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https://hdl.handle.net/2115/84188

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

英語

2022-01-17

Nature Portfolio

Scientific reports

12(1)

809

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.

学術雑誌論文

Version of Record

2045-2322