X-ray Microanalysis of Non-aldehyde-fixed Glycogen Contrast Stained with OsVIIIO4, OsVIIIFeIII, or OsVIFeII Complex in Vitro

Fibrin-enrobed, commercially produced glycogen was treated, without prior glutaraldehyde fixation, to a form of post-fixation with solutions of OsVIIIO4 or with a mixture of either OsVIIIO4 plus K3FeIII(CN)6 or K2OsVIO4 plus K4FeII(CN)6. Only the last mixture gave contrast staining of the glycogen in unstained ultrathin sections. The first mixture rendered the glycogen just barely visible but the glycogen contrast was increased by lead staining. The glycogen treated with the OsVIIIO4 solution was not contrast stained an was just observable after lead staining. Qualitative X-ray microanalysis of the glycogen in the ultrathin sections confirmed the presence of osmium and iron in the glycogen treated with both mixtures. The glycogen treated with OsVIIIO4 alone was difficult to analyse. Quantitative X-ray microanalysis showed that, in the glycogen treated with the OsVIIIO4 mixture plus K3FeIII(CN)6, the mean atomic osmium to iron ratio was 1:5. In the glycogen treated with K2OsVIO4 plus K4FeII(CN)6, this ratio was 1:17. However, th mean net osmium intensity in the latter case was 15 times higher than in the former case and for the iron even 40 times higher.