Multi-focus light-field microscopy for high-speed large-volume imaging

Abstract

High-speed visualization of three-dimensional (3D) processes across a large field of view with cellular resolution is essential for understanding living systems. Light-field microscopy (LFM) has emerged as a powerful tool for fast volumetric imaging. However, one inherent limitation of LFM is that the achievable lateral resolution degrades rapidly with the increase of the distance from the focal plane, which hinders the applications in observing thick samples. Here, we propose Spherical-Aberration-assisted scanning LFM (SAsLFM), a hardware-modification-free method that modulates the phase-space point-spread-functions (PSFs) to extend the effective high-resolution range along the z-axis by ~ 3 times. By transferring the foci to different depths, we take full advantage of the redundant light-field data to preserve finer details over an extended depth range and reduce artifacts near the original focal plane. Experiments on a USAF-resolution chart and zebrafish vasculatures were conducted to verify the effectiveness of the method. We further investigated the capability of SAsLFM in dynamic samples by imaging large-scale calcium transients in the mouse brain, tracking freely-moving jellyfish, and recording the development of Drosophila embryos. In addition, combined with deep-learning approaches, we accelerated the three-dimensional reconstruction of SAsLFM by three orders of magnitude. Our method is compatible with various phase-space imaging techniques without increasing system complexity and can facilitate high-speed large-scale volumetric imaging in thick samples.

Neuron populations across layer 2-6 in the mouse visual cortex exhibit different coding abilities in the awake mice

“…Light field microscopy (LFM) offers a snapshot-based approach to three-dimensional imaging with low phototoxicity on single-photon microscopy. And the development of virtual-scanning LFM (VsLFM) has pushed the resolution to the diffraction limit of a snapshot by introducing periodic optical beam scanning to increase spatial sampling density (Wu et al, 2022 ; Zhang et al, 2022 ; Lu et al, 2023 ). In comparison to single-photon microscopy, two-photon microscopy (Helmchen and Denk, 2005 ) provides several advantages for observing neural activity, such as enhanced tissue penetration, reduced background noise, and improved resolution.…”