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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

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Abstract

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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Acknowledgments

This work was supported by the JSPS bilateral joint research project (SAKURA program) and The NOVARTIS Foundation (Japan) for the Promotion of Science. The authors thank Prof. Hiro-o Hamaguchi for his guidance, support, discussion, and encouragement throughout the present study. The authors thank the LEU KOS company for technical support with the dual-output supercontinuum light source. The authors gratefully acknowledge J. Ukon, UKON CRAFT SCIENCE, Ltd. for assisting with a fruitful collaboration between Japanese and French labs.

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Authors and Affiliations

  1. Department of Chemistry, School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo, 113-0033, Japan

    Hiroki Segawa & Takeaki Ozawa

  2. Department of Chemistry, Graduate School of Pure and Applied Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki, 305-8571, Japan

    Masanari Okuno

  3. Xlim Research Institute, CNRS-University of Limoges, 123 avenue Albert Thomas, 87060, Limoges cedex, France

    Philippe Leproux & Vincent Couderc

  4. Institute of Applied Physics, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki, 305-8573, Japan

    Hideaki Kano

  5. Graduate School of Pure and Applied Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki, 305-8573, Japan

    Hideaki Kano

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  1. Hiroki Segawa
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  2. Masanari Okuno
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  3. Philippe Leproux
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  4. Vincent Couderc
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  5. Takeaki Ozawa
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  6. Hideaki Kano
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Correspondence to Hideaki Kano.

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Segawa, H., Okuno, M., Leproux, P. et al. 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. ANAL. SCI. 31, 299–305 (2015). https://doi.org/10.2116/analsci.31.299

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  • DOI: https://doi.org/10.2116/analsci.31.299

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