Imaging Drosophila brain by combining cryo-soft X-ray microscopy of thick vitreous sections and cryo-electron microscopy of ultrathin vitreous sections

Leforestier, Amélie; Levitz, Pierre; Préat, Thomas; Michot, Laurent J.; Tchénio, P.

doi:10.1016/j.jsb.2014.09.012

Cited by 5 publications

(1 citation statement)

References 35 publications

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“…All cryogenic fixation was performed by high‐pressure freezing (HPF). HPF enables full vitrification within milliseconds of biological specimens that have a higher thickness as compared to what is seen for plunge freezing 30 . Cooling rates for the reported specimens exceeded 15 000 K/s.…”

Section: Methodsmentioning

confidence: 92%

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

et al. 2020

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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.

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Section: Methodsmentioning

confidence: 92%