Adaptive optics enables aberration-free single-objective remote focusing for two-photon fluorescence microscopy

Yuhan Yang; Wei Chen; Jiang Lan Fan; Na Ji

doi:10.1364/BOE.413049

Adaptive optics enables aberration-free single-objective remote focusing for two-photon fluorescence microscopy

Biomed Opt Express. 2020 Dec 15;12(1):354-366. doi: 10.1364/BOE.413049. eCollection 2021 Jan 1.

Authors

Yuhan Yang¹, Wei Chen^{1

2}, Jiang Lan Fan³, Na Ji^{1

2

4

5}

Affiliations

¹ Department of Physics, University of California, Berkeley, CA 94720, USA.
² Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA.
³ Joint Graduate Program in Bioengineering, University of California, Berkeley and University of California, San Francisco, CA 94720, USA.
⁴ Helen Wills Neuroscience Institute, University of California, Berkeley, CA 94720, USA.
⁵ Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.

Abstract

Two-photon fluorescence microscopy has been widely applied to three-dimensional (3D) 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. In this paper, using a liquid-crystal spatial light modulator to control the divergence of the excitation beam through a single objective, we systematically characterized the aberrations introduced by divergence control through microscope objectives of NA 0.45, 0.8, and 1.05. We used adaptive optics to correct the divergence-induced-aberrations and maintain diffraction-limited focal quality over up to 800-µm axial range. We further demonstrated aberration-free remote focusing for in vivo imaging of neurites and synapses in the mouse brain.

Abstract

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