Three-Dimensional Super-Resolved Imaging of Paraffin-Embedded Kidney Samples

Abstract

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