Visualization of the glomerular endothelial glycocalyx by electron microscopy using cationic colloidal thorium...

Area of Science:

• Biomedical Engineering
• Microscopy Techniques
• Cell Biology

Background:

• Biological materials exhibit low contrast in electron microscopy (EM) due to their light element composition.
• Standard EM stains like osmium tetroxide and uranyl acetate poorly contrast extracellular polymeric substances (EPS), including the endothelial glycocalyx.
• The endothelial glycocalyx, primarily proteoglycans, requires specialized heavy metal staining for EM visualization.

Purpose of the Study:

• To introduce and evaluate cationic hydrous thorium dioxide colloids (cThO2) as a novel staining agent for visualizing the endothelial glycocalyx.
• To assess the efficacy of cThO2 in enhancing contrast and enabling fine structural analysis of the glomerular endothelial glycocalyx in mouse kidneys.

Main Methods:

• Developed a method using cationic hydrous thorium dioxide colloids (cThO2) for staining.
• Applied combined perfusion of fixative and stain, including cThO2, to mouse kidneys.
• Utilized electron microscopy to image the stained glomerular endothelial glycocalyx.

Main Results:

• cThO2 demonstrated high electron density, binding to a continuous layer on the glomerular endothelium.
• The stained glycocalyx formed periodic densities with spacing from 50 to 200 nm.
• Layer thickness varied from below 50 nm up to 300 nm, allowing distinct imaging and analysis.

Conclusions:

• cThO2 staining provides excellent contrast for the endothelial glycocalyx in EM.
• This method enables precise fine structural analysis of the endothelial glycocalyx in perfused mouse kidneys.
• cThO2 is a valuable tool for studying the morphology and distribution of the endothelial glycocalyx.