The need to freeze—Dehydration during specimen preparation for electron microscopy collapses the endothelial glycocalyx regardless of fixation method

Abstract

Objective The endothelial glycocalyx covers the luminal surface of the endothelium and plays key roles in vascular function. Despite its biological importance, ideal visualization techniques are lacking. The current study aimed to improve the preservation and subsequent imaging quality of the endothelial glycocalyx. Methods In mice, the endothelial glycocalyx was contrasted with a mixture of lanthanum and dysprosium (LaDy). Standard chemical fixation was compared with high‐pressure frozen specimens processed with freeze substitution. Also, isolated brain microvessels and cultured endothelial cells were high‐pressure frozen and by transmission soft x‐rays, imaged under cryogenic conditions. Results The endothelial glycocalyx was in some tissues significantly more voluminous from chemically fixed specimens compared with high‐pressure frozen specimens. LaDy labeling introduced excessive absorption contrast, which impeded glycocalyx measurements in isolated brain microvessels when using transmission soft x‐rays. In non‐contrasted vessels, the glycocalyx was not resolved. LaDy‐contrasted, cultured brain endothelial cells allowed to assess glycocalyx volume in vitro. Conclusions Both chemical and cryogenic fixation followed by dehydration lead to substantial collapse of the glycocalyx. Cryogenic fixation without freeze substitution could be a way forward although transmission soft x‐ray tomography based solely on amplitude contrast seems unsuitable.

The endothelial glycocalyx in critical illness: A pediatric perspective

“…The microscopic, fragile nature of the EG makes this structure challenging to directly image and quantify [219] . Special techniques are used to attempt tissue EG preservation for electron microscopy [220] but may not consistently prepare the endothelium for high quality imaging [221] . Others have studied tissue-level EG expression using immunohistochemistry and plasma measures of glycocalyx components to characterize EG injury in both humans and animal models.…”

Combined Electron Microscopy Approaches for Arterial Glycocalyx Visualization

“…Soluble components such as proteins, proteoglycans, or other molecules originating from the endothelium or the bloodstream described to preserve the charge selectivity of the permeability barrier, may contribute to the structural organization of the glycocalyx ( 1 ) by maintaining electrostatic interactions or crosslinking. Recently, Hempel et al ( 24 ) reported the collapse of glycocalyx during the dehydration step, whatever the fixation method. Aware of these limitations, we outline the importance to acquire multiscale images.…”

Metabolic Glycoengineering Enables the Ultrastructural Visualization of Sialic Acids in the Glycocalyx of the Alveolar Epithelial Cell Line hAELVi

“…As described in the introduction, there is ongoing debate in the field, how the type of fixation, subsequent dehydration steps and chemical labeling reagents affects the preservation of the glycocalyx. Recently, it was hypothesized that dehydration rather than fixing is critically in terms of volume collapse of the glycocalyx as chemical fixation/dehydration versus cryofixation/freeze-substitution (which includes dehydration) resulted in comparable heights within lung- and heart vessels that were perfused with lanthanum and dysprosium (LaDy) for chemical labeling of the glycocalyx ( Hempel et al, 2020 ). In this study, the authors then tried visualizing the glycocalyx of vessels by combining cryofixation and low-dose x-ray tomography (SXT), a methodology requiring a synchrotron but no dehydration step.…”