Improve Workflow in Kidney Research with 3D STED Deep Nanoscopy

Combining deep nanoscopy and optical clearing, you can now image the ultrastructure of the glomerular filtration barrier from kidney tissue. The Leica TCS SP8 STED 3X (STimulated Emission Depletion) nanoscope sheds light on details deep inside the specimen with unprecedented three-dimensional detail. Using the STED WHITE glycerol objective, 3D nanoscopy in cleared tissue deeper than 150 µm sheds new insights in kidney disease.

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.

Deep nanoscopy 45-65 µm inside cleared adult kidney sample of a rat. 20 µm xyz stack confocal/3D STED. Nephrin visualized with STAR 635P. Clearing by modified CLARITY protocol. STED lens: HC PL APO 93X/1.30 GLYC motCORR – STED WHITE. Sample courtesy of David Unnersjö-Jess, KTH, Stockholm, Sweden.

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.

Nephrin in cleared kidney section. 3D STED at a depth 130-150 um into the tissue, using the STED WHITE 93x glycerol objective lens. Immunostaining performed with Atto594. Courtesy of David Unnersjö Jess, KTH, Stockholm, Sweden.

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.

Order protocol

Download Flyer "3D STED Deep Nanoscopy Workflow"

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.