1932-18 winter college on micro and nano photonics for...
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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)
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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
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CARS microscopy: from principles to applicationsCARS microscopy: from principles to applications
Light moleculeContrast mechanism
Refraction
Scattering
Raman
Fluorescence
Nonlinear+
+Image
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CARS microscopy: from principles to applicationsCARS microscopy: from principles to applications
Light Microscopy
Excitation
Detection
Emission
Laser
NA = n sin
Numerical aperture
2sinn
22.0d2
NA222.1r
Typical NA ~ 0.5 – 1.4
(immersion objective)
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CARS microscopy: from principles to applicationsCARS microscopy: from principles to applications
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
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CARS microscopy: from principles to applicationsCARS microscopy: from principles to applications
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)
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CARS microscopy: from principles to applicationsCARS microscopy: from principles to applications
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
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CARS microscopy: from principles to applicationsCARS microscopy: from principles to applications
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 !
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CARS microscopy: from principles to applicationsCARS microscopy: from principles to applications
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
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CARS microscopy: from principles to applicationsCARS microscopy: from principles to applications
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
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CARS microscopy: from principles to applicationsCARS microscopy: from principles to applications
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
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CARS microscopy: from principles to applicationsCARS microscopy: from principles to applications
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
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CARS microscopy: from principles to applicationsCARS microscopy: from principles to applications
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.
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CARS microscopy: from principles to applicationsCARS microscopy: from principles to applications
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
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CARS microscopy: from principles to applicationsCARS microscopy: from principles to applications
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
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CARS microscopy: from principles to applicationsCARS microscopy: from principles to applications
Raman scattering basics (2)
R
Raman spectrum (Stokes)Optical excitation
p frequency
Main drawback:
Long acquiring time
Excitation
volume~1µm
Source: http://www.aist.go.jp
p- R
p+ R
R=10-14F
Anti-Stokes
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CARS microscopy: from principles to applicationsCARS microscopy: from principles to applications
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
=
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CARS microscopy: from principles to applicationsCARS microscopy: from principles to applications
Bouncing the springs
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CARS microscopy: from principles to applicationsCARS microscopy: from principles to applications
The CARS Hammer
PPump wave
Anti-Stokes
AS= P+ R
R
Stokes S= P- R
P- S= P-( P- R)= R
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CARS microscopy: from principles to applicationsCARS microscopy: from principles to applications
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)
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CARS microscopy: from principles to applicationsCARS microscopy: from principles to applications
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
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CARS microscopy: from principles to applicationsCARS microscopy: from principles to applications
CARS / Raman Scattering
Laser p
Spontaneous RAMAN
Stokes s
AntiStokes as
Stokes s
AntiStokes as
R R
wavelength
frequency
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CARS microscopy: from principles to applicationsCARS microscopy: from principles to applications
CARS microscopy: What do you need?
S
P
High sensitivity detectors
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CARS microscopy: from principles to applicationsCARS microscopy: from principles to applications
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
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CARS microscopy: from principles to applicationsCARS microscopy: from principles to applications
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
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CARS microscopy: from principles to applicationsCARS microscopy: from principles to applications
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
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CARS microscopy: from principles to applicationsCARS microscopy: from principles to applications
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
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CARS microscopy: from principles to applicationsCARS microscopy: from principles to applications
(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
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CARS microscopy: from principles to applicationsCARS microscopy: from principles to applications
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
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CARS microscopy: from principles to applicationsCARS microscopy: from principles to applications
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
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CARS microscopy: from principles to applicationsCARS microscopy: from principles to applications
Raman / CARS spectra
Source: http://www.aist.go.jp
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)
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CARS microscopy: from principles to applicationsCARS microscopy: from principles to applications
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
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CARS microscopy: from principles to applicationsCARS microscopy: from principles to applications
Resonant contribution
can be expressed as a complex number:
Modulus: Phase:
Circle in the complex plane:
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CARS microscopy: from principles to applicationsCARS microscopy: from principles to applications
(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
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CARS microscopy: from principles to applicationsCARS microscopy: from principles to applications
(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
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CARS microscopy: from principles to applicationsCARS microscopy: from principles to applications
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)
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CARS microscopy: from principles to applicationsCARS microscopy: from principles to applications
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
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CARS microscopy: from principles to applicationsCARS microscopy: from principles to applications
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
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CARS microscopy: from principles to applicationsCARS microscopy: from principles to applications
Far-field CARS radiation patterns in direct space
F-CARS
E-CARS
ps
as
as
z
x
Gachet et al., Proc. SPIE (2006)
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CARS microscopy: from principles to applicationsCARS microscopy: from principles to applications
F-CARS emission
more directive
than the excitation beam
along one direction
Far-field CARS
radiation patterns in k
space
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CARS microscopy: from principles to applicationsCARS microscopy: from principles to applications
F-CARS / E-CARS radiation
F-CARS
E-CARS
x 200
E/F
Volkmer et al. PRL (2001)
Gachet et al., Proc. SPIE (2006)
Djaker et al., Appl. Opt. 45, 7005 (2006)
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CARS microscopy: from principles to applicationsCARS microscopy: from principles to applications
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
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CARS microscopy: from principles to applicationsCARS microscopy: from principles to applications
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 )
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CARS microscopy: from principles to applicationsCARS microscopy: from principles to applications
*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
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CARS microscopy: from principles to applicationsCARS microscopy: from principles to applications
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
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CARS microscopy: from principles to applicationsCARS microscopy: from principles to applications
F-CARS / E-CARS radiation
2µm bead
F-CARS
E-CARS
x 200
cl
4
ascl
100nm bead
Gachet et al., Proc. SPIE (2006)
Djaker et al., Appl. Opt. 45, 7005 (2006)
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CARS microscopy: from principles to applicationsCARS microscopy: from principles to applications
CARS instrumentation
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CARS microscopy: from principles to applicationsCARS microscopy: from principles to applications
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
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CARS microscopy: from principles to applicationsCARS microscopy: from principles to applications
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
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CARS microscopy: from principles to applicationsCARS microscopy: from principles to applications
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)
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CARS microscopy: from principles to applicationsCARS microscopy: from principles to applications
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
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CARS microscopy: from principles to applicationsCARS microscopy: from principles to applications
Two oscillators: pico / pico setup
F-CARS
E-CARS
S
P
AS
Sample
MHz
KHz
SynchroLock System
MHz
KHz
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CARS microscopy: from principles to applicationsCARS microscopy: from principles to applications
Two oscillators pico / femto = Multiplex CARS
Pump: 10 ps
Stokes: 80 fs
10 ps
0.8 ps
Muller J. Phys. Chem B 106 2002
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CARS microscopy: from principles to applicationsCARS microscopy: from principles to applications
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)
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CARS microscopy: from principles to applicationsCARS microscopy: from principles to applications
1 oscillator femto + PCF
Kano et al. APL86 (2005)
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CARS microscopy: from principles to applicationsCARS microscopy: from principles to applications
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
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CARS microscopy: from principles to applicationsCARS microscopy: from principles to applications
One oscillator + One OPO pico/pico
Pump ps 532nm
Signal
Idler
>1350 cm-1
>700 cm-1
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CARS microscopy: from principles to applicationsCARS microscopy: from principles to applications
Sensitivity improvement: FM CARS
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CARS microscopy: from principles to applicationsCARS microscopy: from principles to applications
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
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CARS microscopy: from principles to applicationsCARS microscopy: from principles to applications
H(eterodyne) CARS with OPO!
Jurna Opt. Expr 15 (2007)
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CARS microscopy: from principles to applicationsCARS microscopy: from principles to applications
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)
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CARS microscopy: from principles to applicationsCARS microscopy: from principles to applications
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
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CARS microscopy: from principles to applicationsCARS microscopy: from principles to applications
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)
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CARS microscopy: from principles to applicationsCARS microscopy: from principles to applications
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)
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CARS microscopy: from principles to applicationsCARS microscopy: from principles to applications
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
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CARS microscopy: from principles to applicationsCARS microscopy: from principles to applications
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
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CARS microscopy: from principles to applicationsCARS microscopy: from principles to applications
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
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CARS microscopy: from principles to applicationsCARS microscopy: from principles to applications
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)
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CARS microscopy: from principles to applicationsCARS microscopy: from principles to applications
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)
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CARS microscopy: from principles to applicationsCARS microscopy: from principles to applications
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
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CARS microscopy: from principles to applicationsCARS microscopy: from principles to applications
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
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CARS microscopy: from principles to applicationsCARS microscopy: from principles to applications
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
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CARS microscopy: from principles to applicationsCARS microscopy: from principles to applications
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.
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CARS microscopy: from principles to applicationsCARS microscopy: from principles to applications
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