Contrast Mechanism of Osmium Staining in Electron Microscopy of Biological Tissues

12 September 2023, Version 1
This content is a preprint and has not undergone peer review at the time of posting.

Abstract

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.

Keywords

electron microscopy
biological staining
photoemission electron microscopy

Supplementary materials

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Description
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Supporting Information and Figures
Description
Contains details of sample preparation, PEEM imaging, EELS and EDS experiments as well as a spectrum of the Hg lamp used for imaging.
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