The nanoscale organization of biological membranes into structurally and compositionally distinct lateral domains is believed to be central to membrane function. The nature of this organization has remained elusive due to a lack of methods to directly probe nanoscopic membrane features. We show here that cryogenic electron microscopy (cryo-EM) can be used to directly image coexisting nanoscopic domains in synthetic and bioderived membranes without extrinsic probes. Analyzing a series of single-component liposomes composed of synthetic lipids of varying chain lengths, we demonstrate that cryo-EM can distinguish bilayer thickness differences as small as 0.5 Å, comparable to the resolution of small-angle scattering methods. Simulated images from computational models reveal that features in cryo-EM images result from a complex interplay between the atomic distribution normal to the plane of the bilayer and imaging parameters. Simulations of phase-separated bilayers were used to predict two sources of contrast between coexisting ordered and disordered phases within a single liposome, namely differences in membrane thickness and molecular density. We observe both sources of contrast in biomimetic membranes composed of saturated lipids, unsaturated lipids, and cholesterol. When extended to isolated mammalian plasma membranes, cryo-EM reveals similar nanoscale lateral heterogeneities. The methods reported here for direct, probe-free imaging of nanodomains in unperturbed membranes open new avenues for investigation of nanoscopic membrane organization.
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.
more » « less- Award ID(s):
- 2014862
- PAR ID:
- 10523900
- Publisher / Repository:
- ChemRxiv
- Date Published:
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Posted Content:
- https://doi.org/10.26434/chemrxiv-2023-scskq
