Multifocus CARS microscopy for realtime vibrational imaging

We developed a multifocus excitation coherent anti-Stokes Raman scattering microscope using a microlens array scanner for realtime molecular imaging. Two picoseond mode-locked lasers tightly synchronized were splited to a few tens of foci with the microlens array, the foci excited the sample parallely and the generated CARS from each spot was detected with an image sensor at once. By the multifocus excitation, exposure time was prolonged proportionally to the number of the foci because of parallel excitation and detection. The video-rate (frame rate of 30 fps) imaging of polystyrene beads in water was demonstrated, and the Brownian motion of beads were clearly obtained. The three-dimensional reconstructed imaging of living HeLa cells (frame rate of 5 fps, 85 images) was also demonstrated.

[1]  Jun Ye,et al.  Synchronization of two passively mode-locked, picosecond lasers within 20 fs for coherent anti-Stokes Raman scattering microscopy , 2002 .

[2]  Brian J. Bacskai,et al.  Video-Rate Confocal Microscopy , 1995 .

[3]  Natallia Uzunbajakava,et al.  Combined Raman and continuous-wave-excited two-photon fluorescence cell imaging. , 2003, Optics letters.

[4]  Peter H. Bartels,et al.  An Ultrafast Laser Scanner Microscope for Digital Image Analysis , 1982, IEEE Transactions on Biomedical Engineering.

[5]  Satoshi Kawata,et al.  Jitter reduction of two synchronized picosecond mode-locked lasers using balanced cross-correlator with two-photon detectors , 2006 .

[6]  Satoshi Kawata,et al.  Automatic Pulse Duration Control of Picosecond Laser using Two-Photon Absorption Detector , 2005 .

[7]  J. Pawley,et al.  Handbook of Biological Confocal Microscopy , 1990, Springer US.

[8]  Takeo Tanaami,et al.  High-Speed Confocal Fluorescence Microscopy Using a Nipkow Scanner with Microlenses for 3-D Imaging of Single Fluorescent Molecule in Real Time , 1996 .

[9]  M. Diem,et al.  Spectroscopy , 2007, Acta Neuropsychiatrica.

[10]  Satoshi Kawata,et al.  Second-harmonic-generation microscope with a microlens array scanner. , 2002, Optics letters.

[11]  Riyi Shi,et al.  Characterization of photodamage in coherent anti-Stokes Raman scattering microscopy. , 2006, Optics express.

[12]  Satoshi Kawata,et al.  Raman microscopy for dynamic molecular imaging of living cells. , 2008, Journal of biomedical optics.

[13]  W. R. Wiley,et al.  Three-Dimensional Vibrational Imaging by Coherent Anti-Stokes Raman Scattering , 1999 .

[14]  J G Fujimoto,et al.  Attosecond active synchronization of passively mode-locked lasers by balanced cross correlation. , 2003, Optics letters.

[15]  Satoshi Kawata,et al.  Multi-focus excitation coherent anti-Stokes Raman scattering (CARS) microscopy and its applications for real-time imaging. , 2009, Optics express.

[16]  Satoshi Kawata,et al.  Multi-focus CARS microscopy using microlens array scanner for realtime molecular spectral imaging , 2009, BiOS.

[17]  M. Levenson.  The principles of nonlinear optics , 1985, IEEE Journal of Quantum Electronics.

[18]  S. Kawata,et al.  Molecular vibration imaging in the fingerprint region by use of coherent anti-Stokes Raman scattering microscopy with a collinear configuration. , 2000, Optics letters.

[19]  James G. Fujimoto,et al.  Attosecond active synchronization of passively mode-locked lasers by balanced cross correlation. , 2003 .

[20]  Conor L Evans,et al.  Chemical imaging of tissue in vivo with video-rate coherent anti-Stokes Raman scattering microscopy. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[21]  Ian Parker,et al.  Video-rate confocal microscopy. , 2003, Methods in enzymology.

[22]  J. Greve,et al.  Studying single living cells and chromosomes by confocal Raman microspectroscopy , 1990, Nature.