Casper Hempel1,2,3, Sergey Kapishnikov4,5, Ana Joaquina Perez-Berna6, Stephan Werner4, Peter Guttmann4, Eva Pereiro6, Klaus Qvortrup7, Thomas Lars Andresen1. 1. Department of Health Technology, Technical University of Denmark, Lyngby, Denmark. 2. Centre for Medical Parasitology, Department for Clinical Microbiology, Copenhagen University Hospital, Copenhagen, Denmark. 3. Department for Immunology and Microbiology, University of Copenhagen, Copenhagen, Denmark. 4. Department X-Ray microscopy, Helmholtz-Zentrum Berlin für Materialien und Energie GmbH, Berlin, Germany. 5. Niels Bohr Institute, University of Copenhagen, Copenhagen, Denmark. 6. MISTRAL Beamline-Experiments Division, ALBA Synchrotron Light Source, Barcelona, Spain. 7. Core Facility for Integrated Microscopy (CFIM), University of Copenhagen, Copenhagen, Denmark.
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.
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.
Authors: Andrew B Haymet; Nicole Bartnikowski; Emily S Wood; Michael P Vallely; Angela McBride; Sophie Yacoub; Scott B Biering; Eva Harris; Jacky Y Suen; John F Fraser Journal: Front Cardiovasc Med Date: 2021-04-14
Authors: Laurence Chevalier; Jean Selim; Celia Castro; Fabien Cuvilly; Jean-Marc Baste; Vincent Richard; Philippe Pareige; Jeremy Bellien Journal: Front Cardiovasc Med Date: 2022-03-09