Fluorescence radial fluctuation enables two-photon super-resolution microscopy.

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 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 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.