Light sheet and light field microscopy based on scanning Bessel beam illumination

TL;DR

This paper presents a novel Bessel light sheet fluorescence microscopy system for high-speed, wide-field 3D imaging of biological samples, combining light sheet and light field techniques to significantly improve temporal resolution and image quality.

Contribution

The work introduces a combined LSFM and LFM system with an electrically tunable lens, enabling fast, high-resolution 3D imaging with simplified noise reduction, and compares the unique dynamic range features of LFM.

Findings

Achieved confocal-level resolution in 1/40th the time

Enhanced 3D imaging speed by 28-fold using light field microscopy

Identified unique image dynamic range characteristics of LFM

Abstract

We developed a Bessel light sheet fluorescence microscopy (LSFM) system to enable high-speed, wide-field intra-vital imaging of zebrafish and other thick biological samples. This system uses air objectives for the convenient mounting of large samples and incorporates an electrically tunable lens for automatic focusing during volumetric imaging. To enhance the precision of 3D imaging, the impact of the electrically tunable lens on system magnification is investigated and modified through designed experiments. Despite using Bessel beams with side lobes, we achieved satisfactory image quality through a straightforward background noise subtraction method, eliminating the need for further deconvolution. Our system provides zebrafish imaging at a resolution comparable to commercial confocal microscopy but in just 1/40th of the time. We also introduced light field microscopy (LFM) to improve 3D in vivo imaging temporal resolution. Apart from the 28-fold speed enhancement, the comparison of LFM and LSFM results reveals a unique aspect of LFM imaging concerning image dynamic range, which has not been previously reported.