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

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.

Precompensation of 3D field distortions in remote focus two-photon microscopy

“…Compensation for field distortions does not, however, correct for the (largely spherical) aberrations introduced by the objective lens when remote focusing. These have recently been characterized in detail and compensated for in a LC-SLM-remote focus microscope [ 29 ]. Combining field and lens-based aberration corrections would enable non-telecentric remote focus designs to approach the near optimal optical performance achieved with telecentric dual objective 3D microscopes [ 16 , 20 ].…”

Bessel-droplet foci enable high-resolution and high-contrast volumetric imaging of synapses and circulation in the brainin vivo

“…Spatially filtering out stray and undiffracted light, the mask was conjugated to the galvos and objective pupil plane with two lenses (L2, L3), with the transmitted excitation electric field distribution designed to achieve the desired excitation point spread function (PSF). Another phase-only liquid crystal SLM (not shown) was located between and conjugated to the galvos and the objective (21), and can be used to shift the excitation focus axially by displaying a defocus pattern (22).…”

Remote-Focussing for Volumetric Imaging in a Contactless and Label-Free Neurosurgical Microscope

“…There are many ways to achieve volumetric imaging. Remote-focussing (RF) using a dynamic device, such as a deformable mirror (DM), is one of these methods that can permit fast, inertia-free axial refocussing by reshaping of the DM membrane [4].…”