Transcript Vibrational imaging and microspectroscopies based on coherent anti-Stokes Raman scattering (CARS) by Andreas Volkmer Universität Stuttgart 3rd Institute of Physics, University of Stuttgart, Pfaffenwaldring 57 70550 Stuttgart, Germany [email protected] FRISNO-8, Ein.

Vibrational imaging and microspectroscopies
based on coherent anti-Stokes Raman scattering (CARS)
by
Andreas Volkmer
Universität Stuttgart
3rd Institute of Physics,
University of Stuttgart,
Pfaffenwaldring 57
70550 Stuttgart,
Germany
[email protected]
FRISNO-8, Ein Bokek, 20-25 February 2005
AG Volkmer
(Coherent microscopy &
single-molecule spectroscopy)
Ultimate goal in Optical Microscopy
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.
Fluorescence-based microscopy
 Confocal fluorescence laser scanning microscopy
 Two-photon induced fluorescence laser scanning microscopy
knr
abs
!
kfl
Limitations of fluorescence-based spectroscopic studies:
• dye labeling required (photo-toxicity)
• perturbation of structure and dynamics by fluorophore
• photo-stability (# emitted photons)
Fluorescence photobleaching of Rhodamine 6G / water
CW (one-photon)
excitation at 514 nm
NF
Pulsed fs (two-photon)
excitation at 800 nm
Pulsed fs (one-photon)
excitation at 350 nm
10
3
10
3
10
3
10
2
10
2
10
2
10
1
10
1
10
1
10
0
10
0
10
0
10
-1
10
-2
10
NF
2
10
3
4
5
10
10
-2
(I0/2) / W cm
10
6
10
7
10
-1
10
-2
10
NF
2
10
3
4
5
10
10
-2
Iav / kW cm
10
6
10
7
10
-1
10
-2
10
2
10
3
4
5
10
10
-2
(I0/2) / W cm
10
6
10
7
Excited-state
photolysis model:
Eggeling, Volkmer, Seidel, Chem. Phys. Chem. (2005) submitted.
Intrinsic chemical contrast mechanism
Chemical contrast mechanism based on molecular vibrations, which is
intrinsic to the samples: NO requirement of natural or artificial
fluorescent probes!
Raman bands / cm-1
species
DNA backbone,
C-O stretching
~ 1000
Polypeptide backbone,
C=O stretching (Amide I)
1500-1700
Lipids, C-H stretching
2900-3000
[N. Jamin et al., PNAS 95 (1998) 4837-4840 ]
Infrared microscopy:
• low spatial resolution (~2-3 mm)
• low S/B ratio
• water absorption
Spontaneous Raman microscopy:
• weak signal
=> requirement for high excitation power
• fluorescence background
CARS fundamentals


induced third-order polarization:
(3)
 AS    r3  AS    nr3 EP2 P ES* S 
PAS
CARS signal:
I CARS  P
( 3)
AS
 AS 
 AS  2P  S 
2
‘P
P
P
S ‘P AS
v=1
v=0
Resonant CARS

( 3)
r
Non-resonant CARS
1

   p1   s   i

P
S ‘P AS
v=1
v=0

 nr  const.
( 3)
S
AS
Two-photon enhanced
non-resonant CARS
No vibrational contrast !
Development of CARS Microscopy
1982 - Duncan, Reintjes, Manuccia, Optics Lett. 7, 350
Picosecond visible laser,
Noncollinear geometry
Onion-skin cells, soaked in D2O
(CARS image on the 2450-cm-1 band of D20)
1999 - Zumbusch, Holtom, Xie, Phys. Rev. Lett. 82, 4142
Femtosecond near-IR laser,
Collinear geometry,
Forward detection
Sample
Filter

AS

P

S
D
High NA objectives
E-coli
853 nm (100 mW)
1135 nm (100 mW)
 CARS signal at 675 nm
(Raman-shift of 2913 cm-1, on resonance with C-H vibrations)
HeLa cells
Advantages of CARS-microscopy
• 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
Theory of collinear CARS microscopy
Distinct features:
(i)
Under tight focusing conditions -> breakdown of paraxial approximation
(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) Description of a tightly focused Gaussian field
Amplitude distribution
Phase distribution (-kz)
2
1
-2.0
-1.7
-1.4
-1.1
-0.80
-0.50
-0.20
0.10
0.40
0.70
1.0
1.3
1.6
1.9
2.0
z/
z/
1
0
0
-1
-2
-2
-1
0
x/
1
2
-1
-1
0
x/
1
Cheng, Volkmer, Book, Xie, JOSA B, 19 (2002) 1363
(ii) Wave-vector mismatch in collinear CARS microscopy
k  k AS  2k P  k S 
Wave-vector mismatch in collinear beam geometry:
phase matching condition: D   k
(interaction length << coherence length)
F-CARS
detector
L
F
A
k  0
F-CARS
(forward-detected)
F-CARS
kP
kAS
kP
kS
p
AS
Obj
z
Obj
p
sample
x
P
HWP
QWP
AS
P
z
Obj
S
BC F
L
E-CARS
detector
E-CARS
(epi-detected)
Δk  4n s
C-CARS
(counter-propagating)
x
Obj
BS
E-CARS
kP
kAS
kP
kS
S
AS BS
F
L
C-CARS
detector
Δk  4n as
C-CARS
kP
kAS
kP
kS
Cheng, Volkmer, Book, Xie, JOSA B, 19 (2002) 1363
(iii) CARS signal generation for microscopic scatterer
z
Assuming:

ε AS R, r, χ obj r 
• tightly focused incident
Gaussian fields
R

χ solv
χ obj
r
0
x

w0
f
• Incident fields are
polarized along the x axis
• refractive index mismatch
between sample and
solvent is negligible
Einc
Volkmer, Cheng, Xie, Phys. Rev. Lett. 87, 023901 (2001).
Simulated size dependence of CARS signals
ICARS (a.u.)
3
10
1
10
-1
10
-3
10
2
3
F-CARS
E-CARS
0
2
4 6
D / p
8
10
1
10
-1
10
-3
10
F-CARS
E-CARS
0
2
4 6
D / p
8
10
0
10
-2
10
-4
10
C-CARS (forward)
C-CARS (backward)
0
2
4 6
D / p
8
kp
χ solv
χ obj
χ obj
χ solv (reflected)
k p, k S
kp, kS
kS
Volkmer, J. Phys. D : Appl. Phys. 38 (2005) R59
Experimental characterization of CARS microscopy
for a single 500-nm polystyrene bead in water
(Raman shift ~1600 cm-1)
200
150
100
50
0
0.0
(c)
F-CARS xy-image
(b) E-CARS xy- image
signal (cts)
signal (cts)
(a)
FWHM
0.34 mm
0.5
1.0
1.5 2.0
x (mm)
2.5
3.0
20
15
10
5
0
0.0
FWHM
0.34 mm
0.5
1.0
1.5 2.0
x (mm)
2.5
3.0
(d) F-CARS xz- image
C-CARS xy- image
0.0
0.5
FWHM
1.18 mm
1.0
signal (cts)
40
30
20
10
0
0.0
z (mm)
1.5
2.0
2.5
3.0
FWHM
0.36 mm
3.5
4.0
0.5
1.0
1.5 2.0
x (mm)
2.5
3.0
0
500 1000
signal (cts)
Volkmer, J. Phys. D : Appl. Phys. 38 (2005) R59
Picosecond CARS imaging of a live unstained cell
NIH3T3 cells
@ Raman shift ~2860 cm-1
(C-H strectch)
Epithelial cells
@ Raman shift ~1570 cm-1
(amide I)
F-CARS xy- image
100
0
0
20
40
x (mm)
60
80
60
40
20
0
P-CARS xy- image
200
signal (cts)
200
(c)
E-CARS xy- image
(b)
signal (cts)
signal (cts)
(a)
0
20
40
x (mm)
60
100
0
0
10
20
30
x (mm)
40
50
60
Simulation of CARS spectra as a
function of pulse widths
2 = 10 cm1 … line width
A
 3  
  nr3
   p   s   i

I CARS 
 P  
( 3)
as

2
d as
CARS intensity (a.u.)
The CARS intensity is:
 … vibration frequency
 nr A  0.2
pulse width
600 0.5 ps
-1
(29 cm )
X1
400
2 ps
-1
(7.5 cm )
X 10
200 10 ps (1.5 cm-1)
X 100
Raman profie
0
-150 -100
-50
0
50
-1
(p-s)-R (cm )
100
150
CARS intensity vs. excitation pulse spectral width
Pulse temporal width (fs)
0.8
| |
2
150
100
Non-resonant
(  0.01)
CARS intensity (a.u.)
Signal / background
1.0
300
5000

0.6
Resonant
0.4
0.2
Ir/Inr
| |
2
-1
 = 25 cm

0.0
0
3
50
100
-1
Pulse spectral width (cm )
150
Cheng, Volkmer, Book, Xie, J. Phys. Chem. B 105, 1277 (2001).
The CARS microscope
Synchro-Lock
system
microscope
PZT drivers
& galvo’s
Telescopes
Pol
p
6.7 ps Ti:sapphire
mode-locked oscillator
s
6.7 ps Ti:sapphire
mode-locked oscillator
BC
Pol
Multiplex-CARS Microspectroscopy
in the Frequency-Domain
 acquisition of CARS spectrum in one”shot”!
pump
Stokes
CARS
AS
p’

s
p
D
L
L
FM
AS
A
F
Spectrometer +
LN2-CCD array
Obj
Telescopes
P
S
HWP QWP
Obj
p
BC
S
Example: Monitoring the thermodynamic state of
phospholipid membranes in the C-H stretch region
DSPC
Tg=55°C
entropy
DOPC
Tg=-20°C
0.8
1.0
Raman
Intensity (a.u.)
Intensity (a.u.)
1.0
0.6
0.4
0.2
0.0
2800
2900
-1
0.8
0.6
0.4
0.2
0.0
3000
Raman shift / cm
Normalized CARS Int.
Normalized CARS Int.
3
CARS
2
1
0
2800
2900
3000
Raman
1.2
2800
2900
-1
Raman shift (cm )
3000
CARS
0.8
0.4
0.0
2800
ps / cm–1 [Cheng, Volkmer, Book, Xie, J. Phys. Chem. B 2002, 106, 8493-8498]
2900
ps / cm–1
3000
Model system for Stratum Corneum lipids
Raman spectra in CH-stretching mode region
na(CH2)
ns(CH2)
na(CH2)cycl
wavenumbers /cm-1
Hyper-spectral CARS imaging of a Stratum Corneum
Spectrally integrated CARS
image section
Extracted CARS ratio spectra
for each image pixel
1.2
10 mm
1.15
I CARS n~ 
I ref n~ 
CARS
1.1
1.05
1
0.95
0.9
0.85
2700

2900
n~ / cm1
Existence of cholesterol-enriched micro-domains
(see Poster by Nandakumar et al : Mo-4)
3100
CARS microspectroscopy in the time-domain
Raman Free Induction Decay (RFID):
E p t  , E S t 
2
E p ' t 
2
P ( 3) t , t 
t
Three-color CARS set-up:
P’
 AS
I CARS (t )
2
0

2
time
Telescopes P
D
p
L
FD
A
AS
F
S
P
VD
S
|1>
|0>
Obj
S
p’
Obj
BC
BC
Example: RFID imaging of 1-mm polystyrene bead
5
10
4
10
3
3000
residuals
100
10
2
0
-100
-200
2700
2800
2900
3000
3100
3200
3300
Raman shift
500
t / fs
-3
2.0
1.5 t = 0 fs
1.0
2.0
0.5
1.5
0.0
1
2
1.0 0
2.0
0.5
1.5
0.0
1
2
1.00
0.5
0.0
0
1
2
0
-3
-5
-5
3040
3080
-1
Raman shift / cm
200
-500
-5
intensity
cps/ cps
intensity
x10 /x10
cps / x10
intensity
 P1 = 714.6 nm (~85 fs)
S = 914.1 nm (~115 fs)
 P2 = 798.1 nm (~185 fs)
~ || (n~)
intensity (a.u.)
water
bead
/ cps
-3 intensity/x10
intensity
intensity
x10 /x10
cps cps
CARS signal / cps
10
3
x
m
/
3
x
/
m
4
5
m
4
5
m
3
x / mm
4
5
S/B(t=0)  3
4
3
2
1
0
4
3
2
1
0
1000
Quantum beat
recurs at ~ 1280
fs (mode beating
at difference
frequencies of
~ 26 cm-1)
1500
t = 484 fs
4
3
02
1
0
1
2
3
x
0
1
/
m
2
3
x
0
1
4
2
5
m
/
m
4
5
m
3
4
x / mm
5
S/B (t=484 fs)  35
Complete removal of the non-resonant CARS contributions !
Volkmer, Book, Xie, Appl. Phys. Lett. 80 (2002) 1505c
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 scattering (SRS) microscopy
L
P
P
S= S- p+ p
(3)

kS = kS – kP + kp
S
S
Stimulated Raman gain for probe laser in the presence of strong pump laser,
when frequency difference equals Raman frequency
ks
P(2) =  (3) (- 2; 2, -1, 1) E(2) |E(1)|2
Advantages:
kp
kS
kp
Depends only on the Im  (3)
Linear on  (3)
Linear on number density
Linear in pump and Stokes intensities
Automatic Phase matching
Disadvantage: Tiny signal over huge background signal from the Stokes field!
0.5 mm
SRS signal (a.u)
SRS images of a polystyrene 1-mm bead in water
Slope 1.01
Slope 1.005
1.5
5
 Pixel intensity  No. of C=C bonds
 No signal from surrounding water
 No interference effect in image contrast
Nandakumar, Kovalev, Volkmer, manuscript in preparation
SRS intensity (a.u.)
Stoke power / mW
16
1
5
pump power / mW
nas(CH2-aliph.)
ns(CH2-aliph.) 2912 cm-1
2853 cm-1
12
8
4
2800
2900
-1
Raman shift / cm
3000
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 matrix
 SRS spectra qualitatively reproduce the Raman spectra
Acknowledgements
Harvard University
X.S. Xie
J.-X. Cheng
L.D. Book
3. Physikalische Institut, Universität Stuttgart:
P. Nandakumar
A. Kovalev
Roswell Park Cancer Institute, Buffalo, NY:
A. Sen
M. Koehler
€€
$$
Emmy Noether Program
Faculty of Arts and Sciences of
Harvard University