with razor Step 4. Place micro-vice (with sample) on

microscope stage & touch ATR

ATR Contact with sample

STEP 5.

raise stage

to make

contact &

collect data

micro ATR

Microscope Stage

micro-vice

Sample

100 micron

wide (thick)

IR light in IR light out

micro ATR

micro-vice

Microscope Stage

Sample

100 micron

wide (thick)

IR light in IR light out

Standard Live ATR direct FPA IR Image – without correction

Live ATR direct FPA IR Image with Enhanced Chemical Contrast

No Pressure

(before contact)

“Live/Real-Time” ATR contact monitoring

No Pressure

(before contact)

No Pressure

(before contact)


First Contact No Pressure

(before contact)

Stage is

raised

Stage is

raised

First Contact



No Pressure

(before contact) Increasing Pressure

Increasing Pressure


First Contact Complete Contact No Pressure

(before contact)

Stage is

raised

Stage is

raised

First Contact



Sample Presentation

Confidentiality Label

June 12, 2015

39

A small piece of the sample was cut out

and place into a micro-vice sample holder

where it was cut flat with a sharp razor

The micro-vice is placed in a dedicated insert

on the motorised stage and contact is made with the

ATR crystal, using Agilent’s unique “Live ATR contact”

method, without any need for resin embedding

50

0 u

m

670 um

Sausage Packaging (Red): Visible images ATR Imaging Sampling Location

15x obj. vis image

ATR

spot 1

Collection Conditions:

Resolution: 4 cm-1

Scans (time): 32 scans (~30 sec) per spot

Spectral Range: 4000 – 850 cm-1

Collection Mode: Micro ATR & transmission (5 micron microtomed slices)

Pixel Size & FOV: 1.1 microns/pixel, 70x70 microns

ATR

spot 2

ATR

spot 3

Different collection mode comparison

1. Micro ATR (1.1 micron @ 70x70 micron FOV)

2. Transmission with 15x normal mag (5.5 micron pixel size & 700x700 micron FOV)

3. Transmission with 15x HIGH mag (1.1 micron pixel size & 140x140 micron FOV)

4. Transmission with NEW 25x normal mag (3.3 um pixel size & 420x420 um FOV)

5. Transmission with NEW 25x HIGH mag (0.66 um pixel size & 85x85 um FOV)

-


June 12, 2015

41


June 12, 2015

42

15x obj. vis image

70

um

70 um

ATR Image @ 2850cm-1

PE

~46 um thick

70

um

70 um

70

um

70 um

ATR Image @ 1607cm-1 PE (with additive)

~23 um thick

70

um

70 um

ATR Image @ 1725 cm-1 Polycarboxylic acid ester

polymer (possibly)

~5 um thick

70

um

70 um

ATR Image @ 1202cm-1 polyamide

ATR Chemical

RGBY Composite Image

Red: PE

Yellow: PE (with additive)

Green: Polycarboxylic acid ester (possible)

Blue: Polyamide

Layer 1&3

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Layer 2

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ance

Layer 4

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ance

Layer 5

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bsorb

ance

1 2 3 4 5

Sausage Packaging – ATR Chemical Images (Spot 1)



June 12, 2015

43

15x obj. vis image 7

0 u

m

70 um

ATR Chemical Image

RBGWC composite image

Red: PE

Green: Polycarboxylic acid

polymer (possibly)

Blue: Polyamide

White: Polypropylene

Cyan: EVOH

70

um

70 um

Ima

ge

@

28

50

cm

-1

Layer 4

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ance

Layer 5, 8 & 10

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ance

Layer 6

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ance

Ima

ge

@

17

25

cm

-1

Ima

ge

@

12

02

cm

-1

Ima

ge

@

29

51

cm

-1

Ima

ge

@

10

90

cm

-1 Layer 9

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ance

Layer 3, 7

& 11

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ance

PE (with additives)

~6 um thick (centre layer)

Polycarboxylic acid ester

polymer (possibly)

~5 um thick

Polyamide

~7um (left)

~2um (centre)

~3um (right)

Polypropylene

~20um

EVOH

~5um

(spectral overlap from

Polyamide present)

3 4

5 6 7

8 9 10

11


June 12, 2015

44

15x high mag. obj. vis image

70

um

70 um 7

0 u

m

70 um

ATR Chemical Image PP (possible with an

additive as evident by

peak at 1060cm-1)

~37 um thick

RBC composite image

Red: PE

Blue: Polyamide

Cyan: EVOH


Layer 5, 8 & 10

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ance

Polyamide

~2um (left)

~3um (right)

Layer 9

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0.06

0.04

0.02

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Absorb

ance

EVOH

~5um thick

(spectral overlap

from Polyamide

present)

Layer 11

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ance

7 8 9 10 11


June 12, 2015

45

Sausage Packaging – ATR Chemical Images

The sample was measured across its entire width with three

slightly overlapping ATR measurements, with a total

collection time of ~ 1.5 mins.

As extremely low pressures are applied, there is no sample

preparation (via Agilent’s “live ATR imaging” method) with

samples being measured “as is” and no risk of sample

surface deformation, which might otherwise make for

sequential side-by-side, or slightly overlapping

measurements impossible. There was also no evidence of

sample carryover between the measurements.

With the excellent signal-to-noise data collected, quite a

complex sample with at least 11 layers were revealed with

spectral library searches assisting in layer identification.

PE

~11

~130 um

FT

IR A

TR

im

ag

ing

de

term

ine

d id

en

tity

&

ap

pro

xim

ate

th

ickn

ess

(in

mic

ron

s)

PE

with

ad

ditiv

e

Poly

carb

oxylic

acid

este

r

poly

mer

(possib

ly)

P

A

PE

PP

PE

P

A

PA

E

VO

H

PE

~24 ~11 ~5 ~7 ~20 ~6

~3

~5

~3

~37

1 2 3 4 5 6 7 8 9 10 11

40x obj. vis image (polarized)

Pixel size: 1.1micron

Obj mag: 15x, 0.62NA

FOV: 70x70um

Total system mag: 36x

14

0 u

m

180 um


June 12, 2015

46

Sausage Packaging – 25x Transmission High mag Chemical Images

- Even in transmission mode, the ultra high

NA and very small pixel size has allowed for

the resolution of the 3 micron PA layer,

hence rivalling the spatial resolution of Ge

micro ATR

- Most books and papers still talk of ~10um

spatial resolution for transmission imaging!

- The 25x, 0.81NA is a revolution in objective

design


EVOH, layer 3

PA, layer 2

PE, layer 1

3500 3000 2500 2000 1500 1000

Wavenumber

Absorb

ance

Pixel size: 0.66 micron


High mag: ON

FOV: 85x85um


Working distance: 12mm

1

2 3

14

0 u

m

180 um


June 12, 2015

47

The ~3 micron PA layers are only just resolved,

but the spectral differences between PA and

EVOH are now much less, owing to the lower

NA, and hence resolving power, of the 15x

objective compared to the 25x objective.




High mag: ON

FOV: 140x140um


Working distance: 21mm

Sausage Packaging – 15x Transmission High mag Chemical Images

1

2 3

EVOH, layer 3

PA, layer 2

PE layer 1

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Absorb

ance

14

0 u

m

180 um


June 12, 2015

48

With the larger pixel size, now at 3.3um, even

with the high

NA 25x objective, the ~3um PA layers cannot

be spatially resolved




High mag: OFF

FOV: 420x420um


Sausage Packaging – 25x Transmission std mag Chemical Images

14

0 u

m

180 um


June 12, 2015

49

14

0 u

m

180 um




High mag: OFF

FOV: 700x700 um

Total system mag: 7.3x

Sausage Packaging – 15x Transmission std mag Chemical Images

As the pixel size gets larger, now at 5.5um,

layers that are ~5 um now start to blur out

Principal Component Noise Reduction (PCNR)

June 12, 2015

Agilent Confidential

50

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ance

1 scan

1 scan with PCNR

128 scans

- Removes ONLY noise

- Typical S:N

improvement of

~5-10x

- Corresponding time

saving (for equivalent

S:N) is 25-100x !

- Only FTIR imaging

system to include

PCNR

Residual spectrum

1720 cm-1

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ance

640 um

480 u

m ATR

defect, image @ 1402 cm-1

polymer, image @ 1402 cm-1

70

mm

70 mm

Composite (Green/Red) image

GREEN – DEFECT

RED - POLYMER

At initial analysis, it appears that the defect is likely to be an

Inorganic material, most probably a carbonate, or a carbonate

containing mixture

15x obj. vis image – cross-section view ATR Chemical Image

Polymer Film Defects - Visible Images & ATR Imaging Sampling

Location

Micro ATR (FPA) imaging of defects in black rubber sample

Ima

ge

cre

ate

d

at 1

64

4 c

m-1

Im

age

cre

ate

d

at 2

84

8 c

m-1

Vis image IR image

70mm

70

mm

10 um

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0.08

0.06

0.04

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Wavenumber

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ance

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0.05

0.00

Wavenumber

Absorb

ance

3500 3000 2500 2000 1500 1000

0.18

0.16

0.14

0.12

0.10

0.08

0.06

Wavenumber

Ab

so

rba

nce

Polyamide

PE

Polyisoprene (natural rubber)

ATR

640 um

480 u

m

25x, 0.81 NA Objective in “high mag” mode for Transmission Polymer bead analysis

June 12, 2015

Agilent Confidential

53

2915 cm-1 1442 cm-1 1118 cm-1

40x vis image

85mm

85

mm

5 micron polystyrene beads on BaF2 substrate are

clearly chemically distinguished.

Even at relatively long wavelengths (low

wavenumbers), 5 micron beads are clearly resolved

(3.4 microns) (6.9 microns) (8.9 microns)


Obj mag: 25x, 0.81 NA

High mag: ON

FOV: 85x85 um


Working Distance: 12 mm

35

0u

m

470um

Exposed green plastic – Visible images & ATR Imaging Sampling Location

15x obj. vis image

ATR

70

um

70 um

Sample Placement & Measurement


June 12, 2015 55

The large green plastic block was placed directly “as-is” into

the micro-vice holder, which was then placed on the microscope

stage, followed by raising of the stage to make contact with the

ATR for data collection.

3500 3000 2500 2000 1500 1000

0.1

0.0

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Absorb

ance

Results - Exposed Green Plastic


June 12, 2015 56

40x high mag. obj. vis image

70

um

70 um

70

um

70 um

ATR Chemical Image Image @ 1736cm-1

A clear edge containing higher carbonyl content is

clearer visible along the expose side edge.

Pharmaceutical FTIR Imaging

57

Row = 51 Col = 28

3500 3000 2500 2000 1500 1000

0.3

0.2

0.1

0.0

Wavenumber

Absorb

ance

Row = 43 Col = 59

3500 3000 2500 2000 1500 1000

0.3

0.2

0.1

0.0

Wavenumber

Absorb

ance

Row = 12 Col = 10

3500 3000 2500 2000 1500 1000

0.4

0.3

0.2

0.1

0.0

Wavenumber

Absorb

ance

Row = 29 Col = 19

3500 3000 2500 2000 1500 1000

0.20

0.15

0.10

0.05

Wavenumber

Absorb

ance

Tablet – Contamination Location 15x Vis Image

48

0 m

m

640 mm

70

mm

70 mm

Clear and distinct domains from

4 constituents were detected:

- Active, compared to provided standard

- Lactose, identified from library search

- Starch, identified from library search

- Cellulose identified from library search

Active

Lactose

Starch

Cellulose

Defect (next slide)

Row = 10 Col = 26

3500 3000 2500 2000 1500 1000

0.20

0.15

0.10

0.05

Wavenumber

Absorb

ance

Row = 30 Col = 28

3500 3000 2500 2000 1500 1000

0.15

0.10

0.05

Wavenumber

Absorb

ance

15x Vis Image

48

0 m

m

640 mm

70

mm

70 mm

Clear and distinct domains from

4 constituents were detected:

- Contaminant #1 – Possibly a polyester

- Contaminant #2, - Possibly a polyamide

Tablet – Contamination Location 7

0 m

m

70 mm

Contaminant Composite

(RG), part 2

Polyamide

Polyester

From a single FTIR ATR imaging measurement and

without damaging the sample, 4 known constituents

and 2 unknown contaminants were imaged in 1 min

Electronics/Semicon FTIR Imaging

Spacer contamination on LCD filter

3500 3000 2500 2000 1500 1000

0.03

0.02

0.01

0.00

Wavenumber

Absorb

ance

3

50

um

350 um

70

um

70 um

5 mm

Total analysis time = 2 mins

Defects identified as dislodged

Spacers

No sample prep and no sample

damage

1647.960 12.936Row = 6 Col = 30

3800 3600 3400 3200 3000 2800 2600 2400 2200 2000 1800 1600 1400 1200 1000

0.3

0.2

0.1

0.0

Wavenumber

Absorb

ance

LCD Defect – Protein image (1647 cm-1)

70 microns

70 m

icro

ns

Visible Image

IR image

at 1647cm-1 Total analysis time = 1 mins

Defects identified as protein, most

probably flake of dead skin

No sample prep and no sample

damage

480 u

m

640 um

3500 3000 2500 2000 1500 1000

0.20

0.15

0.10

0.05

0.00

Wavenumber

Absorb

ance

350 u

m

70

um

70 um

Contaminated Circuit Board – FTIR ATR Imaging

ATR contact damage

caused by other vendor

Total analysis time = 2 mins

Spectra library search

reveals contaminant to be

polyetherimide

Visible Image

Forensics FTIR Imaging

Vehicle paint cross sectional analysis

36

96

33

99

30

38

29

50

1723.527 21.695

16

95

1602.985 1.303

1561.467 0.11515

10

14

65

13

75

11

33

10

55

Row = 75 Col = 160

3800 3600 3400 3200 3000 2800 2600 2400 2200 2000 1800 1600 1400 1200 1000

0.8

0.6

0.4

0.2

0.0

Wavenumber

Absorbance

36

96 3

39

9

30

38

29

50

1723.527 37.245

16

95

1602.985 -1.255

1561.467 0.10015

10

14

65

13

75

11

33

10

55

Row = 24 Col = 146

3800 3600 3400 3200 3000 2800 2600 2400 2200 2000 1800 1600 1400 1200 1000

0.6

0.4

0.2

0.0

Wavenumber

Absorbance

36

96

33

99

30

38

29

50

1723.527 20.244

16

95

1602.985 -0.465

1561.467 0.301

15

10

14

65 13

75 11

33

10

55

Row = 94 Col = 92

3800 3600 3400 3200 3000 2800 2600 2400 2200 2000 1800 1600 1400 1200 1000

0.8

0.6

0.4

0.2

Wavenumber

Absorbance

36

96

33

99

30

38

29

50

1723.527 13.5691

69

5

1602.985 -2.144

1561.467 0.463

15

10

14

65

13

75

11

33

10

55

Row = 90 Col = 98

3800 3600 3400 3200 3000 2800 2600 2400 2200 2000 1800 1600 1400 1200 1000

0.7

0.6

0.5

0.4

0.3

0.2

0.1

Wavenumber

Absorbance

36

96

33

99

30

38

29

50 1723.527 15.150

16

95

1602.985 3.889

1561.467 0.031

15

10

14

65

13

75

11

33

10

55

Row = 164 Col = 123

3800 3600 3400 3200 3000 2800 2600 2400 2200 2000 1800 1600 1400 1200 1000

0.6

0.5

0.4

0.3

0.2

Wavenumber

Absorbance

36

96

33

99

30

38

29

50

1723.527 21.636

16

951602.985 1.044

1561.467 0.080

15

10

14

65

13

75

1086.493 7.960

10

55

Row = 149 Col = 135

3800 3600 3400 3200 3000 2800 2600 2400 2200 2000 1800 1600 1400 1200 1000

1.0

0.8

0.6

0.4

0.2

0.0

WavenumberA

bsorbance

3696 cm-1

3399 cm-1 1695 cm-1 1561 cm-1 1086 cm-1

3038 cm-1

MIXED POWDER MICRO ATR ANALYSIS – CALCIUM CARBONATE

CaCO3(1420.000)

STPP(1140.010)pyrophosphate(987.127)disodium phosphate(945.393)

Row = 37 Col = 32

3500 3000 2500 2000 1500 1000

0.8

0.6

0.4

0.2

0.0

Wavenumber

Absorb

ance

MIXED POWDER MICRO ATR ANALYSIS – DISODIUM DIPHOSPHATE ANHYDRIDE

CaCO3(1420.000)

STPP(1140.010)

pyrophosphate(987.127)

disodium phosphate(945.393)

Row = 38 Col = 48

3500 3000 2500 2000 1500 1000

0.20

0.15

0.10

0.05

0.00

Wavenumber

Absorb

ance

CaCO3(1420.000)

STPP(1140.010)

pyrophosphate(987.127)disodium phosphate(945.393)

Row = 61 Col = 56

3500 3000 2500 2000 1500 1000

0.2

0.1

0.0

Wavenumber

Absorb

ance

MIXED POWDER MICRO ATR ANALYSIS – SODIUM TRIPOLYPHOSPHATE

CaCO3(1420.000)

STPP(1140.010)

pyrophosphate(987.127)

disodium phosphate(945.393)

Row = 17 Col = 17

3500 3000 2500 2000 1500 1000

0.3

0.2

0.1

0.0

Wavenumber

Absorb

ance

MIXED POWDER MICRO ATR ANALYSIS – SODIUM PYROPHOSPHATE

Food/Cosmetics FTIR Imaging

Imaging of mayonnaise heterogeneity: measured in transmission on BaF2 slide

1648.271 24.136TmpltPk1(3614 0.5)

2999.749 -0.594

2925.612 0.119

2113.148 4.845

oil(1745 0.0)

water(1650 40.7)starch(1046 48.9)

Row = 69 Col = 63

3800 3600 3400 3200 3000 2800 2600 2400 2200 2000 1800 1600 1400 1200 1000

1.5

1.0

0.5

0.0

Wavenumber

Absorbance

1648.271 1.474TmpltPk1(3614 0.0)

2999.749 1.613

2925.612 22.490

2113.148 0.086

oil(1745 9.6)

water(1650 2.3)

starch(1046 0.0)

Row = 28 Col = 106

3800 3600 3400 3200 3000 2800 2600 2400 2200 2000 1800 1600 1400 1200 1000

1.4

1.2

1.0

0.8

0.6

0.4

0.2

Wavenumber

Absorbance

1648.271 24.505TmpltPk1(3614 0.5)

2999.749 -0.2642925.612 0.210

2113.148 4.503

oil(1745 0.1)

water(1650 39.8)

starch(1046 2.0)

Row = 70 Col = 66

3800 3600 3400 3200 3000 2800 2600 2400 2200 2000 1800 1600 1400 1200 1000

1.5

1.0

0.5

0.0

Wavenumber

Absorb

ance

sugar @1046cm-1

oil @1745cm-1

water @1650cm-1

Visible CCD image

650 um

50

0 u

m

Measured on FTS7000+UMA600, using

a 128x128 FPA.

IR measurement area = 700x700 microns

Time of measurement = ~ 2min

Resolution = 8 cm-1

Biological & Biomedical FTIR Imaging

Breast Tissue section

CH image (2943-2893 cm-1) Amide I image

Note: red box, indicates area for single tile high mag collect on next slide

1233 cm-1 image

3800 3600 3400 3200 3000 2800 2600 2400 2200 2000 1800 1600 1400 1200 1000

1.2

1.0

0.8

0.6

0.4

0.2

Wavenumber

Absorb

ance

3800 3600 3400 3200 3000 2800 2600 2400 2200 2000 1800 1600 1400 1200 1000

1.0

0.9

0.8

0.7

0.6

0.5

0.4

0.3

0.2

0.1

Wavenumber

Absorb

ance

Total IR FOV = 1400x1400 um (2x2 mosaic), pixel size = 5.5um, total data collect time ~ 6 mins,

Amide I image 1233 cm-1 image 40x obj vis image

CH image (2943-2893 cm-1)

3800 3600 3400 3200 3000 2800 2600 2400 2200 2000 1800 1600 1400 1200 1000 800

1.0

0.8

0.6

0.4

0.2

0.0

Wavenumber

Absorb

ance

3800 3600 3400 3200 3000 2800 2600 2400 2200 2000 1800 1600 1400 1200 1000 800

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0.0

Wavenumber

Absorb

ance

Breast Tissue section (high mag mode)

Note: red box, indicates area for single tile high mag collect on next slide

Total IR FOV = 280x280 um (2x2 mosaic), pixel size = 1.1um, total data collect time ~ 12 mins,


June 12, 2015

75

Normal Mag (5.5um) – High Mag (1.1um) comparison, CH image

28

0 u

m

280 um

28

0 u

m

280 um

3500 3000 2500 2000 1500 1000

0.6

0.4

0.2

0.0

Wavenumber

Absorb

ance

3500 3000 2500 2000 1500 1000

0.7

0.6

0.5

0.4

0.3

0.2

0.1

Wavenumber

Absorb

ance

Normal Mag (5.5um) High Mag (1.1um)

The 3 features

within this oval

shape are each

about ~3 um

in diameter!

3341.250 71.728

1733.180 -0.889

1654.450 22.9651651.180 28.040

1539.600 7.408

1044.970 27.715

Row = 26 Col = 89

3800 3600 3400 3200 3000 2800 2600 2400 2200 2000 1800 1600 1400 1200 1000

0.8

0.6

0.4

0.2

Wavenumber

Absorb

ance

3341.250 162.961

1733.180 7.765

1654.450 6.1331651.180 8.118

1539.600 -0.261

1044.970 74.983

Row = 68 Col = 104

3800 3600 3400 3200 3000 2800 2600 2400 2200 2000 1800 1600 1400 1200 1000

1.2

1.0

0.8

0.6

0.4

Wavenumber

Absorb

ance

3341.250 104.920

1733.180 2.899

1654.450 9.8421651.180 14.684

1539.600 2.721

1044.970 54.253Row = 57 Col = 100

3800 3600 3400 3200 3000 2800 2600 2400 2200 2000 1800 1600 1400 1200 1000

1.0

0.8

0.6

0.4

Wavenumber

Absorb

ance

Protein – 1654 cm-1

Lipid – 1733 cm-1

Carbohydrate – 1200-950 cm-1

Wheat stem cross section FTIR Image See next set of

data for med.

res. close up

See next set of

data for med.

res. close up

See next set of

data for med.

res. close up

See following set of

data for high.

res. close up


data for high.

res. close up


data for high.

res. close up

1043.570 11.216

Row = 12 Col = 21

3400 3200 3000 2800 2600 2400 2200 2000 1800 1600 1400 1200 1000 800

0.16

0.14

0.12

0.10

0.08

0.06

0.04

0.02

0.00

Wavenumber

Absorb

ance

Visible Image

(red box show field of

view ATR)

2-D IR image

at 1043 cm-1

3-D IR image

at 1043 cm-1

IR spectrum from

cross-hairs above

ATR Imaging has provided

for a 5 fold increase in

magnification and REAL

spatial resolution

enhancement.

~5 micron cells wall have

been CLEARLY resolved

with 10 micron light (~1000

cm-1) and have produced

high quality spectra free of

artefacts due to diffraction

and scattering.

70 microns

70 m

icro

ns

June 12, 2015

78

Visible image C-H image Lipid image Protein image

Composite (RGB) image

Lipid – Red

C-H – Green

Protein - Blue

100 microns 100 microns 100 microns 100 microns 100 microns

Live cell FTIR imaging in water!

Liquid transmission cell pathlength = 7microns

Total measurement time ~ 10mins.

Pixel size : 1.1 microns

Summary of Cary FTIR Imaging Highest Spatial Resolution

with new 25x, 0.81NA obj.

Re-defines, what is possible with actual spatial resolution of <1.5 microns possible in transmission mode!

Largest Field of View

Measure up to 2.4x2.4mm in a single shot

Fastest analysis time

Reduce analysis times by >100x , with higher light throughput & PCNR

June 12, 2015

79

Live FPA Imaging

Removes need for complex, time consuming sample prep & allows for

delicate analysis