Adaptive Optical Two-Photon Microscopy for Surface-Profiled Living Biological Specimens

We developed adaptive optical (AO) two-photon excitation microscopy by introducing a spatial light modulator (SLM) in a commercially available microscopy system. For correcting optical aberrations caused by refractive index (RI) interfaces at a specimen's surface, spatial phase distributions of...

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Bibliographic Details
Published in:ACS omega 2021-01, Vol.6 (1), p.438-447
Main Authors: Yamaguchi, Kazushi, Otomo, Kohei, Kozawa, Yuichi, Tsutsumi, Motosuke, Inose, Tomoko, Hirai, Kenji, Sato, Shunichi, Nemoto, Tomomi, Uji-I, Hiroshi
Format: Article
Language:English
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Summary:We developed adaptive optical (AO) two-photon excitation microscopy by introducing a spatial light modulator (SLM) in a commercially available microscopy system. For correcting optical aberrations caused by refractive index (RI) interfaces at a specimen's surface, spatial phase distributions of the incident excitation laser light were calculated using 3D coordination of the RI interface with a 3D ray-tracing method. Based on the calculation, we applied a 2D phase-shift distribution to a SLM and achieved the proper point spread function. AO two-photon microscopy improved the fluorescence image contrast in optical phantom mimicking biological specimens. Furthermore, it enhanced the fluorescence intensity from tubulin-labeling dyes in living multicellular tumor spheroids and allowed successful visualization of dendritic spines in the cortical layer V of living mouse brains in the secondary motor region with a curved surface. The AO approach is useful for observing dynamic physiological activities in deep regions of various living biological specimens with curved surfaces.
ISSN:2470-1343
2470-1343
DOI:10.1021/acsomega.0c04888