Vibrational imaging and microspectroscopies based on coherent anti-Stokes Raman scattering (CARS) by Andreas Volkmer 3 rd Institute of Physics, University of Stuttgart, Pfaffenwaldring 57 70550 Stuttgart, Germany [email protected]Universität Stuttgart AG Volkmer (Coherent microscopy & single-molecule spectroscopy) ISNO-8, Ein Bokek, 20-25 February 2005
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Vibrational imaging and microspectroscopies based on coherent anti-Stokes Raman scattering (CARS) by Andreas Volkmer 3 rd Institute of Physics, University.
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Vibrational imaging and microspectroscopies based on coherent anti-Stokes Raman scattering (CARS)
by
Andreas Volkmer
3rd Institute of Physics, University of Stuttgart,
Chemical contrast mechanism based on molecular vibrations, which is intrinsic to the samples: NO requirement of natural or artificial fluorescent probes!
(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.