ADAPT-3D: advances in tissue preparation and imaging combined with depth-variant deconvolution – faster, bigger, brighter 4121

Abstract Description Preparative clearing methods enhance light penetration in 3D tissues, revolutionizing research. Here, we develop 2 accelerable complementary approaches across sample and light modalities for applications where cost, speed, and ease-of-use are of the essence. First, we developed a fast-acting refractive-index matching solution, and accompanying protocol, called ADAPT-3D, applicable across species and tissues for immunolabeling or endogenous fluorophore detection, offering speed, sensitivity, and morphology preservation. In the intact mouse skull and brain, the method allowed the specialized channels between skull and underlying tissue to be detected without meningeal tearing. Second, initially successful with confocal and light sheet modalities, it opened new imaging possibilities. Though often overlooked, after optimization, widefield microscopy with advanced deconvolution provided high-quality, large-volume imaging quickly. Pairing depth-variant point spread function deconvolution with a motorized z-acquisition widefield microscope enabled robust deconvolution to 0.5-mm depth in cleared tissues, surpassing previous reports for widefield systems. ADAPT-3D’s rapid clearing can evaluate human donor kidneys for transplant, where timely 3D data is vital. Widefield microscopy with depth-variant deconvolution uniquely suits 3D imaging when speed, accessibility, or cost is limiting. ADAPT-3D and depth-variant deconvolution are ideal for large-volume tissue imaging in immunology. Funding Sources NIH Fellowship to DDL (T32 HL007081) and grant to GJR (P01 AG078106, DP1 K130660, R01 AI168044, R01 DK119147, U01 AI163064) Topic Categories Technological Innovations in Immunology (TECH)