ADAPT-3D:accelerated deep adaptable processing of tissue for 3-dimensional fluorescence tissue imaging for research and clinical settings

Abstract

Light sheet microscopy and preparative clearing methods that improve light penetration in 3D tissues have revolutionized imaging in biomedical research. Here we present ADAPT-3D, a streamlined 3-step approach to turn tissues optically transparent while preserving tissue architecture with the versatility to handle diverse tissue sizes and types across species. Unlike extensive lipid removal utilized by existing protocols, ADAPT-3D only partially removes lipids to preserve cell membranes, yet the non-toxic aqueous refractive indexing solution still rapidly turns tissues transparent while preserving the fluorescence of endogenous and antibody conjugated fluorophores. ADAPT-3D prepares whole mouse brains for light sheet microscopy in a 4-hour refractive indexing step after less than 4 days of preprocessing without changing their size. By maintaining tissue size, ADAPT-3D clears 1-mm thick brain slices in under 24 h without causing damage and facilitates a 3D section-like view of the meandering choroid plexus. We applied ADAPT-3D to overcome challenges of whole mouse skull clearing and visualized the undisturbed brain borders including specialized skull channels after just 8 days of tissue preparation. ADAPT-3D also had utility in clearing and immunolabeling human intestinal tissues in about 5 days. Overall, ADAPT-3D provides a high-speed, non-shrinking, and fluorescence-preserving workflow for 3D imaging that bridges section-based and whole-organ studies, offering new opportunities for biological discovery.