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 microscopyConfocal fluorescence laser scanning microscopyTwo-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!

2900-3000Lipids, C-H stretching

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

~ 1000

DNA backbone,

C-O stretching

Raman bands / cm-1species

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 µm)• low S/B ratio• water absorption

CARS fundamentals

( ) 2)3( ASASCARS PI ω∝

( )[ ] Γ+−−Ω= ispr ωωχ 1)3( 1

( ) ( )( ) ( )[ ] ( ) ( )SSPPnrASrASAS EEP ωωχωχω *233)3( +=

Resonant CARS

ωASωP ωSv=1v=0

ω‘P

CARS signal:

induced third-order polarization:

.)3(

constnr =χ No vibrational contrast !

Two-photon enhancednon-resonant CARS

Non-resonant CARS

ωAS

ω‘P

ωP

ωS

ωASωP ωSv=1v=0

ω‘P

( )SPAS ωωω −= 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 µW)

1135 nm (100 µW)

⇒ 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

Dω

Pω

S

ωAS

• 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 / λ

z / λ

(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 / λ

z / λ

Phase distribution (φ-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

(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).

Φ

w0

Θ

R

r

x

z

f

Einc

γ

0

objχ

( )( )rχrRε objAS ,,

solvχ

Simulated size dependence of CARS signals

0 2 4 6 810-310-1101103

I CA

RS (

a.u.

)

D / λp

F-CARS E-CARS

kp, kS

objχsolvχ

0 2 4 6 810-310-1101103

D / λp

F-CARS E-CARS

kp, kS

)(reflected solvχ

0 2 4 6 810-410-2100102 C-CARS (forward)

C-CARS (backward)

D / λp

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

50100150200

x (µm)

sign

al (

cts)

0.0 0.5 1.0 1.5 2.0 2.5 3.00

5

10

15

20

sign

al (

cts)

x (µm)

0.0 0.5 1.0 1.5 2.0 2.5 3.00

10

20

30

40

sign

al (

cts)

x (µm)

(c) C-CARS xy- image

(a) F-CARS xy-image

FWHM0.34 µm

FWHM0.34 µm

FWHM0.36 µm

(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 (µ

m)

signal (cts)

FWHM1.18 µm

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 (µm )

sig

nal (

cts)

0 20 40 600

100

200

x (µm)

sig

nal (

cts)

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

sign

al (

cts)

(c)

0 10 20 30 40 50 600

100

200

x (µm)

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.)

(ωp-ω

s)-ω

R (cm-1)

Simulation of CARS spectra as afunction of pulse widths

( )( )

( )33nr

sp i

A χωω

χ +Γ−−−Ω

=2Γ = 10 cm−1 … line width

2.0=AnrχΩ … vibration frequency

( )∫+∞

∞−

= asasCARS dPI ωω2)3(

The CARS intensity is:

50 100 1500.0

0.2

0.4

0.6

0.8

1.0

×

03

Resonant

Non-resonant ( 0.01)

Ir/I

nr

Pulse temporal width (fs)

1001503005000S

igna

l / b

ackg

roun

d

CA

RS

inte

nsity

(a.

u.)

Pulse spectral width (cm-1)

Γ = 25 cm-1

ω

| χ |2

|χ |2

ω

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

ω s

ω p

BC

6.7 ps Ti:sapphiremode-locked oscillator

6.7 ps Ti:sapphiremode-locked oscillator

microscope

acquisition of CARS spectrum in one”shot”!

ωAS

ωp

ωs

ωp’

Obj

DL

FA

BC

Spectrometer +LN2-CCD array

Telescopes P SHWP

FM

L

Obj

ωAS

ωp

QWP

ωS

ω

CARSpumpStokes

Multiplex-CARS Microspectroscopyin the Frequency-Domain

Inte

nsity

(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

nsity

(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

Nor

mal

ized

CA

RS

Int. CARS

ωp−ωs / cm–12800 2900 3000

0.0

0.4

0.8

1.2

Nor

mal

ized

CA

RS

Int.

CARS

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

entropy

Model system for Stratum Corneum lipids

νa(CH2)

νa(CH2)cycl

Raman spectra in CH-stretching mode region

νs(CH2)

wavenumbers /cm-1

Spectrally integrated CARSimage section

10 µm

2700 2900 3100

0.85

0.9

0.95

1

1.05

1.1

1.15

1.2

Extracted CARS ratio spectrafor each image pixel

( )( )νν~

~ref

CARS

CARSI

I

1/~ −cmν

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 timeτ

)(τCARSI

( ) ( ) 22 , tt Sp EE ( ) 2' tpE

( ) 2)3( , tτP

Raman Free Induction Decay (RFID):

Obj

D

L

F

A

BC

S

Obj

ωAS

Telescopes P

BC

VD

FD

ωp’

ωp

ωS

ωP ωS

ωP’ ωAS

|0>|1>

Three-color CARS set-up:

-500 0 500 1000 1500

102

103

104

105

water bead

CA

RS

sig

nal /

cps

τ / fs

3000 3040 3080

inte

nsity

(a.

u.)

Raman shift / cm-1

)~(~|| να

0 1 2 3 4 50.00.51.01.52.0

x / µm

0.00.51.01.52.0

inte

nsity

x10

-5 /

cps τ = 0 fs

S/B(τ=0) ≈ 3

inte

nsity

x10

-3 /

cps

0 1 2 3 4 501234

x / µm

01234 τ = 484 fs

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

λ P1 = 714.6 nm (~85 fs) λS = 914.1 nm (~115 fs)

λ 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-µm 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 χ(3)

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

P(ω2) = χ (3) (- ω2; ω2, -ω1, ω1) E(ω2) |E(ω1)|2

ωS= ωS- ωp+ ωpkS = kS – kP + kp

Stimulated Raman scattering (SRS) microscopy

♦ Depends only on the Im χ (3)♦ Linear on χ (3)♦ Linear on number density♦ Linear in pump and Stokes intensities♦ Automatic Phase matching

Advantages:

ks kS

kp kp

χ (3)

LωP ωP

ωS ωS

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 µm

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

SRS images of a polystyrene 1-µm bead in water

2 8 0 0 2 9 0 0 3 0 0 0

4

8

1 2

1 6

SR

S in

tens

ity (

a.u.

)

R a m a n s h if t / c m -1

νs(CH2-aliph.)2853 cm-1

νas(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 matrixSRS spectra qualitatively reproduce the Raman spectra

Harvard University

X.S. XieJ.-X. ChengL.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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