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

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, and monitoring treatment responses.
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.
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.
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.
Competing Interests: T. Benzing reports advisory activity for Otsuka in the field of cystic kidney disease and hyponatremia; support for ADPKD registry by Otsuka; speaker honoraria and travel support from Amgen, Hexal, Novartis, Otsuka, Roche, and Sanofi-Genzyme. He is also on the editorial board of the Journal of the American Society of Nephrology, Nephrology Dialysis Transplantation, and Science Signaling. H. Blom reports other interests/relationships with MedTechLabs, BioClinicum, Karolinska University Hospital, Solna, Sweden. P. Hoyer reports consultancy agreements with Boehringer Ingelheim and is a scientific advisor for Archives of Disease in Childhood. F. Koehler reports other interests/relationships with Else Kröner-FreseniusStiftung, German Research Foundation under Germany’s Excellence Strategy EXC 2030: CECAD—Excellent in Aging Research and Koeln Fortune program/Faculty of Medicine, University of Cologne, Germany. R. Mueller reports consultancy agreements with Alnylam and Sanofi; research funding from Otsuka Pharmaceuticals and Thermo Fisher Scientific, which was paid to the employer (Department II of Internal Medicine); and honoraria from Alnylam and Sanofi. He is also a board member of the WGIKD (ERA-EDTA), Scientific Advisory Board Santa Barbara Nutrients, Editorial Board “Kidney and Dialysis.” All remaining authors have nothing to disclose.
(Copyright © 2022 by the American Society of Nephrology.)