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

Casper Hempel*, Sergey Kapishnikov, Ana Joaquina Perez-Berna, Stephan Werner, Peter Guttmann, Eva Pereiro, Klaus Qvortrup, Thomas Lars Andresen

*Corresponding author for this work

Research output: Contribution to journalJournal articleResearchpeer-review

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.
Original languageEnglish
Article numbere12643
JournalMicrocirculation
Volume27
Issue number7
Number of pages11
ISSN1073-9688
DOIs
Publication statusPublished - 2020

Keywords

  • Electron microscopy
  • Endothelial glycocalyx
  • High-pressure freezing
  • Soft x-ray imaging

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