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

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.