1932-18 Winter College on Micro and Nano Photonics for ...indico.ictp.it/event/a07141/session/53/contribution/35/material/0/0.pdf · 0.2 0.0 E E x E r 0 /V. CARS microscopy: from

1932-18

Winter College on Micro and Nano Photonics for Life Sciences

Herve Rigneault

11 - 22 February 2008

Institut Fresnel MarseilleMarseille, France

Coherent Anti-Stokes Raman Scattering Microscopy (CARS): from fundamentals toapplications (Part I, II and III)

CARS microscopy: from principles to applicationsCARS microscopy: from principles to applications

Coherent anti-Stokes Raman

scattering (CARS) microscopy:

from principles to applications

HervHervéé RigneaultRigneault

Mosaic group, Institut Fresnel Mosaic group, Institut Fresnel –– Marseille, FranceMarseille, France

Thanks to: N. Thanks to: N. DjakerDjaker, D., D. GachetGachet, N. , N. SandeauSandeau, F. , F. BillardBillard


Light moleculeContrast mechanism

Refraction

Scattering

Raman

Fluorescence

Nonlinear+

+Image


Light Microscopy

Excitation

Detection

Emission

Laser

NA = n sin

Numerical aperture

2sinn

22.0d2

NA222.1r

Typical NA ~ 0.5 – 1.4

(immersion objective)


Excitation

Light microscopy: excitation field

z

x

y

0E

F’

max

y

x

zz

x

y F’

Objectif

k

(a)

(b) (c)

0r

0k

Plan

objetPlan focal

image

z

x

y

0E

F’

max

y

x

zz

x

y F’

Objectif

k

(a)

(b) (c)

0r

0k

Plan

objetPlan focal

image

Object

plane

Image

plane

References:

Richards & Wolf, “Electromagnetic diffraction in optical

systems. II. Structure of the image field in an aplanetic

system”, Royal Society of London Proceedings Series A,

Proceedings of the Royal Society of London, 1959, 253,

358-379

Novotny & Hecht, “Principles of Nano-Optics”,

Cambridge University Press, 2006

?

1.0

0.5

0.0

-0.5

-1.0

z (

µm

)

1.0 0.0 -1.0

x (µm)

0.35

0.30

0.25

0.20

0.15

0.10

0.05

0.00

Ez

1.0

0.5

0.0

-0.5

-1.0

z (

µm

)

1.0 0.0 -1.0

1.0

0.8

0.6

0.4

0.2

0.0

Ex

0 /r


Light microscopy: emission field

From J.M. Raimond,

electromagnetism & relativity’s

lesson (2000)

erkir

pkE exp

sin

4

1 0

2

0

Far-field radiation

pattern of a single

z-oriented

Hertzian dipole

Far-field approximation (r >> ):

Contrast mechanism dependent-Fluorescence 1P, 2P

-SHG (TWM), THG, CARS (FWM)


Light emission in incoherent processes

• Incoherent emission => each dipole

emits light with a randomrandom phase.

•The total intensity equals the sum of

individual intensities.

?Equivalent

dipole

……to an assembly to an assembly

of dipoles.of dipoles.

From a single From a single

dipoledipole……

For an incoherent process: Fluorescence


Light emission in coherent process

• Coherent emission => the

phase of each dipole is fixed by

a phase relation.

•Locally, the total field is the

sum of the fields emitted by

each dipole (interference).

•The intensity is the square

modulus of the total field

Adapted from J.X. Cheng and al.,

Biophys. J. 83, 502 (2002)

For a coherent process: SHG (TWM), THG, CARS (FWM)

Radiation pattern

depends on

the phase relationship

between emitters !


1.0

0.5

0.0

-0.5

-1.0

z e

nm

1.00.50.0-0.5-1.0

x en m

160

120

80

40

0

1.0

0.5

0.0

-0.5

-1.0

z e

nm

1.00.50.0-0.5-1.0

x en m

400

300

200

100

0

Ex

Ez


Fluorescence contrast

Excit

ati

on

Emission

Mic

ros

co

pe

ob

jecti

ve

Sample

Dichroic

plate

Ab

so

rpti

on

Em

issio

n

Fluorescence microscopy:

Advantages: Drawbacks:

Chemical specificity Staining step before observation

Very good SNR ratio Staining induced cell’s potential malfunctioning

Photobleaching


Nonlinear contrast

(cf Mario Bertolotti tutorial on nonlinear optics)

(1) (2) (3)

0

(1) (2) (3)

0

( ) ( : : : ...)

( ...)i ij j ijk j k ijkl j k lP E E E E E E

P E E E E E E

Einstein notation

Introduction / mixing of frequencies

Need to be strong

Linear optics

Non linear tensor

Symmetry dependant

Non linear optics requires strong optical field

-Hydogen atom,

- Sun on earth: 103 V.m-1 , linear optics regime

- 10kW laser focused on a 10 m spot: 108 V.m-1, non linear optics regime

- Non linear microscopy needs to focus the incident fields!!

11 1 -11

2

0

5.10 . ; Bohr radius a=5.10 m4

at

eE V m

a


NLO contrasts in microscopy

Débarre et al., Opt. Lett. 30, 2134 (2005)Muscle tissue (SHG)

(Webb lab)

32

SHG microscopy THG microscopy

(1) () )

0

2 3( :( . ):) ( . .:E E E EEP E

SHG and THG microscopy

Advantages: Drawbacks:

Useless staining Non-Centrosymmetric media required (SHG)

No photobleaching No chemical specificity


The challenge: a chemical selectivity without staining

2-propanol molecule

CH3-CH(OH)-CH3

C

O

R

Modeling:

Assembly of oscillators with

mode frequency R and

mode energy h R.

Specificity:

R specific to each

vibrational mode.


Detecting vibrational levels: IR absorption microscopy

R

IR excitation

IR absorption spectrum

Source: http://www.aist.go.jp

Excitation

volume 10

-

100µm

1/ =3300 cm-1

=100 THz

=3 m

1/ =1000 cm-1

=30 THz

=10 m

Main drawback:

bad spatial resolution


Detecting vibrational levels: Raman scattering basics (1)

R

V=1

V=0

p s

Stokes scattering

Vibrational level

Anti-Stokes scattering

Fundamental level

p as

R

Spontaneous Raman scattering


Raman scattering basics (2)

R

Raman spectrum (Stokes)Optical excitation

p frequency

Main drawback:

Long acquiring time

Excitation

volume~1µm


p- R

p+ R

R=10-14F

Anti-Stokes


Coherent anti-Stokes Raman Scattering

- Can we excite a specific molecular bond efficiently?

- Can we make an image at a sub-cellular level?

Coherent Anti-Stokes Raman Scattering

Microscopy

CARSCARS

=


Bouncing the springs


The CARS Hammer

PPump wave

Anti-Stokes

AS= P+ R

R

Stokes S= P- R

P- S= P-( P- R)= R


Coherent anti-Stokes Raman scattering: Energy view

Coherent anti-Stokes

Raman scattering (CARS)

V=1

V=0R

p

s s

p as

CARS=106R=10-8

F (in microscopy)


Coherent anti-Stokes Raman scattering: (3) view

(1) (2) (3)

0 :( ) ( : ...):P E E E E EE

Four waves mixing(3) ( )

= (3) (2 P S P P S )

f

v

P

P

SP P S


CARS / Raman Scattering

Laser p

Spontaneous RAMAN

Stokes s

AntiStokes as

Stokes s

AntiStokes as

R R

wavelength

frequency


CARS microscopy: What do you need?

S

P

High sensitivity detectors


A first experimental set-up

p = 730 nm

Pulse width 3 psBC: beam combiner

BS: beam splitter

C: condenser (NA=0.5)

F: filter

L: lens

Forward

CARS

detectorNA 1.2

Epi

CARS

detector

p

s

as

Ep

Sample

Objective C

Es

LELF

F

BC

BS

as

p 780 - 920 nm

Pulse width 3 ps

x

zy

F


CARS microscopy: let’s do a first experiment on GUV

Deuterated lipids

C-D bond Raman spectrumElectroformation

Giant unilamellar vesicles (GUV):diameter: 5 -100 µm


CARS microscopy: a first experiment on GUV

F-CARS images of GUV (Giant Unilamellar vesicle ) DMPC[D54]

F-CARS GUV (DMPC-D54):

(60×60) pixels, 1ms/pixel.

Pump 730nm, Stokes 862nm: Power 800µW : rep rate: 4MHz


CARS: Resonant and non Resonant contribution

Two contributions to CARS

generation

Resonant contribution (Resonant contribution ( RR(3)(3)))

(vibrational origin)

V=1

V=0

ps as

R

p

Nonresonant contribution Nonresonant contribution (( NRNR(3)(3)))

(electronic response of the medium)

RV=1

V=0

s

as

p

p

Presence of molecules with oscillating vibrational mode R

Enhancement of the signal at frequency as


(3) (3)

( ) 2

tRNR

R p s R t p t

AA

i i

R)3(

Far from two photons absorption

f

e

p

s

as

v

p

f

v

pas

s

p

2as p p s p sDegenerated FWM

P

f

e

p

t

v

Stokes

S

p

AS

AntiStokes

s

f

v

p

p

s

CARS: Resonant and non Resonant contribution


Spectral behaviour of the (3) tensor: (3)R and (3)

NR

(3) (3) *

2 2(3) (3)

, : , : , : ,

( ) ( )

as as p p p p s s

as as

P r E r E r E r

I P

CARS as a third-order nonlinear process:

constant&realis:tindependenspectrallyresponseElectronic

:lineRamanisolatedanFor

:partstwointoiondecomposit

(3)

(3)

NR

Rsp

R

NRR

i

a

)(

)3(

)3()3()3(

Raman line

half-width

Oscillator

strength

Vibrational

frequency

(3) spectral behaviour


Spectral behaviour of the (3) tensor: Interference term

Potma et al., J. Raman Spectr. 34, 642 (2003)

CARS resonance lineshape

)3()3(

2)3(2)3(

2)3()3(

Re2NRR

NRRCARS

NRRCARS

I

I

Homodyne terms

Heterodyne terms


Raman / CARS spectra


Polystyrene spontaneous

Raman spectrum Polystyrene CARS spectrum

500

400

300

200

100

0

CA

RS

inte

nsity (

kcps)

108010401000960

Raman shift (cm-1)

104

102

100

98

96 Y p

os

itio

n (

µm

)

1041021009896 X position (µm)

250

200

150

100

50

CA

RS

inte

nsity

(kcp

s)

CARS resonance

Off-resonance

104

102

100

98

96 Y p

os

itio

n (

µm

)

1041021009896 X position (µm)

70

65

60

55

50

45

40

35

CA

RS

inte

nsity

(kcp

s)


Experimental evidence

6

5

4

3

2

1

CA

RS

in

ten

sit

y (

AU

)

10810410096Scan position (µm)

1097cm-1

104

102

100

98

96 Y p

os

itio

n (

µm

)

1041021009896 X position (µm)

250

200

150

100

50

CA

RS

inte

nsity

(kcp

s)

Raman shift (cm-1)

400

300

200

100

CA

RS

in

ten

sity (

kcp

s)

10801060104010201000980960

1035cm-11030cm-11024cm-11018-1024cm-11018cm-11013cm-11007-1013cm-11007cm-11002cm-1945cm-1

Gachet et al., Optics

Express 15, 10408

(2007)

Bead experimental 1D scans


Resonant contribution

can be expressed as a complex number:

Modulus: Phase:

Circle in the complex plane:


(3) in the complex plane

(3)/ (3)NR modulus

01 2 3 4 5

1

2

3

4

5

0

15

30

45

60

7590105

120

135

150

165

180

(3) phase (°)

ORPM

P

D

RP

OR1 Bef. Res.

P CARS Res.

RP Raman Res.

PM Phase max.

D Spect. dip

OR2 Aft. Res.

15

10

5

0

|(3

) /(3

) NR|2

-20 -10 0 10 20Normalized Raman resonance detuning

OR

P

DPM

OR

RP

~ NR/ a

Representation of the resonance in the complex plane


(3) drives the CARS antiStokes field

(3) (3) *

(3)

, : , : , : ,

( )

as as p p p p s s

as as

P r E r E r E r

P E

The amplitude and phase of (3) drives the amplitude and phase of EAS


CARS step by step

Induced polarization(3) (3) *, : , : , : ,as as p p p p s sP r E r E r E r

Incoming fields , ; ,p p s sE r E r

1.0

0.5

0.0

-0.5

-1.0

z (

µm

)

1.0 0.0 -1.0

x (µm)

0.35

0.30

0.25

0.20

0.15

0.10

0.05

0.00

Ez

1.0

0.5

0.0

-0.5

-1.0

z (

µm

)

1.0 0.0 -1.0

1.0

0.8

0.6

0.4

0.2

0.0

Ex

Phase of (3)


CARS step by step

1. Pump & Stokes fields

2. Induced nonlinear polarization

3. Dipolar emission

4. Summation over far-fields

emitted in a particular direction

)r(Es

Stokes

laserPump

laser

)r(E p

ssppppasas rErErErP ,:,:,:, *)3()3(

Medium Exciting fields

Induced nonlinear

polarization)r(P )3(

erkir

pkE exp

sin

4

1 0

2

0

Far-field approximation

fields

rticular direction

)r(s

mpeses

)))r))rr(rr((p((ss ))

ced nonlinear ppeseseseslarizationerer

E

asE asE


CARS field in direct and reciprocal spaces

Electric field EAS in

the reciprocal space

(kx,ky)

Electric field EAS in the

direct space (x,y,z)

)r(ECoherent summation!

)k(E

Coherent summation!

Induced

polarizationInduced

polarization


Far-field CARS radiation patterns in direct space

F-CARS

E-CARS

ps

as

as

z

x

Gachet et al., Proc. SPIE (2006)


F-CARS emission

more directive

than the excitation beam

along one direction

Far-field CARS

radiation patterns in k

space


F-CARS / E-CARS radiation

F-CARS

E-CARS

x 200

E/F

Volkmer et al. PRL (2001)


Djaker et al., Appl. Opt. 45, 7005 (2006)


Epi-detected CARS: a way to visualize small objects

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

A. Volkmer, id.

Excitation

volume

Solvent

Forward

Epi

Epi detection

Forward detection

Small

object


Phase matching in NLO (SHG example)

Cf lecture NLO Mario Bertolotti

(2k )

PNL(2 )

(2) 2(2 ) ( )NLP E

2 / 2

2 nk

(k2 )

2 2

2 / 2

nk2 2

2 2

(2 ) 2( )n nk k k lc

I

x

clk

E(2 )


*2)3( )()()( SSPPAS

NL EEP

Phase matching in CARS

(2kP-kS)

)2(

1

2

2

PS

SP

SP nkk

PNL( AS)

kAS

E( AS)

n

AS

ASk

2

)2(

22

SPAS kkkk

(2 )c

AS P S

lk k k k

lc

IAS

x


CARS phase matching: intuitive approach

Epi-CARS

Fwd-CARS

ps

as

as

z

x

kas,Fwd

kp kp

-ks

z

Fwd-CARS

k= k=2kas,Epi

kp kp

z

Epi-CARS

-kskas,Epi

Phase-matching & CARS generation

2kp-ks 2kp-ks

clk

Ias max

Fwd-CARS: 0;

Epi-CARS: 24

;2 4 ( )

c

aasas c

as as

s

k l

k k lk k n


F-CARS / E-CARS radiation

2µm bead

F-CARS

E-CARS

x 200

cl

4

ascl

100nm bead


Djaker et al., Appl. Opt. 45, 7005 (2006)


CARS instrumentation


A first experimental set-up

p = 730 nm

Pulse width 3 psBC: beam combiner

BS: beam splitter

C: condenser (NA=0.5)

F: filter

L: lens

Forward

CARS

detectorNA 1.2

Epi

CARS

detector

p

s

as

Ep

Sample

Objective C

Es

LELF

F

BC

BS

as

p 780 - 920 nm

Pulse width 3 ps

x

zy

F


How to choose pulses temporal length?

J.-X. Cheng et al., J. Phys. Chem. B 108, 827 (2004)

The dilemma:

•• Long pulsesLong pulses::

++ good spectral selectivity

- poor CARS generation efficiency

••Short pulsesShort pulses:

+ efficient CARS generation

- low spectral resolution

Solutions:

• To spectrally match the studied Raman line ps range


Laser Synchronization

M

Saphir

Titane

839

nm

Saphir

Titane

740

nm

Délai (ps)

Correlation

Cross-correlation

CARS signal

FD: filtre dichroïque, M: miroir

- Fast photodiodes

-/ Autocorrelator (SHG)

-/ 2P detector

- CARS signal

Synchro electronic

Potma Opt Lett 27 (2002)


Nd:Vd VERDI 10W

MIRA

Saphir

Titane

(Master)

MIRA

Saphir

Titane

(Slave)

Pulse

Select

Pulse

Select

Synchro

Lock

APD

PZT stage

XYZ

Sample

Microscope

Objective NA1.2

Dichroic

FilterM

BC

M

M

M

Delay

( /2)+Glan

P+ S AS

PZT

Filters

Monochromator

Telescope

APD

Filters

Collection objective

NA 0.5

E-CARS

F-CARS

M BS

APDAPD

PS

AS

Retroreflector

BC: beam combiner

M : Miroir

PZT : Piezo

APD : Avalanche Photodiode

Setup scheme pico/pico


Two oscillators: pico / pico setup

F-CARS

E-CARS

S

P

AS

Sample

MHz

KHz

SynchroLock System

MHz

KHz


Two oscillators pico / femto = Multiplex CARS

Pump: 10 ps

Stokes: 80 fs

10 ps

0.8 ps

Muller J. Phys. Chem B 106 2002


Multiplex CARS

Identification of the Thermodynamic State of Lipids in Multi-lamellar Membranes

Gel phase DSPC

1128 cm-1

Liquid phase DOPC

1087 cm-1

1128 cm-11087 cm-1

From Müller, J. Phys. Chem. B. (2002)


1 oscillator femto + PCF

Kano et al. APL86 (2005)


What is an OPO (Optical Parametric Oscillator)

Recent advances in Optical Parametric Oscillators

Berlin

Parametric generation (2) ( ).

Idler

f

e

Signal

Parametric amplification (2) ( ).

Signal

Idler


One oscillator + One OPO pico/pico

Pump ps 532nm

Signal

Idler

>1350 cm-1

>700 cm-1


Sensitivity improvement: FM CARS


Sensitivity improvement: H(eterodyne) CARS

Potma Opt. Lett. 31 (2006): LO generated in DMSO

Enable to recover real and imaginary part of (3)

Lipid resonance 2845cm-1

Raman

Off resonance


H(eterodyne) CARS with OPO!

Jurna Opt. Expr 15 (2007)


HCARS with interfaces

z

x

Obj.

C.

NA :

1.2

NA :

0.5

BS

BC

E-CARS

Detector

Sample

S

P

AS

AS

F

F

EP

ES

F-CARS

Detector

LF

LE

3.0

2.5

2.0

1.5

1.0

0.5

0.0

CA

RS

inte

nsity

(UA

)

1500145014001350

Raman shift (cm-1

)

15

10

5

0

Ram

an In

tensity

(UA

)

I(Fwd)

(bulk)

I(Fwd)

I(Fwd)

3x(I(Fwd)

-I(Fwd)

)

Raman spectrum

zI (Fwd) I (Fwd)

p s

p s

glass

glass

DMF

A

B

(a)

)3(R1

)3(NR2

)Fwd( 4I

1. Field symmetry permits to use non

resonant CARS as a local oscillator

2. Raman spectrum recovery and

heterodyne detectionGachet et al., PRL (submitted)


Single pulse CARS

Dudovich, Oron, Sylberberg Nature 418 (2002)

Other scheme with a control of the probe beam: Oron PRL 89 2002

Can excite only a vibration with

CH2Br2 (CH2Cl)2

Number of oscillation across the SLM


CARS Application: a stain free microscopy

Scanning

directions

Advantages:

1. Fluorescent staining useless.

2. Chemical selectivity of the contrast.

3. Intrinsic 3D imaging.

p

as

s

as

Forward

detected signal

(F-CARS)

Backward

detected signal

(E-CARS)

Sample

x

y

z

Microscope

objective

E-CARS F-CARS

Fish gills

Courtesy Julian Moger

Exeter- UK

(2008)


Imaging lipids in cell

NIH 3T3 cells in interphase. Aliphatic C-H stretching 2970 cm-1

Pump 14054 cm-1(711nm) and the Stokes 11184 cm-1(894nm). P: 40mW; S: 20mW

Cheng et al Biophys. J. 83, 502 (2002)

N.Djaker et al, Medecine & science – (2006)


3D sectioning capability of CARS

Three dimensional

distribution of lipids in epithelial cells.

CH2 stretching vibration (2845 cm-1).

Lipid granules and plasma membranes.

http://bernstein.harvard.edu/research/cars.html


Raman Spectrum of the cell

Potma, E.O. et al. Optics and Photonics News, 2004, 15

CH3 (protein)CH2 (lipid)

Amide I (protein)Phosphate

(ADN)

500 15001000 2500 35002000 3000 4000

Raman frequency (cm-1)

PO2-symmetric stretching

vibrational frequency at 1090 cm-1

Lipid droplets in 3T3 cells (Xie group)

CARS image


From Potma PNAS 98, 1577 (2001)

OH strech 3300 cm-1

OD strech 2800 cm-1

living D. discoideum cells

3300 cm-1 OH strech

H2OH2OD2O

Permeability of the plasma membrane Pd=2.2 m/s

Dw=5 m2/s (10%-20% of the cell diameter)

Dw>500 m2/s (central cell region)

t=0

H2O

D2O

Dw

Exceptionally low Dw due to the presence of densely packed actin

filaments in this region that provide an additional barrier in the

process of water diffusion.

P S

Imaging H2O in cell


F-CARS back reflected in scattering tissue

Microscope

objective

Sample

z

Excitation

Back-scattered

photonsCARS imaging in

scattering media

Evans et al., PNAS 102, 16807 (2005)


Imaging Tissue

Coherent anti-Stokes Raman scattering imaging of

adipocytes (red) and second harmonic generation

imaging of collagen fibrils (green)

to evaluate the impact of obesity on mammary

gland and tumor stromal composition.

Le et al., Molecular Imaging 6 (2007)

Experimental Setup and in vivo E-CARS images. (A) Experimental

setup for combined E-CARS and SHG imaging of a live mouse. (B) E-CARS

image of parallel myelinated axons in the sciatic nerve and the surrounding

fat cells. Scale bar = 25 µm.

Huff, Cheng, J of Microscopy 225 (2007)


Imaging the skin: Rat skin

Depth: 0 µm

Depth 15 µm

Stratum corneum

Adipocytes of the dermis

Non resonantE-CARS rat Skin. CH strech 2845cm-1

(200×200) pixels - 1ms/pixel.

Pump 730nm, Stokes 920nm: Power 800µW, rep rate: 4MHz

0 m

15 m 15 m

Marseille


E-CARS Stratum Corneum with depth.

R=2829cm-1 (C-H bond)

(200×200) pixels - 1ms/pixel.

Pump 730nm, Stokes 920nm: Power 800µW, rep rate: 4MHz

1

mm

1

cm

Corneocytes

Imaging the skin: Stratum Corneum


A polarization sensitive technique

Djaker et al., Médecine / Science 22, 853 (2006)

Forthcoming

Investigation in

polarization CARS

microscopy

F-CARS GUV (DMPC-D54):

(60×60) pixels, 1ms/pixel.

Pump 730nm, Stokes 860nm: Power 800µW : rep rate: 4MHz


Conclusion

1. CARS addresses molecular intrinsic vibrational transition and does not require staining with fluorophore or radioactivity.

2. CARS is a coherent process which builds an anti-Stokes wave on a large number ofmolecular bonds. This coherent process permits to obtain a signal orders of magnitude larger than spontaneous Raman scattering. Small laser powers (1mw) can be used which are compatible with biological samples.

3. CARS is selective of a certain molecular bond (by adjusting the detuning between laser and Stokes beam) (spectral selectivity)

4. CARS is a non linear process which takes place only at the focal point of the microscope objective (diffraction limited) . Therefore no confocal pinhole is needed to obtain 3D imaging of biological samples.

5. Working in IR limits the absorption and diffusion of bio- tissue. Images as deep as 0.3mm can be obtained in living tissues.

6. CARS is an elastic process which does not store energy into the system. It is thereforeinsensible to photobleaching.

7. Finally, CARS is not affected by endogenous fluorescence because the anti-Stokessignal is at lower wavelength than the pump lasers.


CARS

Single Particle

Detection

FCS

Dynamic Multiple

Optical Tweezers

NanostructuresMicro-stereolithographyLaser nanoscissors

Dynamic organization

of living cells and tissues

http://www.fresnel.fr/mosaic

Pulse shaping

imaging

1932-18 Winter College on Micro and Nano Photonics for ...indico.ictp.it/event/a07141/session/53/contribution/35/material/0/0.pdf · 0.2 0.0 E E x E r 0 /V. CARS microscopy: from

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