Vibrational imaging and microspectroscopies based on coherent anti-stokes Raman scattering (CARS)

Transcription

1 Vibrational imaging and microspectroscopies based on coherent anti-stokes Raman scattering (CARS) by Andreas Volkmer Universität Stuttgart 3 rd Institute of Physics, University of Stuttgart, Pfaffenwaldring Stuttgart, Germany a.volkmer@physik.uni-stuttgart.de AG Volkmer (Coherent microscopy & single-molecule spectroscopy) FRISNO-8, Ein Bokek, February 2005

2 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.

3 Fluorescence-based microscopy Confocal fluorescence laser scanning microscopy Two-photon induced fluorescence laser scanning microscopy k nr abs k fl! Limitations of fluorescence-based spectroscopic studies: dye labeling required (photo-toxicity) perturbation of structure and dynamics by fluorophore photo-stability (# emitted photons)

4 Fluorescence photobleaching of Rhodamine 6G / water CW (one-photon) excitation at 514 nm Pulsed fs (two-photon) excitation at 800 nm Pulsed fs (one-photon) excitation at 350 nm N F 10 0 N F 10 0 N F (I 0 /2) / W cm I av / kw cm -2 (I 0 /2) / W cm Excited-state photolysis model: Eggeling, Volkmer, Seidel, Chem. Phys. Chem. (2005) submitted.

5 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! species DNA backbone, C-O stretching Polypeptide backbone, C=O stretching (Amide I) Raman bands / cm -1 ~ Lipids, C-H stretching [N. Jamin et al., PNAS 95 (1998) ] Infrared microscopy: low spatial resolution (~2-3 µm) low S/B ratio water absorption Spontaneous Raman microscopy: weak signal => requirement for high excitation power fluorescence background

6 induced third-order polarization: CARS fundamentals P (3) AS [ ] 2 * E ( ω ) E ( ω ) ( 3 ( ) ) ( 3) ω = χ ( ω ) + χ AS r AS nr P P S S CARS signal: I (3) CARS P AS ( ω ) 2 AS ( ω = 2ω ω ) AS P S ω P ω S ω AS ω AS ω P ω S ω P v=1 ω P ω S ω P ω AS v=1 ω P Resonant CARS χ v=0 ( 3) 1 r = p1 [ Ω ( ω ω )] + iγ s v=0 Non-resonant CARS Two-photon enhanced non-resonant CARS (3) χ nr = const. No vibrational contrast!

7 Development of CARS Microscopy Duncan, Reintjes, Manuccia, Optics Lett. 7, 350 Picosecond visible laser, Noncollinear geometry Onion-skin cells, soaked in D 2 O (CARS image on the 2450-cm -1 band of D 2 0)

8 Zumbusch, Holtom, Xie, Phys. Rev. Lett. 82, 4142 Femtosecond near-ir laser, Collinear geometry, Forward detection Sample Filter ω AS ω P ω S D High NA objectives E-coli HeLa cells 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)

9 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

10 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

11 (i) Description of a tightly focused Gaussian field z / λ Amplitude distribution x / λ z / λ 1 0 Phase distribution (φ-kz) x / λ Cheng, Volkmer, Book, Xie, JOSA B, 19 (2002) 1363

12 (ii) Wave-vector mismatch in collinear CARS microscopy Wave-vector mismatch in collinear beam geometry: phase matching condition: D << π k (interaction length << coherence length) L F F-CARS detector F-CARS k P k AS ω p k = k 2 AS F-CARS (forward-detected) ( k k ) P k S = 0 A ω AS Obj z k P k S Obj E-CARS (epi-detected) k = 4πn λ as P HWP QWP ω p Obj x sample Obj z x C-CARS k = 4πn λ s (counter-propagating) ω S ω AS BS E-CARS ω S ωas BS C-CARS P BC F L k P k AS F L k P k AS E-CARS detector k P k S C-CARS detector k P k S Cheng, Volkmer, Book, Xie, JOSA B, 19 (2002) 1363

13 (iii) CARS signal generation for microscopic scatterer z ε ( R, r χ () r ) AS, obj Φ Assuming: R tightly focused incident Gaussian fields χ solv Θ r χ obj Incident fields are polarized along the x axis 0 γ f x refractive index mismatch between sample and solvent is negligible w 0 E inc Volkmer, Cheng, Xie, Phys. Rev. Lett. 87, (2001).

14 Simulated size dependence of CARS signals I CARS (a.u.) F-CARS E-CARS F-CARS E-CARS C-CARS (forward) C-CARS (backward) D / λ p D / λ p D / λ p k p χ solv χ obj χ obj χ solv (reflected) k p, k S k p, k S k S Volkmer, J. Phys. D : Appl. Phys. 38 (2005) R59

15 Experimental characterization of CARS microscopy for a single 500-nm polystyrene bead in water (Raman shift ~1600 cm -1 ) (a) F-CARS xy-image (b) E-CARS xy- image signal (cts) signal (cts) x (µm) FWHM 0.34 µm (c) C-CARS xy- image FWHM 0.36 µm x (µm) signal (cts) FWHM 0.34 µm x (µm) (d) F-CARS xz- image z (µm) FWHM 1.18 µm signal (cts) Volkmer, J. Phys. D : Appl. Phys. 38 (2005) R59

16 Picosecond CARS imaging of a live unstained cell Epithelial Raman shift ~1570 cm -1 (amide I) NIH3T3 Raman shift ~2860 cm -1 (C-H strectch) (a) F-CARS xy- image (b) E-CARS xy- image (c) P-CARS xy- image signal (cts) x (µm) signal (cts) x (µm) signal (cts) x (µm)

17 Simulation of CARS spectra as a function of pulse widths χ ( 3) A = χ Ω iγ + ( ω ω ) p s ( 3) nr 2Γ = 10 cm 1 line width Ω vibration frequency A = 0.2 χ nr The CARS intensity is: I CARS + (3) = P ( ω as ) 2 dω as CARS intensity (a.u.) pulse width 0.5 ps (29 cm -1 ) 2 ps (7.5 cm -1 ) 10 ps (1.5 cm -1 ) Raman profie X 1 X 10 X (ω p -ω s )-ω R (cm -1 )

18 CARS intensity vs. excitation pulse spectral width Signal / background χ 2 Pulse temporal width (fs) I r /I nr Non-resonant ( 0.01) ω χ 2 Resonant Γ = 25 cm Pulse spectral width (cm -1 ) ω -1 CARS intensity (a.u.) Cheng, Volkmer, Book, Xie, J. Phys. Chem. B 105, 1277 (2001). 0

19 The CARS microscope Synchro-Lock system microscope PZT drivers & galvo s Telescopes Pol 6.7 ps Ti:sapphire mode-locked oscillator ω p 6.7 ps Ti:sapphire mode-locked oscillator ω s Pol BC

20 Multiplex-CARS Microspectroscopy in the Frequency-Domain acquisition of CARS spectrum in one shot! Stokes pump CARS ω AS ω s ω p ω ω p L D L ω AS FM Spectrometer + LN 2 -CCD array A F Obj Telescopes P HWP QWP S Obj ω p BC ω S

21 Example: Monitoring the thermodynamic state of phospholipid membranes in the C-H stretch region DSPC T g =55 C entropy DOPC T g =-20 C Normalized CARS Int. Intensity (a.u.) Raman Raman shift / cm -1 CARS Intensity (a.u.) Raman Raman shift (cm -1 ) ω p ω s / cm 1 [Cheng, Volkmer, Book, Xie, J. Phys. Chem. B 2002, 106, ] ω p ω s / cm 1 Normalized CARS Int CARS

22 Model system for Stratum Corneum lipids Raman spectra in CH-stretching mode region ν a (CH 2 ) ν s (CH 2 ) ν a (CH 2 ) cycl wavenumbers /cm -1

23 Hyper-spectral CARS imaging of a Stratum Corneum Spectrally integrated CARS image section Extracted CARS ratio spectra for each image pixel 10 µm I CARS ref I CARS ~ ( ν ) ( ~ ν ) ~ 1 ν / cm Existence of cholesterol-enriched micro-domains (see Poster by Nandakumar et al : Mo-4)

24 CARS microspectroscopy in the time-domain Raman Free Induction Decay (RFID): 2 E () t, E () t 2 () 2 p S E p ' t P (3) ( τ, t ) 2 I CARS (τ ) Three-color CARS set-up: 0 time τ Telescopes P D ω P ω P ω S ω AS 1> 0> ω p ω S ω p FD VD ω AS Obj Obj L A F S BC BC

25 Example: RFID imaging of 1-µm polystyrene bead intensity x10-5 / cps CARS signal / cps water bead τ = 0 fs intensity (a.u.) ~ α ( ~ ν ) Raman shift / cm τ / fs x / µm S/B(τ=0) 3 intensity x10-3 / cps τ = 484 fs x / µm S/B (τ=484 fs) 35 λ P1 = nm (~85 fs) λ S = nm (~115 fs) λ P2 = nm (~185 fs) Quantum beat recurs at ~ 1280 fs (mode beating at difference frequencies of ~ 26 cm -1 ) Complete removal of the non-resonant CARS contributions! Volkmer, Book, Xie, Appl. Phys. Lett. 80 (2002) 1505c

26 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)

27 Stimulated Raman scattering (SRS) microscopy L ω P ω P χ (3) ω S = ω S - ω p + ω p ω S ω S k S =k S k P +k p Stimulated Raman gain for probe laser in the presence of strong pump laser, when frequency difference equals Raman frequency k s k S P(ω 2 ) = χ (3) (- ω 2 ; ω 2, -ω 1, ω 1 ) E(ω 2 ) E(ω 1 ) 2 k p k p Advantages: Depends only on the Im χ (3) Linear on χ (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!

28 SRS images of a polystyrene 1-µm bead in water 0.5 µm SRS signal (a.u) Slope Slope Stoke power / mw 1 5 pump power / mw Pixel intensity No. of C=C bonds No signal from surrounding water No interference effect in image contrast SRS intensity (a.u.) ν s (CH 2 -aliph.) 2853 cm -1 ν as (CH 2 -aliph.) 2912 cm -1 Nandakumar, Kovalev, Volkmer, manuscript in preparation R am an shift / cm -1

29 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

30 Acknowledgements Harvard University X.S. Xie J.-X. Cheng L.D. Book 3. Physikalische Institut, Universität Stuttgart: Roswell Park Cancer Institute, Buffalo, NY: P. Nandakumar A. Kovalev A. Sen M. Koehler $$ Emmy Noether Program Faculty of Arts and Sciences of Harvard University