Background Diseases of the glomeruli, the renalfiltration units, are a leading cause of progressive kidney disease. Assessment of the ultrastructure of podocytes at the glomerularfiltration barrier is essential for diagnosing diverse disease entities, providing insight into the disease pathogenesis, and monitoring treatment responses.& nbsp;Methods Here we apply previously published sample preparation methods together with stimulated emission depletion and confocal microscopy for resolving nanoscale podocyte substructure. The protocols are modified and optimized in order to be applied to formalin-fixed paraffin-embedded (FFPE) samples.& nbsp;Results We successfully modified our protocols to allow for deep three-dimensional stimulated emission depletion and confocal imaging of FFPE kidney tissue with similar staining and image quality compared with our previous approaches. We further show that quantitative analysis can be applied to extract morphometrics from healthy and diseased samples from both mice and humans.& nbsp;Conclusions The results from this study could increase the feasibility of implementing optical kidney imaging protocols in clinical routines because FFPE is the gold-standard method for storage of patient samples.& nbsp;