An Extended DIY Light-sheet Microscope Enabling Four-color Volumetric Imaging of Whole Biological Samples

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Abstract
Objectives A combination of tissue clearing technology and light-sheet fluorescence microscopy enables volumetric analysis of intact biological specimens. To analyze complex spatial organizations of molecules and cells, the number of imaging channels available for simultaneous visualization from a single sample is a key parameter. This study aimed to expand the previously reported DIY compact light-sheet microscope, descSPIM, for four-color volumetric imaging of whole biological samples.Materials A four-color-labeled optical phantom and an entire E12.5 mouse embryo labeled with a nuclear stain and three distinct antibodies were used.Methods We modified the previously reported DIY compact light-sheet microscope, descSPIM, for four-color volumetric imaging. Chromatic aberration was evaluated across the visible wavelength range (400-700 nm) using the four-color-labeled optical phantom. Four-color three-dimensional imaging was then performed on the entire E12.5 mouse embryo.Results The chromatic aberration of the extended descSPIM was mostly negligible across the visible wavelength range (400-700 nm). The extended system also enabled four-color three-dimensional imaging covering the entire E12.5 mouse embryo.Conclusions Together, these results validate the reliability and expandability of the extended system, supporting its broader use for multicolor imaging of cleared tissue samples.

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