Uniform volumetric single-cell processing for organ-scale molecular phenotyping

Extending single-cell analysis to intact tissues while maintaining organ-scale spatial information poses a major challenge due to unequal chemical processing of densely packed cells. Here we introduce Continuous Redispersion of Volumetric Equilibrium (CuRVE) in nanoporous matrices, a framework to address this challenge. CuRVE ensures uniform processing of all cells in organ-scale tissues by perpetually maintaining dynamic equilibrium of the tissue's gradually shifting chemical environment. The tissue chemical reaction environment changes at a continuous, slow rate, allowing redispersion of unevenly distributed chemicals and preserving chemical equilibrium tissue wide at any given moment. We implemented CuRVE to immunologically label whole mouse and rat brains and marmoset and human tissue blocks within 1 day. We discovered highly variable regionalized reduction of parvalbumin immunoreactive cells in wild-type adult mice, a phenotype missed by the commonly used genetic labeling. We envision that our platform will advance volumetric single-cell processing and analysis, facilitating comprehensive single-cell level investigations within their spatial context in organ-scale tissues.