Fourier-transform coherent anti-Stokes Raman scattering microscopy

1. Fourier-transform coherent anti-Stokes Raman scattering microscopy Jennifer P. Ogilvie et al. Opt. Lett. 31, 480 (2006) Kazuya MORI MIYASAKA Lab.

2. Contents • Introduction ・ Raman Scattering ・ Coherent Anti-Stokes Raman Scattering (CARS) ・ Nonresonant background problem ・ Motivation • Fourier-transform CARS (FTCARS) method • Experimental Setup using a femtosecond laser • Results and Discussion ・CARS imaging of polystyrene bead • Summary

3. Introduction Raman Scattering ラマン散乱 Molecular vibration Energy diagram 分子振動 Anti-Stokes Raman Scattering ωvib Virtual state Incident light ν0+ωvib ν0 Rayleigh scattering ν0 ωvib Stokes Raman Scattering Stokes Anti-Stokes ν0-ωvib excellent ”molecular fingerprint” for their identification Raman Scattering + protein nucleic acid lipid microscopy Raman spectra http://www.nanophoton.jp/raman/raman-11.html

4. Introduction But Raman scattering is・・・ ・very weak signal (~10-6 of incident radiation)・difficult to separate from fluorescence Coherent Anti-Stokes Raman Scattering (CARS) ω1 Molecular vibration ωvib ω1 ω2 beam1 (pump, probe) ω3 ω1 ω3= 2ω1 – ω2 ωvib ω2 beam2 (stokes) ・significant signal enhancement over Raman scattering・Anti-Stokes field → fluorescence-free

5. Ramanspectra of polystyrene beads conventionalRaman microscope CARS microscope Nonresonant (NR) background Introduction Problem 非共鳴 low resolution（30cm-1）and spectral shift NR background signal diagram ω’vib ωvib ωvib Near two-photon resonance far from two-photon resonance M. Hashimoto et. al. Opt. Lett.25, 1768-1770 (2000)

6. Motivation Problem • NR background • Limited fingerprint region imaging • Complex setup a new method for CARS microscopy with high resolution using a single femtosecond pulse laser

7. Time delay τ/ fs Fourier-transform CARS (FTCARS) method • Single femtosecond pulse laser two femtosecond pulse probe time-domain pump stokes CARS ωvib ① ② τ delay vibrational polarization + ② Time-domain CARS signal 振動分極

8. Fourier-transform CARS (FTCARS) method • Fourier-transform frequency-domain time-domain Fourier-transform ＝Molecular vibration frequencies frequency / cm-1 Time delay / fs NR background vibrational polarization (electronic response) = CARS spectra frequency-domain spectra

9. ω2 ω1 2k1 k2 ω3= 2ω1 – ω2 k3 Experimental Setup ~820nm Pulse duration ~20fs PC: prism compressor LPF: long-pass filter(>780nm) SPF: short-pass filter(<760nm) BS: beam splitter DBS: dichroic beam splitter PMT: photomultiplier tube fluorescence delay 光電子増倍管 CARS signal CARS microscopy Conventional CARS Phase matching ω1 ω3 spectral filtering ω2 (CARS) Filter spatially-resolved

10. Results and Discussion Sample 20μm polystyrene bead in 2-propanol polystyrene 2-propanol time-domain amplitude (a.u.) vibrational polarization NR background Time delay (fs)

11. Results and Discussion FTCARS imaging Scan 1005cm-1 → C-C stretching mode in polystyrene 816cm-1 → C-C stretching mode in 2-propanol FTCARS offers high spectral resolution ~13cm-1

12. Summary • A Fourier-transform technique for CARS microscopy that employs a single laser source and time-delay setup was proposed. • FTCARS method was demonstrated by spectrally imaging a polystyrene bead in 2-propanol. • FTCARS microscopy offers a compact optical setup and high spectral resolusion(～13cm-1).

14. femtosecond pulse probe ω1 pump stokes ω2 ω2 ω1 CARS ① ② time/ fs Frequency / cm-1 uncertainty principleΔEΔt~ħ

15. Sample Spontaneous Raman spectrum of polystyrene http://riodb01.ibase.aist.go.jp/sdbs/cgi-bin/direct_frame_top.cgi

16. Sample Spontaneous Raman spectrum of 2-propanol http://riodb01.ibase.aist.go.jp/sdbs/cgi-bin/direct_frame_top.cgi

17. Sample Fluorescent dye http://www.bangslabs.com/cgi-bin/PSGFind.pl?return=6.2&code=FC07F

18. photomultiplier tube　（光電子増倍管） http://ja.wikipedia.org/wiki/%E7%94%BB%E5%83%8F:Photomultipliertube.png