Contact & Support

3D STED Deep Nanoscopy

Visualize details at the nanoscale in cleared tissue with optimal fluorescence labeling. Investigate key players in kidney disease. Improve your 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.

Top image: Cleared kidney sample, revealing the podocyte cell layer; magnified podocytes by STED and electron microscopy (EM). Cleared kidney sample courtesy: David Unnersjö-Jess, KTH, Stockholm, Sweden. Scanning EM image of kidney podocytes courtesy: Center for Microscopy and Image Analysis, Univ. of Zurich, Dr. Urs Ziegler. Scale bar is 1 µm.

Contact us for STED imaging solutions!

Please contact us for personal expert advice on our microscopy solutions for STED imaging.

Related products 2

Filter by Area of Application
Leica motCORR Microscope Objective

Leica motCORR Objectives

Fast adjustment to varying RI for best optical performance in 3D

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.

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. 

7. Visualization

Display your 3D STED results with the LAS X 3D visualization tool – easy and straightforward.

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

Additional fields of research

  • Neuroscience
  • Pathology
  • Renal diseases
  • Glomerulopathies
  • Clinical research
  • Pathology research
  • Pharmaceutical research

News & Related articles

Image Gallery: THUNDER Imager

To help you answer important scientific questions, THUNDER Imagers eliminate the out-of-focus blur that clouds the view of thick samples when using…

Leica Science Lab

Where to go? Cellular Migration requires coordinated Transitions of Actin Cortex

Plants, Bacteria, and Fungi possess a rigid cell wall that protects the cell and gives it shape. Animal cells, such as mammalian cells, have no outer…

Leica Science Lab
Cover glomerulus

The Guide to STED Sample Preparation

This guide is intended to help users optimize sample preparation for stimulated emission depletion (STED) nanoscopy, specifically when using the TCS…

Leica Science Lab

How can PAULA help streamline the workflow for my wound healing assay?

The wound healing assay, often used as a synonym for scratch assay or migration assay, is an important tool for the evaluation and measurement of cell…

Leica Science Lab

THUNDER Technology Note

So far, widefield microscopy was not suitable to image larger volumes, since the contrast of the recorded fluorescence image is reduced by the…

Leica Science Lab
MDCK cells, Phase Contrast, red outlines are marking the cell borders. Courtesy of Prof. Ralf Jacob, University of Marburg, Germany

Confluency Check with PAULA Cell Imager

Many cell-based experiments require cells in a certain state. This can include their morphology,  fluorescent protein expression, and the confluency…

Leica Science Lab

Extending Nanoscopy Possibilities with STED and exchangeable fluorophores

When it comes to STED Nanoscopy, keeping high signal-to-noise is key to achieve the best possible resolution in fixed and living cells. This can be…

Leica Science Lab

Webinar: See More Than Just Your Image

Despite the emergence of new imaging methods in recent years, true 3D resolution is still achieved by Confocal Laser Scanning Microscopy (CLSM).…

Leica Science Lab

Dynamic Endoplasmic Reticulum

The Endoplasmic Reticulum (ER) is a dynamic, reticular or net-like structure consisting of tubules and cisternae that extend throughout the cell and…

Leica Science Lab

Simultaneously Measuring Image Features and Resolution in Live-Cell STED Images

Reliable interpretation and quantification of cellular features in fluorescence microscopy requires an accurate estimate of microscope resolution.…

Leica Science Lab
SP8 LIGHTNING confocal microscope

Next generation confocal platform for super-resolution live cell imaging in multicolor

Detailed observations of fast biological processes with the SP8 LIGHTNING

White Blood Cell Recruitment to Bacterial Infection

Zebrafish In Vivo Study Using Leica Fluorescence Microscopes

Tile scan image of Mouse brain tissue section with multicolor immunofluorescent labeling

Find the Missing Links in Advanced Live Cell Research - Leica launches DMi8 S live cell imaging solution

Wetzlar, Germany – Leica introduces the DMi8 S, a new complete solution designed to give researchers the ultimate tool for fast, versatile microscopic...

Full Spectral Freedom for Multi-color Deep Tissue In Vivo Imaging

Leica Microsystems launches SP8 DIVE – The World's First Tunable Deep Imaging Solution

Explore life’s true nature with super-resolution and nanoscopy

With HyVolution 2 and the Leica TCS SP8 STED ONE, Leica Microsystems paves the way to access imaging beyond the diffraction limit

GSD Around the World – A Travel Diary on Facebook

Tamara Straube and Peter Laskey will take you on a journey around the world: Together with a team of specialists, they visited 10 countries, organized...

EMBL Advanced Course – Intensive Super-Resolution Microscopy Course for Localization Microscopy and STED

From 20th to 25th July 2015 the EMBL (European Molecular Biology Laboratory), Heidelberg will be holding a six-day intensive course on the subject of...

New Leica TCS SP8 at Bates College: „Second of its kind in Maine, new microscope is a game-changer“

According to Bates College, the confocal offers new imaging capabilities and supports research in biology, neuroscience, nanotechnology and...

Free Infographic: Which Super-Resolution Microscopy Method is Right for You?

This illustrated guide to super-resolution microscopy helps you compare technologies to best fulfill your individual research needs.

The Leica TCS SP8 STED 3X was chosen among The Scientist Top 10 Innovations 2014

The Scientist Top 10 Innovations Award for Leica TCS SP8 STED 3X

Leica Microsystems’ STED Super-Resolution Microscope Again Awarded for its Innovative Technology