<p>Innovations in 3D tissue imaging have revolutionized research, but limitations stemming from lengthy protocols and equipment accessibility persist. Widefield microscopy is fast and accessible but often excluded from 3D imaging workflows due to its lack of optical sectioning. Here we combine tissue clearing with a commercial depth-variant deconvolution approach that we optimized for large-volume widefield imaging. By implementing prefiltering with z-brick splitting, we achieve subnuclear axial resolution in tissues to a depth of 500 µm in multi-tile scan images. We illustrate the utility of this method in a model of ileitis and to gain a 3D perspective in thick brain slices from a model of cerebral amyloid angiopathy, where we resolve amyloid deposits along small blood vessels, attaining resolution that compared favorably to confocal microscopy. Finally, we leverage our approach to image hundreds of consecutive z planes for richer evaluation of cleared human kidney biopsies in a simulated, transplant time window and visualized atrophic tubules and winding arterioles associated with glomeruli in 3D. Having achieved subnuclear z-resolution in sections hundreds of microns thick, coupling widefield microscopy of cleared tissue to robust deconvolution now emerges as an accessible and viable method to gain 3D insight in research or clinical evaluations.</p>