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

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.