Analytical Sciences

Multimodal Imaging of Living Cells with Multiplex Coherent Anti-stokes Raman Scattering (CARS), Third-order Sum Frequency Generation (TSFG) and Two-photon Excitation Fluorescence (TPEF) Using a Nanosecond White-light Laser Source

Hiroki SEGAWA,*1 Masanari OKUNO,*2 Philippe LEPROUX,*3 Vincent COUDERC,*3 Takeaki OZAWA,*1 and Hideaki KANO*4,*5

*1 Department of Chemistry, School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-0033, Japan
*2 Department of Chemistry, Graduate School of Pure and Applied Sciences,University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8571, Japan
*3 Xlim Research Institute, CNRS-University of Limoges, 123 avenue Albert Thomas, 87060 Limoges cedex, France
*4 Institute of Applied Physics, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8573, Japan
*5 Graduate School of Pure and Applied Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8573, Japan

The subnanosecond “white-light laser” source has been applied to multimodal, multiphoton, and multiplex spectroscopic imaging (M³ spectroscopic imaging) with coherent anti-Stokes Raman scattering (CARS), third-order sum frequency generation (TSFG), and two-photon excitation fluorescence (TPEF). As the proof-of-principle experiment, we performed simultaneous imaging of polystyrene beads with TSFG and TPEF. This technique is then applied to live cell imaging. Mouse L929 fibroblastic cells are clearly visualized by CARS, TSFG, and TPEF processes. M³ spectroscopic imaging provides various and unique cellular information with different image contrast based on each multiphoton process.

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