Fluorescence radial fluctuation enables two-photon super-resolution microscopy

Frontiers in Cellular Neuroscience

Cellular Neuroscience

Taiga Takahashi¹  Motosuke Tsutsumi¹  Tomomi Nemoto²  Kentaro Kobayashi³ 

[1] Biophotonics Research Group, Exploratory Research Center on Life and Living Systems, National Institutes of Natural Sciences, Okazaki, Japan;Research Division of Biophotonics, National Institute for Physiological Sciences, National Institutes of Natural Sciences, Okazaki, Japan;Biophotonics Research Group, Exploratory Research Center on Life and Living Systems, National Institutes of Natural Sciences, Okazaki, Japan;Research Division of Biophotonics, National Institute for Physiological Sciences, National Institutes of Natural Sciences, Okazaki, Japan;Nikon Imaging Center, Research Institute for Electronic Science, Hokkaido University, Sapporo, Japan;Nikon Imaging Center, Research Institute for Electronic Science, Hokkaido University, Sapporo, Japan;

关键词: two-photon microscopy;    super-resolution;    SRRF;    in vivo;    spine morphology;   

DOI  :  10.3389/fncel.2023.1243633

 received in 2023-06-21, accepted in 2023-09-26,  发布年份 2023

来源: Frontiers

【 摘 要 】

Despite recent improvements in microscopy, it is still difficult to apply super-resolution microscopy for deep imaging due to the deterioration of light convergence properties in thick specimens. As a strategy to avoid such optical limitations for deep super-resolution imaging, we focused on super-resolution radial fluctuation (SRRF), a super-resolution technique based on image analysis. In this study, we applied SRRF to two-photon microscopy (2P-SRRF) and characterized its spatial resolution, suitability for deep observation, and morphological reproducibility in real brain tissue. By the comparison with structured illumination microscopy (SIM), it was confirmed that 2P-SRRF exhibited two-point resolution and morphological reproducibility comparable to that of SIM. The improvement in spatial resolution was also demonstrated at depths of more than several hundred micrometers in a brain-mimetic environment. After optimizing SRRF processing parameters, we successfully demonstrated in vivo high-resolution imaging of the fifth layer of the cerebral cortex using 2P-SRRF. This is the first report on the application of SRRF to in vivo two-photon imaging. This method can be easily applied to existing two-photon microscopes and can expand the visualization range of super-resolution imaging studies.

【 授权许可】

Unknown   
Copyright © 2023 Tsutsumi, Takahashi, Kobayashi and Nemoto.