New Views of the Glomerulus: Advanced Microscopy for Advanced Diagnosis.

New technologies are ready to revolutionize glomerular imaging and significantly improve or replace immunofluorescence and electron microscopy, which have driven research and diagnosis of glomerular diseases for over 50 years. Advanced forms of transmission and scanning electron microscopy have revealed the detailed spatial relationships of the glomerular basement membrane, podocytes, and endothelial cells. These may be overshadowed by super resolution microscopy (SRM), which combines the advantages of immunofluorescence and electron microscopy, offers high resolution identification of specific molecules, and images large, physiologically relevant volumes of the glomerulus. Rapidity, ease of use and low cost with some types of SRM make them potentially suitable for routine diagnosis. SRM visualizes structures below the classical diffraction limit of conventional light microscopy by adding a time variable to either the illumination of the specimen, or to the fluorescence signal emitted by it. Ensemble techniques vary illumination and include Structured Illumination Microscopy (SIM) and Stimulation Emission Depletion Microscopy (STED). Single molecule localization techniques vary the light emission by fluorescence labels in the specimen, and include Photoactivated Localization Microscopy (PALM) and Stochastic Optical Reconstruction Microscopy (STORM). Technologies such as expansion microscopy and genetic labeling can also create effective super resolution imaging by non-optical, specialized preparation techniques. All technologies require dark field fluorescence and some require computer image analysis and reconstruction. Replicating successful application in other areas of biology, SIM, STED, and STORM have visualized normal and nephrotic disease podocytes, and have confirmed their appearances to be similar to those seen by electron microscopy, but with added new information on cell configuration and protein localization. STORM has also localized podocyte cytoskeleton and adhesion proteins, and glomerular basement membrane proteins at a resolution never before possible. These pioneering efforts show the promise of super resolution microscopy, and lay the groundwork for future study and new diagnostic tools for glomerular diseases.