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Performance trade-offs for single- and dual-objective light-sheet microscope designs

By Kevin W. Bishop, Adam K. Glaser, Jonathan T.C. Liu

Posted 12 May 2020
bioRxiv DOI: 10.1101/2020.05.10.087171

Light-sheet microscopy (LSM) has emerged as a powerful tool for high-speed volumetric imaging of live model organisms and large optically cleared specimens. When designing cleared-tissue LSM systems with certain desired imaging specifications (e.g. resolution, contrast, and working distance), various design parameters must be taken into consideration. In order to elucidate some of the key design trade-offs for LSM systems, we present a diffraction- based analysis of single- and dual-objective LSM configurations where Gaussian illumination is utilized. Specifically, we analyze the effects of the illumination and collection numerical aperture (NA), as well as their crossing angle, on spatial resolution and contrast. Assuming an open-top light-sheet (OTLS) architecture, we constrain these parameters based on fundamental geometric considerations as well as those imposed by currently available microscope objectives. In addition to revealing the performance tradeoffs of various single- and dual-objective LSM configurations, our analysis showcases the potential advantages of a novel, non-orthogonal dual-objective (NODO) architecture, especially for moderate-resolution imaging applications (collection NA of 0.5 to 0.8). ### Competing Interest Statement JTCL and AKG are co-founders and shareholders of Lightspeed Microscopy Inc., of which JTCL is a board member. Technology developed by JTCL and AKG at the University of Washington has been licensed by Lightspeed Microscopy Inc.

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