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Two-photon fluorescence microscopy has been widely applied to three-dimensional imaging of complex samples. Remote focusing by controlling the divergence of excitation light is a common approach to scanning the focus axially. However, microscope objectives induce distortion to the wavefront of non-collimated excitation beams, leading to degraded imaging quality away from the natural focal plane. We characterized the aberrations introduced by remote focusing and used adaptive optics to correct the remote-focusing-induced aberrations. Diffraction-limited focal quality over up to 800-µm axial range can be maintained. We further demonstrated aberration-free remote focusing for in vivo imaging of neurites and synapses in mouse brain.
Yuhan Yang,Wei Chen,Jiang Lan Fan, andNa Ji
"Adaptive optics enables aberration-free remote focusing for two-photon fluorescence microscopy", Proc. SPIE 11652, Adaptive Optics and Wavefront Control for Biological Systems VII, 116520G (5 March 2021); https://doi.org/10.1117/12.2578926
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Yuhan Yang, Wei Chen, Jiang Lan Fan, Na Ji, "Adaptive optics enables aberration-free remote focusing for two-photon fluorescence microscopy," Proc. SPIE 11652, Adaptive Optics and Wavefront Control for Biological Systems VII, 116520G (5 March 2021); https://doi.org/10.1117/12.2578926