Abstract: SA-OR007
Confocal Imaging of the Glomerular Filtration Barrier in Expanded Kidney Tissue Samples
Session Information
- A View on the Glomerulus
November 04, 2017 | Location: Room 294, Morial Convention Center
Abstract Time: 05:42 PM - 05:54 PM
Category: Glomerular
- 1002 Glomerular: Basic/Experimental Pathology
Authors
- Unnersjö-Jess, David, Royal Institute of Technology, Solna, Sweden
- Scott, Lena, Karolinska Institutet, Solna, Sweden
- Zambrano Sevilla, Sonia, Karolinska Institutet, Solna, Sweden
- Patrakka, Jaakko, Karolinska Institutet, Solna, Sweden
- Blom, Hans, Royal Institute of Technology, Solna, Sweden
- Brismar, Hjalmar, Royal Institute of Technology, Solna, Sweden
Background
When studying the glomerular filtration barrier (GFB), one is normally restricted to electron microscopy (EM). However, using EM, it is not trivial to perform volumetric imaging and multiple labelling of different epitopes. We have recently demonstrated an optical clearing protocol that in combination with super-resolution STED microscopy enables epitope-specific nanoscale imaging of the GFB. STED microscopy is presently an expensive and complex technique, and thus it would be highly relevant to develop more conventional bioimaging methods which resolve the GFB.
Methods
We apply a sample preparation protocol which isotropically expands kidney tissue samples approximately 5 times, while making them optically transparent. We then use immunofluorescence and confocal microscopy to image components of the GFB.
Results
Kidney samples were sufficiently expanded to allow for nanoscale localizations of different parts of the GFB (e.g. glomerular basement membrane, podocyte foot processes and the slit diaphragm), at an effective resolution below 70 nm. 2-color z-stacks of glomeruli were acquired, giving the unique possibility to study the 3D localizations of GFB proteins in intact tissue samples, including the detection of foot process effacement in mice with induced glomerulonephritis. Aditionally, by applying super-resolution STED microscopy on expanded kidney samples, a resolution below 20 nm could be obtained.
Conclusion
We show that an expansion protocol in combination with confocal microscopy can be used to perform sub-70 nm resolution imaging of the GFB using standard lab equipment and reagents. This finding has an impact for researchers and clinical pathologists, since conventional light microscopy can for the first time be used to study kidney fine-morphology and protein topology on the effective nanometer-scale.
Expanded kidney sample stained for podocin (magenta) and cytosolic podocyte-specific tdTomato (pod-cre-tdTomato, green), showing podocyte foot processes and the filtration slit. The sample was imaged using confocal microscopy.