Light Sheet and Light Field Microscopy Based on Scanning Bessel Beam Illumination

Abstract

We developed a Bessel light sheet fluorescence microscopy (LSFM) system to enable high-speed, wide field of view (~0.73 * 3.16 * 0.6 mm) intra-vital imaging of zebrafish and other thick biological samples. The system employs air objectives for the convenient mounting of large samples and integrates an electrically tunable lens for automatic focusing during volumetric imaging. To refine the precision of 3D imaging, we systematically investigate and characterize the impact of the electrically tunable lens on system magnification through designed experiments. Despite the existence of side lobes in Bessel beams, we have successfully attained satisfactory image quality via a straightforward background noise subtraction method, obviating the necessity for additional deconvolution steps. Our developed system affords zebrafish imaging at a resolution comparable to commercial confocal microscopy, accomplishing this feat in merely 1/40 of the confocal’s acquisition time. Furthermore, we introduce light field microscopy (LFM) to enhance the temporal resolution of 3D in vivo imaging. In addition to the 28-fold acceleration in imaging speed, a comparative analysis of LFM and LSFM results discovers a distinctive aspect of LFM imaging about image dynamic range, a phenomenon hitherto unreported in the literature.

Keywords: Light sheet microscopy, light field microscopy, zebrafish imaging, Bessel beam, Autofocus