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Abstract 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. OsO2has a metallic electronic structure that drastically increases the electron density of states near the Fermi level. Depositing metallic OsO2in 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.
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Rehydration of Freeze Substituted Brain Tissue for Pre-embedding Immunoelectron Microscopy
Abstract Electron microscopy (EM) volume reconstruction is a powerful tool for investigating the fundamental structure of brain circuits, but the full potential of this technique is limited by the difficulty of integrating molecular information. High quality ultrastructural preservation is necessary for EM reconstruction, and intact, highly contrasted cell membranes are essential for following small neuronal processes through serial sections. Unfortunately, the antibody labeling methods used to identify most endogenous molecules result in compromised morphology, especially of membranes. Cryofixation can produce superior morphological preservation and has the additional advantage of allowing indefinite storage of valuable samples. We have developed a method based on cryofixation that allows sensitive immunolabeling of endogenous molecules, preserves excellent ultrastructure, and is compatible with high-contrast staining for serial EM reconstruction.
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- Award ID(s):
- 2014862
- PAR ID:
- 10442250
- Publisher / Repository:
- Oxford University Press
- Date Published:
- Journal Name:
- Microscopy and Microanalysis
- Volume:
- 29
- Issue:
- 5
- ISSN:
- 1431-9276
- Format(s):
- Medium: X Size: p. 1694-1704
- Size(s):
- p. 1694-1704
- Sponsoring Org:
- National Science Foundation
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