by two-photon excitation spinning disk confocal … resolution 3D and 4D bio-imaging by two-photon...
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High-temporal resolution 3D and 4D bio-imaging by two-photon excitation spinning disk confocal microscopy Kohei Otomo 1 , Terumasa Hibi 1 , Hirotaka Watanabe 1, 2 , Yumi Yamanaka 1, 2 , Hiroshi Nakayama 3 , Tomomi Nemoto 1, 2 ( 1 Research Institute for Electronic Science, Hokkaido University, Kita 20 Nishi 10, Kita-ku, Sapporo 001-0020, Japan, 2 Graduate School of Information Science and Technology, Hokkaido University, Kita 14 Nishi 9, Kita, Sapporo 060–0814, Japan, Japan, 3 Yokogawa Electric Corporation, 2-3 Hokuyodai, Kanazawa 920–0177, Japan) E-mail: [email protected] KEY WORDS: spinning disk, multi-photon microscopy, 3D, 4D-imaging, in vivo imaging Two-photon excitation laser scanning fluorescence microscopy (TPLSM) has been widely used as a powerful technique for elucidation of dynamical molecular and cellular functions in live specimens. This is because of its superior penetration depth and less-invasiveness for specimens due to the near-infrared excitation laser wavelength, compared with single photon excitation based systems. The temporal resolution of a TPLSM system is limited by the excitation laser beam’s scanning speed. To improve the temporal resolution, the TPLSM system is equipped with a spinning-disk confocal scanning unit. However, the insufficient energy of a conventional Ti-Sapphire laser source restricts the field of view (FOV) for TPLSM images to a narrow region [1]. Therefore, we introduced a high-peak-power Yb-based laser in order to enlarge the FOV [2] (Fig. 1). This system provided three-dimensional imaging of a sufficiently deep and wide region of fixed mouse brain slices, clear four-dimensional imaging of actin dynamics in live mammalian cells and microtubule dynamics during mitosis and cytokinesis in live plant cells, and 108 fps in vivo imaging of blood vessel of a live mouse (Fig. 2). Acknowledgements This work was supported by Core Research for Evolutional Science and Technology, Japan Science and Technology Agency; JSPS KAKENHI of the Ministry of Education, Culture, Sports, Science and Technology (MEXT); Nano-Macro Materials, Devices and System Research Alliance (MEXT); Network Joint Research Center for Materials and Devices (MEXT), the Brain Mapping by Integrated Neurotechnologies for Disease Studies from Japan Agency for Medical Research and development, AMED and Grants in Aid for Regional R&D Proposal Based Program from Northern Advancement Center for Science & Technology of Hokkaido Japan. References [1] Shimozawa, T., et al., Proc. Natl. Acad. Sci. U.S.A., 110, 3399-3404 (2013). [2] Otomo, K., et al., Anal. Sci., 31, 307-313 (2015).
Transcript of by two-photon excitation spinning disk confocal … resolution 3D and 4D bio-imaging by two-photon...
High-temporal resolution 3D and 4D bio-imaging by two-photon excitation spinning disk confocal microscopy
Kohei Otomo 1, Terumasa Hibi 1, Hirotaka Watanabe 1, 2,
Yumi Yamanaka 1, 2, Hiroshi Nakayama 3, Tomomi Nemoto 1, 2 (1 Research Institute for Electronic Science, Hokkaido University, Kita 20 Nishi 10, Kita-ku, Sapporo 001-0020, Japan, 2 Graduate School of Information Science and
Technology, Hokkaido University, Kita 14 Nishi 9, Kita, Sapporo 060–0814, Japan, Japan, 3 Yokogawa Electric Corporation, 2-3 Hokuyodai, Kanazawa 920–0177, Japan)
E-mail: [email protected] KEY WORDS: spinning disk, multi-photon microscopy, 3D, 4D-imaging, in vivo imaging Two-photon excitation laser scanning fluorescence microscopy (TPLSM) has been widely used as a powerful technique for elucidation of dynamical molecular and cellular functions in live specimens. This is because of its superior penetration depth and less-invasiveness for specimens due to the near-infrared excitation laser wavelength, compared with single photon excitation based systems. The temporal resolution of a TPLSM system is limited by the excitation laser beam’s scanning speed. To improve the temporal resolution, the TPLSM system is equipped with a spinning-disk confocal scanning unit. However, the insufficient energy of a conventional Ti-Sapphire laser source restricts the field of view (FOV) for TPLSM images to a narrow region [1]. Therefore, we introduced a high-peak-power Yb-based laser in order to enlarge the FOV [2] (Fig. 1). This system provided three-dimensional imaging of a sufficiently deep and wide region of fixed mouse brain slices, clear four-dimensional imaging of actin dynamics in live mammalian cells and microtubule dynamics during mitosis and cytokinesis in live plant cells, and 108 fps in vivo imaging of blood vessel of a live mouse (Fig. 2).
Acknowledgements This work was supported by Core Research for Evolutional Science and Technology, Japan Science and Technology Agency; JSPS KAKENHI of the Ministry of Education, Culture, Sports, Science and Technology (MEXT); Nano-Macro Materials, Devices and System Research Alliance (MEXT); Network Joint Research Center for Materials and Devices (MEXT), the Brain Mapping by Integrated Neurotechnologies for Disease Studies from Japan Agency for Medical Research and development, AMED and Grants in Aid for Regional R&D Proposal Based Program from Northern Advancement Center for Science & Technology of Hokkaido Japan. References [1] Shimozawa, T., et al., Proc. Natl. Acad. Sci. U.S.A., 110, 3399-3404 (2013). [2] Otomo, K., et al., Anal. Sci., 31, 307-313 (2015).
