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

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.

Enucleation of theC. elegansembryo revealed dynein-dependent spacing between microtubule asters

“…The localization of fluorescent proteins was observed using a spinning-disk confocal microscope (CSU-MP; Yokogawa Electric) ( Otomo et al, 2015 ; Kamada et al, 2022 ) equipped with an EM-CCD camera (iXon; Andor) mounted on an IX71 microscope (Olympus) and controlled using NIS-elements software (Nikon). Details of the system and the examination of phototoxicity will be published elsewhere (in preparation).…”

Improving two-photon excitation microscopy for sharper and faster biological imaging

“…Moreover, the pinhole-array disk in the detection optics provides the TPLSM-SD system confocality, resulting in superior axial spatial resolution compared to the conventional TPLSM system. By utilizing the developed TPLSM-SD system, several biological phenomena have been visualized [28][29][30][31][32]. As a representative example, Figure 3b showed time-lapse xyz images of three kinds of organelle in a tobacco BY-2 cell undergoing the mitotic progression [32].…”

“…By utilizing the developed TPLSM-SD system, several biological phenomena have been visualized [28][29][30][31][32]. As a representative example, Figure 3b showed time-lapse xyz images of three kinds of organelle in a tobacco BY-2 cell undergoing the mitotic progression [32]. Since mitotic control is susceptible to photodamage, intensive excitation laser light irradiations often cause mitotic progression blockage, especially for detailed multidimensional measurements.…”

Enucleation of theC. elegansembryo revealed the mechanism of dynein-dependent spacing between microtubule asters

“…The localization of the fluorescent proteins was observed using a spinning-disk confocal microscope (CSU-MP; Yokogawa Electric, Tokyo, Japan) (Otomo et al, 2015;Kamada et al, 2022) equipped with an and an EM-CCD camera (iXon; Andor, Belfast, UK) mounted on an IX71 microscope (Olympus, Tokyo, Japan) and controlled using NISelements software (Nikon, Tokyo, Japan). Details of the system and the examination of phototoxicity will be published elsewhere (in preparation).…”