Date of Award
Open Access Thesis
Medical Doctor (MD)
Virtually all electron density observable in membranous elements in thin sections of buffered OsO4 -fixed tissue with electron microscopy was removed by the action of K4Fe(CN)6 in acetic acid. This reagent reacted only with Os(VIII) and not with lower oxidation states of osmium to give an insoluble crystalline precipitate, K2Fe0s(CN)6. The osmate esters originally synthesized by Criegree (8,9) were unreactive with acetic ferrocyanide. Acetic acid alone decreased membrane density of fixed sections but did not remove osmium from osmate esters.
Further, fresh tissue incubated in a saturated urea solution, then fixed in buffered OsO4 not only failed to blacken, as routinely observed after OsO4 fixation, but also tissue structure was poorly preserved and membranous structures were almost completely electron lucent. Tissue fixed in a 1:1 solution of urea and OsO4 also demonstrated competition between the two compounds for binding sites in membrane.
Thus, osmium in the Os(VIII) state appears responsible for most of the membrane density, and is probably hydrogen bound in its native state as OsO4 to protein and to aliphatic side chains of lipid in membrane. Theoretical calculations pertaining to molecular interactions of OsO4, urea, and model proteins and lipids indicate that the above mechanistic hypothesis of the interaction of OsO4 with tissue components is favored over the mechanism of osmate ester formation with the double bonds in lipid as has been believed to date.
Litman, Robert Barry, "The Mechanism of the Fixation of Tissue Components by Osmium Tetroxide" (1970). Yale Medicine Thesis Digital Library. 3557.
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