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

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

microscopy Molecular vibration Anti-Stokes Raman Scattering ω vib Stokes Raman Scattering Rayleigh scattering Incident light ν0ν0 ν 0 +ω vib ν0ν0 ν 0 -ω vib ω vib Virtual state + Raman Scattering StokesAnti-Stokes Energy diagram Raman Scattering Introduction excellent ”molecular fingerprint” for their identification 分子振動 ラマン散乱 nucleic acid protein lipid Raman spectra

Introduction Coherent Anti-Stokes Raman Scattering (CARS) But Raman scattering is ・・・ ・ very weak signal (~10 -6 of incident radiation) ・ difficult to separate from fluorescence Molecular vibration ω vib ω1ω1 beam2 beam1 ω vib ω2ω2 ω 3 = 2ω 1 – ω 2 (pump, probe) (stokes) ω1ω1 ω1ω1 ω2ω2 ω3ω3 ・ significant signal enhancement over Raman scattering ・ Anti-Stokes field → fluorescence-free

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

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

Single femtosecond pulse laser Fourier-transform CARS (FTCARS) method two femtosecond pulse ω vib time-domain Time delay τ/ fs vibrational polarization+ ② Time-domain CARS signal 振動分極 stokespump probe ①② CARS τ delay

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

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 CARS signal delay 光電子増倍管 ω 3 = 2ω 1 – ω 2 ω1ω1 ω2ω2 Phase matching 2k 1 k2k2 k3k3 Conventional CARS CARS microscopy ω3ω3 ω1ω1 ω2ω2 (CARS) Filter spatially-resolved spectral filtering

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

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

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 ).

ω2ω2 Frequency / cm -1 femtosecond pulse ω1ω1 time/ fs uncertainty principle ΔEΔt~ ħ stokespump probe ①② CARS ω1ω1 ω2ω2

Spontaneous Raman spectrum of polystyrene Sample

Spontaneous Raman spectrum of 2-propanol

Fluorescent dye Sample

photomultiplier tube （光電子増倍 管）