Cryogenic electron microscopy (cryo-EM) is among the most powerful tools available for interrogating the nanoscale structure of biological structures. We recently showed that cryo-EM can be used to measure the bilayer thickness of lipid vesicles and biological membranes with sub-angstrom precision, resulting in the direct visualization of nanoscopic domains of different thickness in multicomponent lipid mixtures and giant plasma membrane vesicles. Despite the great potential of cryo-EM for revealing the lateral organization of biomembranes, a large parameter space of experimental conditions remains to be optimized, and analytical tools for extracting information about domain area fractions and sizes have yet to be developed. To this end, we investigated a variety of factors that can influence the primary data; these include pre-acquisition hardware options and instrument settings, post-acquisition image processing steps, and data analysis algorithms. We report on how these factors influence measurements including radial bilayer intensity profiles, distributions of membrane thickness, spatial autocorrelation of the bilayer thickness, and spatially-resolved phase state. Complementary analysis of synthetic images generated from atomistic molecular simulations corroborated experimental observations and yielded further insights into the molecular origins of image features. These efforts demonstrate how data collection can be systematically optimized to maximize the potential of cryo-EM for resolving variation in bilayer thickness within biological membranes.

Development of cryo-EM for probing lateral organization in biomembranes

ACS Spring 2022

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