Fluorescence radial fluctuation enables two-photon super-resolution microscopy

Motosuke Tsutsumi; Taiga Takahashi; Kentaro Kobayashi; Tomomi Nemoto

doi:10.3389/fncel.2023.1243633

Fluorescence radial fluctuation enables two-photon super-resolution microscopy

Front Cell Neurosci. 2023 Oct 10:17:1243633. doi: 10.3389/fncel.2023.1243633. eCollection 2023.

Authors

Motosuke Tsutsumi^{1

2}, Taiga Takahashi^{1

2}, Kentaro Kobayashi³, Tomomi Nemoto^{1

2

3}

Affiliations

¹ Biophotonics Research Group, Exploratory Research Center on Life and Living Systems, National Institutes of Natural Sciences, Okazaki, Japan.
² Research Division of Biophotonics, National Institute for Physiological Sciences, National Institutes of Natural Sciences, Okazaki, Japan.
³ Nikon Imaging Center, Research Institute for Electronic Science, Hokkaido University, Sapporo, Japan.

Abstract

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.

Keywords: SRRF; in vivo imaging; spine morphology; super-resolution; two-photon microscopy.

Grants and funding

This work was supported by MEXT/JSPS KAKENHI Grant Numbers JP16H06280, JP22H04926, 19K15406, 20H05669, 22K14578, 22KK0100; Brain/MINDS (AMED) JP19dm0207078; JST, ACT-X Grant Number JPMJAX2228; Cooperative Research Program of “NJRC Mater. & Dev.”