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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,
Pfaffenwaldring 5770550 Stuttgart,
Germany
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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