Tissue Processing of Mammalian Vascular Endothelium for Scanning or Scanning-Transmission Electron Microscopy

Abstract

Samples of mammalian vascular endothelium,processed for surface scanning electron microscopy in the secondary electron emission mode or for transmission-scanning electron microscopy, were found to be susceptible to considerable artifacts during specimen preparation resulting in severe distortions in shape, size and spatial relationships.Of all methods tested, freeze-drying or freeze substitution induced most severe artifactual problems due to differential shrinkage rates of underlying elastic layers. While critical point drying with carbon dioxide or Freon 13 proved to be consistently superior to the above methods, significant cellular lipid losses during processing were unavoidable. Use of nitrous oxide as suggested by Koller and Bernhard reduced delipidation but was found to give inconsistent results.As an alternative, the following processing technique was developed and found to reduce morphological artifacts to a minimum while still preserving surface lipids and intercellular relationships.