Changes in kidney substructures reveal renal pathologies
The podocyte cell layer in the glomerular filtration barrier plays a central role in kidney disease. Alterations in the architecture of the podocytes are linked to the onset of renal failure. Therefore, the ability to image this layer at high resolution is key to identify healthy and unhealthy tissue.
The size of the podocyte layer requires imaging techniques with a resolution well below the diffraction limit. This has been typically addressed with electron microscopy techniques, demanding complex sample preparation protocols and where three dimensional information is missing.
3D STED Deep Nanoscopy opens a new door for kidney research. The combination of 3D STED Nanoscopy with the Leica TCS SP8 STED 3X and optical clearing enables imaging the kidney with unprecedented three-dimensional detail in light microscopy, and with protein specificity.
Visualize the ultrastructure of the glomerular filtration barrier by 3D STED Deep Nanoscopy
Even deeper than 150 µm within the tissue, the ultrastructural features of the specimen come to light powered by the new STED WHITE glycerol objective lens. This is achieved not only at 23°C, but also under live cell imaging conditions at 37°C with a comfortable working distance of 300 µm.
Forget the hassle of physical sectioning. Enjoy spectral freedom and keep up with the growing palette of fluorescent biomarkers.
Be at the forefront of clinical research.
3D STED Deep Nanoscopy Workflow
For Cleared Kidney Samples
Nanoscopy with the TCS SP8 STED system equipped with the HC PL APO 93X/1.30 GLYC motCORR – STED WHITE objective enables deep insights into the ultrastructure of healthy and diseased tissue.
1. Sample Preparation
The very first step is to dissect the kidney and stabilize its structure and biomolecular content with the help of a hydrogel. Therefore it is incubated at 4°C in an acrylamid-based gel to allow gel penetration into the tissue. For polymerization, the temperature is shifted to 37°C.
2. Sectioning
After stabilization in a hydrogel, the kidney is cut in sections of up to 500 µm thickness using a vibratome.
3. Clearing
Optical clearing helps to make thick biological samples transparent and therefore accessible to deep imaging. Moreover, clearing increases antibody penetration depth and staining quality. For optimal results clearing, the kidney sections are transferred into a clearing solution and incubated at 50°C for several days.
4. Fluorescence Immunolabeling
Standard immunolabelling protocols deliver excellent signal to noise ratio in cleared samples. The primary and secondary antibodies are incubated for a whole day to allow efficient penetration into the thick sample.
5. Mounting
Light scattering occurs due to the mismatch of refractive indeces (RI) in the penetrating media. Thus, in the mounting step the RI of the sample and the mounting medium have to be considered. The RI of fixed tissues is around 1.45, and water has an RI of 1.33, a Fructose solution matches the RI of fixed tissue. Therefore, samples are mounted in fructose to match the refractive index, allowing imaging with high penetration depth.
6. Nanoscopy
The samples are ready for imaging at the TCS SP8 STED 3X. Image the 3D ultrastructure of the glomerular filtration barrier at high resolution and at great depth. With the HC PL APO 93X/1.30 GLYC motCORR – STED WHITE Objective, nanoscopy more than 100 µm deep is possible.
7. Visualization
Display your 3D STED results with the LAS X 3D visualization tool – easy and straightforward.
Additional fields of research
- Neuroscience
- Pathology
- Renal diseases
- Glomerulopathies
- Clinical research
- Pathology research
- Pharmaceutical research
Further Reading
Blom et al., Chem. Rev., 2017. Stimulated Emission Depletion Microscopy.
Randles et al., Sci. Rep., 2016. Three-dimensional electron microscopy reveals the evolution of glomerular barrier injury.
Unnersjö-Jess et al., Kidney International, 2015: Super-resolution stimulated emission depletion imaging of slit diaphragm proteins in optically cleared kidney tissue
