Stain-free three-dimensional organ imaging by serial optical coherence microscopy and tissue clearing: toward comprehensive histopathology

Abstract Histopathology is advancing rapidly with technologies such as slide scanners and deep learning that improve diagnostic efficiency and enable digital workflows. However, conventional methods rely on stained thin tissue sections, limiting comprehensive access to three-dimensional (3D) pathological information. Here, we introduce a histological approach integrating optical coherence microscopy (OCM) with tissue clearing and serial sectioning, eliminating the need for staining. We optimized the processing conditions of OptiMuS for OCM to accelerate clearing while preserving structural backscattering contrast. Applying serial OCM (SOCM), we visualized and quantified key anatomical features, including 3D vascular networks, in five optically cleared organs from eight-week-old male C57BL/6 mice. This approach supports large-scale data acquisition and statistical analysis across whole mouse organs. By delivering quantitative, spatially resolved data, it provides new insights and enhances the scalability and objectivity of histopathological evaluation beyond the capabilities of conventional digital pathology.