Tissue fixation and osmium black formation with nonvolatile octavalent osmium compounds

Several compounds of osmiumVIII, including potassium osmiamate and coordination complexes of OsO4 with ammonia and various heterocyclic nitrogen compounds, have been synthesized and characterized. They have also been evaluated as substitutes for OsO4 in postfixation of biological specimens and in light and electron microscopic cytochemical methods resulting in osmium black formation. The most useful of these osmic compounds, a molecular addition complex of hexamethylenetetramine (methenamine) with OsO4, has a negligible vapor pressure of OsO4. It has the molecular formula C6H12N4.2OsO4 and has been designated osmeth. Although it has only limited solubility, aqueous solutions of the compound (or of OsO4) can be rapidly prepared by dissolution in a minimal amount of dimethylformamide and subsequent dilution with distilled water or buffer. Although stable in the solid state, the complex in solution undergoes partial dissociation releasing OsO4, and the odor of OsO4 becomes apparent. Such solutions of osmeth are (approximately 0.25%) considerably less concentrated with respect to OsO4 than solutions (1-2%) ordinarily employed for ultrastructural preservation or in cytochemical studies. Osmeth has limited value for postosmication after glutaraldehyde fixation because the generation (release) of OsO4 appears to be slow. Adequate osmication of tissue blocks exists only at the surface, but effective osmication can be achieved throughout tissue sections. In cytochemical reactions resulting in the formation of osmium blacks, the osmeth solutions are as effective as OsO4 solutions of equivalent concentrations. Our findings indicate that OsO4 solutions of less than 1% may be satisfactorily utilized in many cytochemical studies. Osmeth is safer and more convenient to handle than OsO4 because small amounts may be solubilized as needed. It should be considered as a substitute for OsO4 in ultrastructural cytochemistry. These results suggest that the effectiveness of OsO4 as a fixative may, in part, be related to its nonpolarity. The infrared spectra indicate that the OsO4 molecule is tetrahedral, perfectly symmetrical and, therefore, as a whole nonpolar. As a consequence, it could be expected to readily penetrate charged surfaces of tissues, cells, and organelles. The spectral studies show that osmeth is much less symmetrical and, to that extent, polar; thus, it penetrates biomembranes less readily.