The need to freeze—Dehydration during specimen preparation for electron microscopy collapses the endothelial glycocalyx regardless of fixation method
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The need to freeze—Dehydration during specimen preparation for electron microscopy collapses the endothelial glycocalyx regardless of fixation method. / Hempel, Casper; Kapishnikov, Sergey; Perez-Berna, Ana Joaquina; Werner, Stephan; Guttmann, Peter; Pereiro, Eva; Qvortrup, Klaus; Andresen, Thomas Lars.
I: Microcirculation, Bind 27, Nr. 7, e12643, 2020.Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › fagfællebedømt
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TY - JOUR
T1 - The need to freeze—Dehydration during specimen preparation for electron microscopy collapses the endothelial glycocalyx regardless of fixation method
AU - Hempel, Casper
AU - Kapishnikov, Sergey
AU - Perez-Berna, Ana Joaquina
AU - Werner, Stephan
AU - Guttmann, Peter
AU - Pereiro, Eva
AU - Qvortrup, Klaus
AU - Andresen, Thomas Lars
PY - 2020
Y1 - 2020
N2 - 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.
AB - 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.
KW - electron microscopy
KW - endothelial glycocalyx
KW - high-pressure freezing
KW - soft x-ray imaging
U2 - 10.1111/micc.12643
DO - 10.1111/micc.12643
M3 - Journal article
C2 - 32542908
AN - SCOPUS:85088437904
VL - 27
JO - Microcirculation
JF - Microcirculation
SN - 1073-9688
IS - 7
M1 - e12643
ER -
ID: 249772461