Volumetric fluorescence microscopy-based quantitative comparison of murine tissue clearing using CUBIC protocols

Abstract A wide variety of protocols have been proposed for optical clearing of tissues, whole-mount organs, and other bulky specimens to enable their volumetric fluorescence imaging. However, quantitative comparisons of tissue clearing protocols that take into account the fluorescence of the final specimens remain rare. Here, we propose a volumetric fluorescence image–based workflow for evaluating tissue clearing and fluorescence staining protocols. The workflow calculates depth-dependent fluorescence attenuation coefficients using data from entire 3D images, thereby avoiding spatial sampling bias and eliminating reliance on simple readouts, such as light transmittance, to predict fluorescence image quality. By combining autofluorescence signal with the signal from a specific fluorescence label, we independently evaluated transparency and the quality of fluorescence staining in cleared specimens. Using the proposed workflow, we systematically compared clearing and staining performance of three CUBIC-based protocols in murine liver, kidney, spleen, thymus, and intestine, and revealed differences in final fluorescence image quality across protocol–organ combinations.