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Novel tissue clearing method for preservation of morphology, fluorescence and epitope integrity permits quantitative cellular analysis in intact organs

Spatial organization of cells within tissues is critical for most biological processes, such as cellular differentiation, signal relay, and localized function. Our understanding of such events is historically based on section imaging, which discards 3-dimensional (3D) information and is problematic for understanding structure-function relationships of complex tissues and rare events. Although recent innovations in tissue clearing methods have paved way for whole organ microscopy, they suffer from various technique-specific artifacts, such as fluorophore quenching, poor epitope preservation, or morphological distortions. Here we report a new clearing method, Clearing-enhanced 3D (C e 3D), which generates excellent tissue transparency, preserves cellular morphology and protein fluorescence, and retains epitopes for antibody-based labeling. We use C e 3D to demonstrate whole-tissue microscopy of various cell types in lymphoid and non-lymphoid organs. Further, when coupled with highly multiplexed antibody labeling and Histo-Cytometry, C e 3D permits quantitative analysis of phenotypically complex cells in intact tissues. Collectively, this methodology provides a comprehensive strategy for whole-organ quantitative imaging and analysis, paving the way for improved understanding of cellular organization and physiology. This research was supported by the Intramural Research Program of the NIH, NIAID.