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are co-founders and shareholders of Lightspeed Microscopy Inc. A. Madabhushi is an equity holder in Elucid Bioimaging and in Inspirata Inc. In addition, he has served as a scientific advisory board member for Inspirata Inc, Astrazeneca, Bristol Meyers-Squibb and Merck. Currently, A. Madabhushi serves on the advisory board of Aiforia Inc. He also has sponsored research agreements with Philips, AstraZeneca, Boehringer-Ingelheim and Bristol Meyers-Squibb. A. Madabhushi's technology has been licensed to Elucid Bioimaging. He is also involved in a NIH U24 grant with PathCore Inc, and 3 different R01 grants with Inspirata Inc.Research.

Light-sheet microscopy has emerged as the preferred means for high-throughput volumetric imaging of cleared tissues. However, there is a need for a user-friendly system that can address imaging applications with varied requirements in terms of resolution (mesoscopic to sub-micrometer), sample geometry (size, shape, and number), and compatibility with tissue-clearing protocols and sample holders of various refractive indices. We present a 'hybrid' system that combines a novel non-orthogonal dual-objective and conventional (orthogonal) open-top light-sheet architecture for versatile multi-scale volumetric imaging. Main TextRecent advances in tissue-clearing protocols greatly reduce optical scattering, aberrations, and background uorescence, enabling deep-tissue imaging with high resolution and contrast. These approaches have yielded new insights in many elds, including neuroscience, developmental biology, and anatomic pathology [1][2][3][4][5][6][7][8][9][10][11]. Light-sheet microscopy has emerged as a preferred means for highresolution volumetric imaging of cleared tissues due to its unrivaled speed and low photobleaching [12,13]. Many variants of light-sheet microscopes have been developed in recent years by academic researchers and commercial entities to tackle a diverse range of imaging applications (Error! Reference source not found. and Error! Reference source not found.) [14][15][16][17][18]. Whereas individual light-sheet systems are well-suited for a subset of cleared-tissue applications, trade-offs are inevitable. In particular, no current light-sheet microscope can satisfy all of the following requirements: (1) user-friendly mounting

Slide-free digital pathology techniques, including nondestructive 3D microscopy, are gaining interest as alternatives to traditional slide-based histology. In order to facilitate clinical adoption of these fluorescence-based techniques, software methods have been developed to convert grayscale fluorescence images into color images that mimic the appearance of standard absorptive chromogens such as hematoxylin and eosin (H&E). However, these falsecoloring algorithms often require manual and iterative adjustment of parameters, with results that can be inconsistent in the presence of intensity nonuniformities within an image and/or between specimens (intra-and inter-specimen variability). Here, we present an open-source (Python-based) rapid intensity-leveling and digital-staining package that is specifically designed to render two-channel fluorescence images (i.e. a fluorescent analog of H&E) to the traditional H&E color space for 2D and 3D microscopy datasets. However, this method can be easily tailored for other false-coloring needs. Our package offers (1) automated and uniform false coloring in spite of uneven staining within a large thick specimen, (2) consistent color-space representations that are robust to variations in staining and imaging conditions between different specimens, and (3) GPU-accelerated data processing to allow these methods to scale to large datasets. We demonstrate this platform by generating H&E-like images from cleared tissues that are fluorescently imaged in 3D with open-top light-sheet (OTLS) microscopy, and quantitatively characterizing the results in comparison to traditional slide-based H&E histology.