OsO₂ as the Contrast-Generating Chemical Species of Osmium-Stained Biological Tissues in Electron Microscopy

Electron imaging of biological samples stained with heavy metals has enabled visualization of subcellular structures critical in chemical-, structural-, and neuro-biology. In particular, osmium tetroxide (OsO₄) has been widely adopted for selective lipid imaging. Despite the ubiquity of its use, the osmium speciation in lipid membranes and the process for contrast generation in electron microscopy (EM) have continued to be open questions, limiting efforts to improve staining protocols and therefore high-resolution nanoscale imaging of biological samples. Following our recent success using photoemission electron microscopy (PEEM) to image mouse brain tissues with synaptic resolution, we have used PEEM to determine the nanoscale electronic structure of Os-stained biological samples. Os(IV), in the form of OsO₂, generates nanoaggregates 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 and conductivity of biological materials. The identification of the chemical species and understanding of the membrane contrast mechanism of Os-stained biological specimens provides a new opportunity for the development of staining protocols for high-resolution, high-contrast EM imaging.