Super-resolution stimulated emission depletion imaging of slit diaphragm proteins in optically cleared kidney tissue

Abstract

The glomerular filtration barrier, consisting of podocyte foot processes with bridging slit diaphragm, glomerular basement membrane, and endothelium, is a key component for renal function. Previously, the subtlest elements of the filtration barrier have only been visualized using electron microscopy. However, electron microscopy is mostly restricted to ultrathin two-dimensional samples, and the possibility to simultaneously visualize multiple different proteins is limited. Therefore, we sought to implement a super-resolution immunofluorescence microscopy protocol for the study of the filtration barrier in the kidney. Recently, several optical clearing methods have been developed making it possible to image through large volumes of tissue and even whole organs using light microscopy. Here we found that hydrogel-based optical clearing is a beneficial tool to study intact renal tissue at the nanometer scale. When imaging samples using super-resolution STED microscopy, the staining quality was critical in order to assess correct nanoscale information. The signal-to-noise ratio and immunosignal homogeneity were both improved in optically cleared tissue. Thus, STED of slit diaphragms in fluorescently labeled, optically cleared, intact kidney samples is a new tool for studying the glomerular filtration barrier in health and disease.

Visualization of podocyte substructure with structured illumination microscopy (SIM): a new approach to nephrotic disease

“…We have also tested other nephrotic diseases, and they also have podocin staining patterns that correspond to enlarged or effaced foot processes, although not as dramatically as minimal change disease does (data not shown). A recent study using STED to image optically cleared rat kidney tissue also showed similar changes using an experimental model of nephrotic disease, Heymann nephritis [7].…”

“…Adding an extra step to tag podocin for IF does not significantly increase the preparation time for samples. The optical clearing technique used in the recent STED imaging study of rat kidney [7] eliminates the step of tissue sectioning but adds a significant time delay (up to 2 weeks).…”

“…This is a very promising study, but is not yet directly applicable to routine diagnosis on standardly prepared kidney tissue samples as the technique requires specific fluorophores, additional sample preparations, and cannot capture the full 3D structure over a sufficiently large volume. Recently, stimulated emission depletion (STED) microscopy, typically capable of resolving structures on the order of 50 nm, has been utilized in conjunction with optical clearing protocols to visualize the structure of foot processes throughout whole, intact glomeruli [7]. While this technique yields detailed images of podocyte substructure, this is only achieved after a lengthy sample preparation protocol (over 2 weeks) [7] which is not useful for routine diagnosis.…”

“…Recently, stimulated emission depletion (STED) microscopy, typically capable of resolving structures on the order of 50 nm, has been utilized in conjunction with optical clearing protocols to visualize the structure of foot processes throughout whole, intact glomeruli [7]. While this technique yields detailed images of podocyte substructure, this is only achieved after a lengthy sample preparation protocol (over 2 weeks) [7] which is not useful for routine diagnosis.…”

New structural insights into podocyte biology

“…Helium ion SEM technology has the power to provide superb new insights into the topographical ultrastructure of the glomerulum (Rice et al 2013). Lastly, an improvement of the available clearance techniques might enhance our possibilities to localize proteins in health and disease when combined with high- or super-resolution microscopy (Richardson and Lichtman 2015; Unnersjo-Jess et al 2016). …”