Backscattered electron imaging to visualize arterial endothelial detachment in the scanning electron microscope

The endothelium of the central ear artery of an anesthetized rabbit was damaged by placing artery forceps on the ear over the vessel for 30 min. After removal of the forceps, blood was allowed to flow through the injured vessel for 30 min. Blood flow was then stopped, blood was washed out of the artery in situ and initial fixation with 1% glutaraldehyde was begun. Specimens of the vessel containing the damaged region were postfixed in 3% cacodylate buffered glutaraldehyde and prepared for SEM examination after methenamine silver staining or following post fixation with osmium. Backscattered electron imaging (BEI) of silver stained specimens with inverted signal polarity showed "black" endothelial nuclei similar to darkly stained nuclei of conventional light microscope slide preparations. Secondary electron imaging (SEI) of corresponding sites stained with silver also showed endothelial nuclei. An interruption of the normal pattern of nuclei indicated a detached area of endothelium where subendothelium was exposed. Platelets were observed adhering to the surface of the exposed subendothelium. Injury to the endothelium was seen more readily in silver stained preparations as voids in the nuclear pattern as compared with conventional SEI preparations, postfixed with osmium, which did not show well defined nuclei.