Vibrational imaging and microspectroscopies based on coherent anti-Stokes Raman scattering (CARS)

by

Andreas Volkmer

3rd Institute of Physics, University of Stuttgart,

Pfaffenwaldring 5770550 Stuttgart,

Germany

a.volkmer@physik.uni-stuttgart.de

Universität Stuttgart

AG Volkmer(Coherent microscopy &

single-molecule spectroscopy)

FRISNO-8, Ein Bokek, 20-25 February 2005

Noninvasive three-dimensional characterization of mesoscopic objects within complex heterogeneous systems

• with high spatial resolution,• with high spectral resolution,• with high temporal resolution,• and with high sensitivity.

Ultimate goal in Optical Microscopy

Fluorescence-based microscopy Confocal fluorescence laser scanning microscopy Two-photon induced fluorescence laser scanning microscopy

Limitations of fluorescence-based spectroscopic studies:

• dye labeling required (photo-toxicity)

• perturbation of structure and dynamics by fluorophore

• photo-stability (# emitted photons)

!

knr

abs kfl

102 103 104 105 106 10710-2

10-1

100

101

102

103

NF

(I0/2) / W cm-2

CW (one-photon)excitation at 514 nm

102 103 104 105 106 10710-2

10-1

100

101

102

103

NF

Iav

/ kW cm-2102 103 104 105 106 107

10-2

10-1

100

101

102

103

NF

(I0/2) / W cm-2

Pulsed fs (one-photon)excitation at 350 nm

Pulsed fs (two-photon)excitation at 800 nm

Fluorescence photobleaching of Rhodamine 6G / water

Excited-statephotolysis model:

Eggeling, Volkmer, Seidel, Chem. Phys. Chem. (2005) submitted.

Chemical contrast mechanism based on molecular vibrations, which is intrinsic to the samples: NO requirement of natural or artificial fluorescent probes!

species Raman bands / cm-1

DNA backbone,

C-O stretching ~ 1000

Polypeptide backbone, C=O stretching (Amide I) 1500-1700

Lipids, C-H stretching 2900-3000

Intrinsic chemical contrast mechanism

[N. Jamin et al., PNAS 95 (1998) 4837-4840 ]

Spontaneous Raman microscopy:

• weak signal=> requirement for high excitation

power• fluorescence background

Infrared microscopy:

• low spatial resolution (~2-3 m)• low S/B ratio• water absorption

CARS fundamentals

2)3(ASASCARS PI

ispr

1

)3( 1

SSPPnrASrASAS EEP *233)3(

Resonant CARS

ASPS

v=1v=0

‘P

CARS signal:

induced third-order polarization:

.)3(

constnr No vibrational contrast !

Two-photon enhancednon-resonant CARS

Non-resonant CARS

AS

‘P

P

S

ASP S

v=1v=0

‘P

SPAS 2

1982 - Duncan, Reintjes, Manuccia, Optics Lett. 7, 350

Picosecond visible laser,Noncollinear geometry

Development of CARS Microscopy

(CARS image on the 2450-cm-1 band of D20)

Onion-skin cells, soaked in D2O

853 nm (100 W)

1135 nm (100 W)

CARS signal at 675 nm

(Raman-shift of 2913 cm-1, on resonance with C-H vibrations)

HeLa cellsE-coli

1999 - Zumbusch, Holtom, Xie, Phys. Rev. Lett. 82, 4142

Femtosecond near-IR laser,Collinear geometry,Forward detection

High NA objectives

FilterSample

D

P

S

AS

• Intrinsic sensitivity to specific chemical bonds

=> No dye labeling

• Coherent signal enhanced by orders of magnitudes

=> Less laser power required compared to conventional Raman

compared to spontaneous Raman signal microscopy

• No population of higher electronic states

=> No photobleaching

• Confinement of nonlinear excitation to confocal volume

=> Inherent 3D spatial sectioning capability

Advantages of CARS-microscopy

Theory of collinear CARS microscopy

Distinct features:

(ii) Actual extent of wave-vector mismatch is controlled by geometry for

propagation directions of both incident beams and the CARS radiation

(iii) Heterogeneous sample of Raman scatterers of arbitrary shape and size

embedded in nonlinear medium

(i) Under tight focusing conditions -> breakdown of paraxial approximation

Amplitude distribution

-2 -1 0 1 2-2

-1

0

1

2

x /

z /

(i) Description of a tightly focused Gaussian field

-1 0 1-1

0

1

-2.0-1.7-1.4-1.1-0.80-0.50-0.200.100.400.701.01.31.61.92.0

x /

z /

Phase distribution (-kz)

Cheng, Volkmer, Book, Xie, JOSA B, 19 (2002) 1363

(ii) Wave-vector mismatch in collinear CARS microscopy

Wave-vector mismatch in collinear beam geometry:

phase matching condition:

(interaction length << coherence length)

kD

SPAS kkkk 2

kP kAS

kP kS

BS

p

L

Obj

Obj

S AS

F

C-CARSdetector

samplex

z

C-CARS

kP kAS

kP kS

kP kAS

kP kS

BS

L

Obj

S AS

F

E-CARSdetector

AS

Obj

LF

F-CARSdetector

x

z

F-CARS

E-CARS

BCP

P

HWPQWP

p

A

F-CARS(forward-detected)

0k

E-CARS(epi-detected)

asn 4Δ k

C-CARS(counter-propagating)

sn 4Δ k

Cheng, Volkmer, Book, Xie, JOSA B, 19 (2002) 1363

(iii) CARS signal generation for microscopic scatterer

Assuming:

• tightly focused incident Gaussian fields

• Incident fields are polarized along the x axis

• refractive index mismatch between sample and solvent is negligible

Volkmer, Cheng, Xie, Phys. Rev. Lett. 87, 023901 (2001).

w0

R

r

x

z

f

Einc

0

objχ

rχrRε objAS ,,

solvχ

Simulated size dependence of CARS signals

0 2 4 6 810-3

10-1

101

103

I CA

RS (

a.u.

)

D / p

F-CARS E-CARS

kp, kS

objχ

solvχ

0 2 4 6 810-3

10-1

101

103

D / p

F-CARS E-CARS

kp, kS

)(reflected solvχ

0 2 4 6 810-4

10-2

100

102 C-CARS (forward) C-CARS (backward)

D / p

kS

kp

objχ

Volkmer, J. Phys. D : Appl. Phys. 38 (2005) R59

0.0 0.5 1.0 1.5 2.0 2.5 3.00

50

100

150

200

x (m)

sig

na

l (ct

s)

0.0 0.5 1.0 1.5 2.0 2.5 3.00

5

10

15

20

sig

na

l (ct

s)

x (m)

0.0 0.5 1.0 1.5 2.0 2.5 3.00

10

20

30

40

sig

na

l (ct

s)

x (m)

(c) C-CARS xy- image

(a) F-CARS xy-image

FWHM0.34 m

FWHM0.34 m

FWHM0.36 m

(b) E-CARS xy- image

(d) F-CARS xz- image

0 500 1000

4.0

3.5

3.0

2.5

2.0

1.5

1.0

0.5

0.0

z (

m)

signal (cts)

FWHM1.18 m

Experimental characterization of CARS microscopyfor a single 500-nm polystyrene bead in water

(Raman shift ~1600 cm-1)

Volkmer, J. Phys. D : Appl. Phys. 38 (2005) R59

P-CARS xy- image

0 20 40 600

20406080

x (m)

sig

na

l (c

ts)

0 20 40 600

100

200

x (m)

sig

na

l (c

ts)

(a) (b)F-CARS xy- image E-CARS xy- image

sign

al (

cts)

(c)

0 10 20 30 40 50 600

100

200

x (m)

Picosecond CARS imaging of a live unstained cell

Epithelial cells@ Raman shift ~1570 cm-1

(amide I)

NIH3T3 cells@ Raman shift ~2860 cm-1

(C-H strectch)

-150 -100 -50 0 50 100 1500

200

400

600

2 ps

(7.5 cm-1) X 10

Raman profie

X 1

X 100

pulse width0.5 ps

(29 cm-1)

10 ps (1.5 cm-1)

CA

RS

inte

nsity

(a.

u.)

(p-

s)-

R (cm-1)

Simulation of CARS spectra as afunction of pulse widths

33nr

sp i

A

2 = 10 cm1 … line width

2.0Anr … vibration frequency

asasCARS dPI 2)3(

The CARS intensity is:

= 25 cm-1

| |2

| |2

CARS intensity vs. excitation pulse spectral width

Cheng, Volkmer, Book, Xie, J. Phys. Chem. B 105, 1277 (2001).

Synchro-Locksystem

PZT drivers& galvo’s

The CARS microscope

Telescopes Pol

Pol

s

p

BC

6.7 ps Ti:sapphiremode-locked oscillator

6.7 ps Ti:sapphiremode-locked oscillator

microscope

acquisition of CARS spectrum in one”shot”!

AS

p

s

p’

Obj

DL

FA

BC

Spectrometer +LN2-CCD array

Telescopes P SHWP

FM

L

Obj

AS

p

QWP

S

CARSpumpStokes

Multiplex-CARS Microspectroscopyin the Frequency-Domain

Inte

nsi

ty (

a.u

.)

Raman shift / cm-12800 2900 3000

0.0

0.2

0.4

0.6

0.8

1.0

Raman

2800 2900 30000.0

0.2

0.4

0.6

0.8

1.0

Inte

nsi

ty (

a.u

.)

Raman shift (cm-1)

Raman

Example: Monitoring the thermodynamic state of phospholipid membranes in the C-H stretch region

DSPCTg=55°C

DOPCTg=-20°C

2800 2900 30000

1

2

3

No

rma

lize

d C

AR

S I

nt.

CARS

ps / cm–12800 2900 3000

0.0

0.4

0.8

1.2

Nor

mal

ized

CA

RS

Int.

CARS

ps / cm–1[Cheng, Volkmer, Book, Xie, J. Phys. Chem. B 2002, 106, 8493-8498]

entropy

Model system for Stratum Corneum lipids

a(CH2)

a(CH2)cycl

Raman spectra in CH-stretching mode region

s(CH2)

wavenumbers /cm-1

Spectrally integrated CARS image section

10 m

2700 2900 3100

0.85

0.9

0.95

1

1.05

1.1

1.15

1.2

Extracted CARS ratio spectra for each image pixel

~

~ref

CARS

CARSI

I

1/~ cm

Hyper-spectral CARS imaging of a Stratum Corneum

Existence of cholesterol-enriched micro-domains

(see Poster by Nandakumar et al : Mo-4)

CARS microspectroscopy in the time-domain

0 time

)(CARSI

22, tt Sp EE 2

' tpE

2)3( , tP

Raman Free Induction Decay (RFID):

Obj

D

L

F

A

BC

S

Obj

AS

Telescopes P

BC

VD

FD

p’

p

S

P

S

P’

AS

|0>|1>

Three-color CARS set-up:

)~(~ ||

= 0 fs

S/B(=0) 3

= 484 fs

S/B (=484 fs) 35Complete removal of the non-resonant CARS contributions !

P1 = 714.6 nm (~85 fs)

S = 914.1 nm (~115 fs)

P2 = 798.1 nm (~185 fs)

Quantum beat recurs at ~ 1280 fs (mode beating at difference frequencies of~ 26 cm-1)

Volkmer, Book, Xie, Appl. Phys. Lett. 80 (2002) 1505c

Example: RFID imaging of 1-m polystyrene bead

Coherent Vibrational Imaging beyond CARS

Simplifying coherent Raman microscopy by use of a nonlinear optical imaging technique which

maps only the imaginary part of (3)

Stimulated Raman gain for probe laser in the presence of strong pump laser, when frequency difference equals Raman frequency

P(2) = (3)

(- 2; 2, -1, 1) E(2) |E(1)|2

S= S- p+ p

kS = kS – kP + kp

Stimulated Raman scattering (SRS) microscopy

Depends only on the Im (3)

Linear on (3)

Linear on number densityLinear in pump and Stokes intensitiesAutomatic Phase matching

Advantages:

ks kS

kp kp

(3)

LP P

S S

Disadvantage: Tiny signal over huge background signal from the Stokes field!

1 5

Slope 1.01

pump power / mW1.5 5

Slope 1.005SR

S s

igna

l (a.

u)

Stoke power / mW

0.5 m

Pixel intensity No. of C=C bonds No signal from surrounding water No interference effect in image contrast

SRS images of a polystyrene 1-m bead in water

2800 2900 3000

4

8

12

16

SR

S in

ten

sity (

a.u

.)

Raman shift / cm-1

s(CH2-aliph.)2853 cm-1

as(CH2-aliph.)2912 cm-1

Nandakumar, Kovalev, Volkmer, manuscript in preparation

Summary

• Under tight focusing conditions, size-selectivity in CARS signal generation is introduced by wave-vector mismatch geometries, e.g. epi-detected CARS (E-CARS) microscopy

allows efficient rejection of bulk solvent signal

E-CARS is easily implemented with a commonly used confocal epi-fluorescence microscope

• Combination of CARS microscopy with spectroscopic techniques provides wealth of chemical and physical structure information within a femto-liter volume in both the frequency-domain (multiplex CARS microspectroscopy) and time-domain (RFID imaging)

allows rejection of nonresonant background contributions by polarization-sensitive and time-delayed detection schemes

• Highly sensitive tool for the chemical mapping of unstained live cells in a spectral region for DNA, membranes and proteins.

[J. Phys. D : Appl. Phys. 38 (2005) R59 (Topical review)]

• First demonstration of Stimulated Raman Scattering (SRS) microscopy on model systems of polystyrene beads embedded in water

No interference effects with nonresonant contributions from both object and matrix SRS spectra qualitatively reproduce the Raman spectra

Harvard University

X.S. Xie  J.-X. Cheng

L.D. Book

3. Physikalische Institut, Universität Stuttgart:

P. NandakumarA. Kovalev

Roswell Park Cancer Institute, Buffalo, NY:

A. SenM. Koehler

Acknowledgements

Emmy Noether Program Faculty of Arts and Sciences of Harvard University

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