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Low-invasive 5D visualization of mitotic progression by two-photon excitation spinning-disk confocal microscopy

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Title: Low-invasive 5D visualization of mitotic progression by two-photon excitation spinning-disk confocal microscopy
Authors: Kamada, Takafumi Browse this author
Otomo, Kohei Browse this author →KAKEN DB
Murata, Takashi Browse this author →KAKEN DB
Nakata, Kaito Browse this author
Hiruma, Shota Browse this author
Uehara, Ryota Browse this author →KAKEN DB
Hasebe, Mitsuyasu Browse this author
Nemoto, Tomomi Browse this author →KAKEN DB
Issue Date: 17-Jan-2022
Publisher: Nature Portfolio
Journal Title: Scientific reports
Volume: 12
Issue: 1
Start Page: 809
Publisher DOI: 10.1038/s41598-021-04543-7
Abstract: Non-linear microscopy, such as multi-photon excitation microscopy, offers spatial localities of excitations, thereby achieving 3D cross-sectional imaging with low phototoxicity even in thick biological specimens. We had developed a multi-point scanning two-photon excitation microscopy system using a spinning-disk confocal scanning unit. However, its severe color cross-talk has precluded multi-color simultaneous imaging. Therefore, in this study, we introduced a mechanical switching system to select either of two NIR laser light pulses and an image-splitting detection system for 3- or 4-color imaging. As a proof of concept, we performed multi-color fluorescent imaging of actively dividing human HeLa cells and tobacco BY-2 cells. We found that the proposed microscopy system enabled time-lapse multi-color 3D imaging of cell divisions while avoiding photodamage. Moreover, the application of a linear unmixing method to the 5D dataset enabled the precise separation of individual intracellular components in multi-color images. We thus demonstrated the versatility of our new microscopy system in capturing the dynamic processes of cellular components that could have multitudes of application.
Type: article
Appears in Collections:電子科学研究所 (Research Institute for Electronic Science) > 雑誌発表論文等 (Peer-reviewed Journal Articles, etc)

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