Optimizing and extending light-sculpting microscopy for fast functional imaging in neuroscience

TL;DR

This paper enhances light-sculpting microscopy by optimizing scanning modalities and synchronization techniques, enabling faster, deeper, and higher-contrast functional imaging of neuronal activity in brain tissue.

Contribution

It introduces strategies to improve volume speed, depth penetration, and image contrast in light-sculpting microscopy through theoretical, experimental, and synchronization innovations.

Findings

Optimized scanning modalities increase imaging volume and speed.

Synchronization with rolling-shutter reduces scattering and improves contrast.

Experimental calcium imaging demonstrates practical improvements in mouse brain slices.

Abstract

A number of questions in systems biology such as understanding how dynamics of neuronal networks are related to brain function require the ability to capture the functional dynamics of large cellular populations at high speed. Recently, this has driven the development of a number of parallel and high speed imaging techniques such as light-sculpting microscopy, which has been used to capture neuronal dynamics at the whole brain and single cell level in small model organism. However, the broader applicability of light-sculpting microscopy is limited by the size of volumes for which high speed imaging can be obtained and scattering in brain tissue. Here, we present strategies for optimizing the present tradeoffs in light-sculpting microscopy. Various scanning modalities in light-sculpting microscopy are theoretically and experimentally evaluated, and strategies to maximize the obtainable volume speeds, and depth penetration in brain tissue using different laser systems are provided. Design-choices, important parameters and their trade-offs are experimentally demonstrated by performing calcium-imaging in acute mouse-brain slices. We further show that synchronization of line-scanning techniques with rolling-shutter read-out of the camera can reduce scattering effects and enhance image contrast at depth.