Axially overlapped multi-focus light sheet with enlarged field of view

Abstract

Light sheet fluorescence microscopy provides optical sectioning and is widely used in volumetric imaging of large specimens. However, the axial resolution and the lateral Field of View (FoV) of the system, defined by the light sheet, typically limit each other due to the spatial band product of the excitation objective. Here, we develop a simple multi-focus scheme to extend the FoV, where a Gaussian light sheet can be focused at three or more consecutive positions. Axially overlapped multiple light sheets significantly enlarge the FoV with improved uniformity and negligible loss in axial resolution. By measuring the point spread function of fluorescent beads, we demonstrated that the obtained light sheet has a FoV of 450 μm and a maximum axial FWHM of 7.5 μm. Compared with the conventional single-focus one, the multi-focus Gaussian light sheet displays a significantly improved optical sectioning ability over the full FoV when imaging cells and zebrafish. Light sheet fluorescence microscopy (LSFM) has become an indispensable tool in volumetric imaging, with the advances in high spatiotemporal resolution and low photo-toxicity to the fluorescent sample. The open-source design with detailed instructions encourages DIY setups, which has significantly accelerated the wide-range adoption and applications of LSFM. Pioneering works, including OpenSPIM,1 OpenSpin,2 and the recent mesoSPIM,3 provide detailed protocols for building and using the microscopes. These joint efforts further allow the biology labs to build their LSFM system for a specific application, including more complex schemes, such as multiview excitation or detection configurations.4

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