Chemical aspects of glycogen contrast-staining by potassium osmate

To obtain contrast-staining of glycogen in electron microscopy, various contrast-enhancing additives can be used in combination with potassium osmate. Examples are potassium ferrocyanide and certain nitrogen heterocyclic compounds, such as triazoles. In the reaction sequence leading to contrast-stained glycogen, a primary reaction is the formation of glycogen osmate. This reaction was studied with isolated glycogen. On the basis of the stoichiometric findings, a molecular structure of the reaction product is proposed; apparently, osmate is bound by glycogen because of the presence of suitably located hydroxyl groups. The resulting compound is not itself sufficiently electron dense, but it binds 1,2,4-triazole as an additional ligand. Secondary reactions can result in additional osmium binding, and finally to osmium (IV) deposits, leading to contrast.