1932-17

Winter College on Micro and Nano Photonics for Life Sciences

Martina Havenith

11 - 22 February 2008

Ruhr University BochumBochum, Germany

Scanning IR-nearfield optical microscopy (SNIM)

IR-nearfield microscopyM. Havenith

Physical Chemistry, Ruhr-Universität Bochum, GermanyMünster, 17.12.07

www.rub.de/pc2

wav

elen

gth

ener

gy

OpticalOptical

FIRFIR

THzTHz

MIRMIR

Taking a closer look on surfaces and interfaces: Spectral Fingerprints

Determination of structure

Skeleton motionsBreathing modesLarge amplitude vibrations

Intermolecular modesPhonons

Biology: a “physicist” view

Cytosol: High concentration of Protein (1650 cm-1 Amid I band)

Membrane: Strong absorption of lipids at 2920 cm-1

Nucleus with DNA in high concentration: Strong absorption at 1240 cm-1

Water: percentage of water 60-98%: high absorption coefficient in the IR and NIR

DNACytosol

Lipids

Absorption

wavenumber [cm-1]

Fingerprint region of infrared vibration bands

A cheap chemical microscope

Measurement of intracellular water

• Intracellular water concentration is important for cell function

• Hormone, Oxidative stress, miniomla dosis of pharmaceuticals can alter the intracellular water concentration

• Change of osmotic pressure causes cell swelling or cell shrinkage

• Intracellular water concentration influences protein syntheses, cell proliferation, cell death (apotheosis)

• Dream of medicine: control of intracellular water concentration

Insulin + cAMPcAMP/HypoosmolarityGlucagon/HypoosmolarityGlucagon/InsulinHyperosmolarity/InsulincAMP + VasopressinHypoosmolarityGlutamineGlycineAlanineGlutamine + GlycineInsulinInsulin/BumetanideEthanolAcetaldehydeEthanol/BumetanideEthanol/MethylpyrazolTaurocholateGlycerolIGF1BaCl2Glucagon + HypoosmolarityGlucagon + Insulin

Liver Cell Hydration and ProteolysisStudy of Prof. Häussinger (medicine)

% change of cell water

% inhibition of proteolysis

Influence of minimum dosages of pharmaceuticals on single cell

A non-invasive method to quantify water in living cells

• Water: most abundant substance in organisms

• Chemical reactions of all organism take place in aqueous solution

• Transport of solutes• Temperature regulation• NIR overtone transition

(∆v=2), weaker than ∆v=1Absorption, but still penetrating

Experimental Results:

reference: absorption by HBSS

absorption by intra-cellularwater + HBSS

Swelling: volume increases by a factor of 3Intracellular water concentration: first increase (dilution), followed by a decreaseExplanation: Onset of protein synthesis yields a decreasein relative water concentration

I(ν) = I0 exp(-α(ν)l)

E. Bründermann, E. Bründermann, A. Bergner, F. Petrat, R. Schiwon, G. Wollny,I. Kopf, H. de Groot, M. Havenith, Analyst, 129, 893 (2004)

Future potential for medical applications:Monitoring the influence of pharmaceuticals on a single cell

level in real time

Future improvements:

Tomography

Tracking drugs in single cells„A non-invasive methodTo quantify water in livingcells is being pioneerd in Germany“

by the Royal Society, November 2004

Spectralfingerprints

Confocal Raman microscopyof human skin (WITEC)

measurementof hydrationin human skin

hydrationin dependenceof depth

„Chemical Microscopy“ in the IR-and THz :advantage: Marker free

disadvantage: diffraction limited

Abbe Limit: lateral resolution > λ/2

Abs

orba

nce

1800 1500wavenumber cm-1

Senterra Raman microscope

Atomic force field microscopy (AFM)measurement of topography

nm lateral resolution

1986:G. Binning, C. Quate,Ch. Gerber

Infrared Near field microscopyChemical imaging beyond the wave length limit

Far fieldPlane wave

Infrared Near field microscopyChemical imaging beyond the wave length limit

Near field

Infrared Near field microscopyChemical imaging beyond the wave length limit

Nano antennaNano aperature

evanescent field withexponential decay

cut-offpointλ/2

e.g. glass fiber coatedwith metal

z max. 0.1·d

angle : 10 - 20°d : 100 - 50 nm

10 -3 - 10 -6transmissionVIS

IR d : 100 nm 10 -25

transmission

Near field microscopyMicroscopy beyond the wave length limit

Aperture d

Pump laser: Master Oscillator Power Amplifier: output power: 20 W at 1064 nm Periodically Poled Lithium Niobate (quasiphase matched) 19 poling areasend caps are tilted to prevent back reflection; tripe band antireflection coatingcw IR radiation 2.9 Wresonant for signal wavelength (1485-1.665 nm) ; idler (3000-4000 nm)Faraday isolator; three lens system for proper phase matching

High power IR-radiationIR-cw Opto Parametric Oscillator (2.9 W)

35 db

Apertureless near-fieldusing our high powerIR-cw OPO (2.9 W) !

[1] Knoll, B. and Keilmann, F.“Near-field probing of vibrational absorption for chemical microscopy“ Nature 399 134-137 (1999)[2] A. Lahrech, R. Bachelot, P. Gleyzes, A.C. Boccara, “Infrared-reflection-mode near-field microscopy using an apertureless probe with a resolution of λ/600”, Optics Letters 21, 1315-1317 (1996).

OPO

160 - 190 kHz

MCTdetector

lock-inamplifier

AFM

amplifier

pp

ps

εprobe

probe dipole (∅ = a)

sample dipole

r = distance

OPO( )( )pincss

sincpp

EEp

EEp

+=

+=

α

α

p-polarization

62

3

14

1

1

r

rsp

spsp

eff

⋅−

⋅++=

πααπαα

ααα

62

3

116

1

1

r

rsp

spsp

eff

⋅−

⋅−+=

πααπαα

ααα

dipole

• r >> a, sum of the polarizabilities

• otherwise, dipole-dipole interaction

• p-polarization : increase

• s-polarization : decrease

• σscatt ∝ k4 αeff׀ 2׀ • absorption ∝ Im (αeff)

of α ∝ Esca

s-polarization

A chemical nanoscope: contrast mechanism

Scanning near-field infrared microscopy (SNIM)Chemical fingerprints beyond the diffraction limit

(λ = 3,22 µm; ν = 3100 cm-1)30 nm gold balls on glas

chemical nanoscopeNear field

physical nanoscopetopography

PolylysineAu balls

30nm

Resolution is limited only by the tip of the cantilever not by the wavelengthLateral resolution correpsonds to λ/100 !!

Scanning nearfield infrared microscopy (SNIM)Imaging of sub-surface structures

PeriodicallyimplantedGa ionsin a Si wafer

5 µm

1 µm 1 µm

5 µma) b)

c) d)

2004e) f)

AFM SNIM

Ga:SiN(Ga)=4,4.1019cm3

λ=3,22 µm

Measurement of implanted Ga-ionsin a Si wafer

Imaging with a „chemical nanoscope“Observation of subsurface structures(Phys. Chem. Chem. Phys. 8, 753 - 758 (2006))

Chemical imaging of monolayers

S S

CH3CH3

S S

CH3CH3

S SS S

biotin

biotin

biotin

biotin

S SS SS S

CH3CH

S S

CH3CH33 biotin

biot

in

biotin

biot

in

AuTiSi

Electron microscope image of stamp and structured surface

PDMS stamp

biotinylated alkylthiol (BAT)

1-octadecanthiol (ODT)

biotinylated alkylthiol(BAT)

1-octadecanthiol(ODT)

IR spectra of distinct functional groups

C=O bandof ureidogroup at1710 cm-1

1.8µm 1.8µm

Experimental results

0 1 2 3 4 5 6 7 8 90.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

heig

ht (n

m)

x (µm)

topographynear-field

(1711 cm-1 – ureido absorption)

SEM

0 1 2 3 4 5 6 7 8 90.0

0.2

0.4

0.6

0.8

1.0

rel.

IR s

igna

l (ar

b. u

.)

x (µm)

A „chemical nanoscope“Fingerprint spectra with nm lateral resolution

(Kopf, Samson, Wollny, Grunwald,Bründermann and Havenith,

J. Phys. Chem. C 111, 8166-8171 (2007)

Detection limit: 5 10-20 mol/90 mm2

27 attogramm

Contrast mechanism:Dipole- mirror dipole coupling:αmirror = β αtip ; Β = (εprobe-1) / (εprobe +1)

K. Jacobsen, C. Dietrich,Trends in cell biology, 9, 88 (1999)

New project: Taking a closer look (an infared image) at lipid rafts ?

Artifical membranes

00.020.040.060.08

0.10.12

1680 1700 1720 1740 1760 1780Frequenz (1/cm)

Abs

0% Chol

20% Chol

40% Chol

Dimeristylphosphatidylcholin (DMPC) und Cholesterin

DMPC CholesterinZ. Arsov et al. Chem. Phys. Lipids, 150:35–48, 2007.

XChol

proteinlipid

fluid lipid raft

5 nm lipiddouble layer

alcyl chains

Experimental set-up

CantileverLinse

MCT-Detektor

Piezo-ScannerProbe

Diodenlaser4-QuadrantenDetektor

11kV

CO

-laser

4 squaredetector

lense

FTIR Spektren: DMPC

κε in +=

Frequenz [cm ]-1

Abs

orpt

ion

1000 1500 2000 2500 3000 3500 40000,0

0,2

0,4

0,6

0,8 FTIR

ATR-FTIR

κ n

effαπα

ασ6

)Im(24

eff

eff

kk +=

abs. scattering

absorption refraction

IR-near field microscopy of DMPC multilayer

Topography

1744 cm-1

on resonance

DMPC multilayer and 30 nm gold ball on glimmer

1658 cm-1

off resonance

350nm

6004002000

403020100

-10-20-30

DMPC

Gold

2001000

40200

-20

DMPC

Gold

X [nm]

X [nm]

Nah

feld

sign

al [m

V]N

ahfe

ldsi

gnal

[mV]

750nm750nm

350nm

Lateral resolution 30 nm

IR near field

IR near field: Lipid double layerG. Wollny, E. Bründermann et al.

1500 1600 1700 1800 1900 20000,000,010,020,030,040,050,06

Abs

orpt

ion

Frequenz [cm-1]

FTIR-Spektrum von DMPC

DMPC

IR near field images

1.0µm

300nm

0,80,40

8

4

0

X[µm]

Z [n

m]

near field-contrast: DMPC on silicon

1650 1700 1750 1800 1850 1900 1950

0,5

0,0

0,5

1-I

/ILi

pid

Si

Frequenz [cm ]-1

Experimental result

Si

DMPC

IIC −=1

contrast

1650 1700 1750 1800 1850 1900 1950

() L

ipid

σN

Frequenz [cm ]-1

9

10

11

12

predictedscattering cross section

πα

ασ6

)Im(24

eff

eff

kk +=

Chemical mapping of a surface

green: two lipid double layer (DMPC) area: (90 nm)²Corresponds to 320000 molecules

IR near field spectrum of DMP double layer

K. Jacobsen, C. Dietrich,Trends in cell biology, 9, 88 (1999)

Taking a closer look (an infared image) of proteins in membranes

Topograpy31 31 dots

1658 cm-1 1740 cm-1 1804 cm-1

512 512 dots

15 m

15 m

Near field-signals recorded with a tapping-mode-frequency of 172,8 kHz

The bright spot has an extension of ca. 300-400 nm and a height of ca. 50 nm

3.0µm

190nm

190nm

190nm 190nm

Each dot correspondsto ca. 30 nm

Imaging proteins inartificial membranes

Cooperation withHeberle group

0 100 200 300 400 500 600

0.6

0.7

0.8

0.9

1.0

nm

brig

htne

ssHa.u.L

Grundfrequenz 172.8 kHz

Topography

Protein absorption (1658 cm-1)

Lipid absorption (1740 cm-1)

Off resonance 1804 cm-1

DMPC Cytochrome C Oxidase

Ni-NTA

First experimental resultsF. Ballout, I. Kopf, et al

IR mapping of a single tobacco virusBrehm, Taubner, Hillebrand, Keilmann

Nanoletter 6, 1308 (2006)

Imaging of DNA nano arrayscooperation withELETTRA, Trieste

Distinction between single and double stranded DNA on a nano scaleAu surface

IR-nearfield Measurementsof single and double stranded DNA

-0.11

-0.09

-0.07

-0.05

-0.03

-0.01

0.01

0.03

0.05

0.07

1635 1640 1645 1650 1655 1660 1665 1670 1675 1680

wavenumber (cm-1)

near

-fiel

d co

ntra

st

Patch9 (ss)Patch10 (sshybr)dsDNASAM

710nm

dsDNApatch 10

ssDNApatch 9

ssDNApatch 11

dsDNApatch 8

Au surface

I. Kopf et al

Core:- Diamond-like stucture (DL) - Hard material- No water- 50-100 nm diameter

Shell:- Polymer-like structure (PL)- Soft material- Intersticial water- Diameter depends on growing time (450 nm)

50 nm

250 nm

Chemical mapping of nano particlesCooperation with plasma physics (J. Winter, RUB)

PL nanoparticles (450 nm)

630nm610nm

450nm

3016 nm2981 nm 3141 nm

PL particle

Measurement volume

Absorption of the N-H band of PL carbonaceous nanoparticles

-1

-0,8

-0,6

-0,4

-0,2

0

0,2

2900 2950 3000 3050 3100 3150 3200

w avelength (nm)

IR in

tens

ity (A

. U.)

normalized contrast

J. Sebastian et al

3051 nm2998 nm 3148 nm

DL nanoparticles (56 nm)

DL particle

Measurement volume

140nm120nm 150nm

absorption of the N-H band of DL carbonaceous nanoparticles

-1

-0,8

-0,6

-0,4

-0,2

0

0,2

0,4

2900 2950 3000 3050 3100 3150 3200

w avelength (nm)

IR in

tens

ity

normalized contrast

IR absorption of N-H band of DL and PL nanoparticles

-140

-120

-100

-80

-60

-40

-20

0

20

40

2900 2950 3000 3050 3100 3150 3200

wavelength (nm)

IR in

tens

ity (A

. U.)

PLDL

In comparison: IR spectra of diamond like and polymer like nano particels

J. Sebastian et al. cooperation with J. Winter , RUB

SummaryChemical Imaging on the nm scale

• Set-up of a „chemical nanoscope“ (SNIM) using new IR laser sources in the amid, C-H and O-H spectral region

• Chemical Imaging without the need of extrinsic labels• Demonstration of high lateral resolution (10 nm)• Sub-surface structures can be imaged (up to 100nm)

penetration depth scales with wavelength• IR spectra of 30 nm x 30 nm area can be recorded• High detection sensitivity:

Observation of monolayers• Detection limit:

4 molecules/nm2 (6.6 ·10-24 mol/nm2 )• Observation of protein in membranes or lipid rafts ?

Pump laser: Master Oscillator Power Amplifier: output power: 20 W at 1064 nm Periodically Poled Lithium Niobate (quasiphase matched) cw IR radiation 2.9 Wresonant for signal wavelength (1485-1.665 nm) ; idler (3000-4000 nm)

High power IR-radiationIR-cw OPO (2.9 W)

Aufbau einer leistungstarken Strahlungsquelle im IR Bereich