Contrast Mechanism of Osmium Staining in Electron Microscopy of Biological Tissues

Electron imaging of biological samples stained with heavy metals has enabled visualization of nanoscale subcellular structures critical in chemical-, structural-, and neuro-biology. In particular, osmium tetroxide has been widely adopted for selective lipid imaging. Despite the ubiquity of its use, the osmium speciation in lipid membranes and the mechanism for image contrast in electron microscopy (EM) have continued to be open questions, limiting efforts to improve staining protocols and improve high-resolution imaging of biological samples. Following our recent success using photoemission electron microscopy (PEEM) to image mouse brain tissues with a subcellular resolution of 15 nm, we have used PEEM to determine the chemical contrast mechanism of Os staining in lipid membranes. Os (IV), in the form of OsO2, generates aggregates in lipid membranes, leading to a strong spatial variation in the electronic structure and electron density of states. OsO2 has a metallic electronic structure that drastically increases the electron density of states near the Fermi level. Depositing metallic OsO2 in lipid membranes allows for strongly enhanced EM signals of biological materials. This understanding of the membrane contrast mechanism of Os-stained biological specimens provides a new opportunity for the exploration and development of staining protocols for high-resolution, high-contrast EM imaging.