Optimization of cryo-electron microscopy for quantitative analysis of lipid bilayers

Cryogenic electron microscopy (cryo-EM) is among the most powerful tools available for interrogating 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. Here, we systematically investigate the influence of instrument parameters and image post-processing steps on the ability to accurately measure bilayer thickness and discriminate regions of different thickness within unilamellar liposomes. We also demonstrate a spatial autocorrelation analysis to extract additional information about lateral heterogeneity. Significance Raft domains in unstimulated cells have proven difficult to directly visualize owing to their nanoscopic size and fleeting existence. The few techniques capable of nanoscopic spatial resolution typically rely on interpretation of indirect spectroscopic or scattering signals or require stabilizing the membrane on a solid support. In contrast, cryo-EM yields direct images of nanoscale domains in probe-free, unsupported membranes. Here, we systematically optimize key steps in the experimental and analysis workflow for this new and specialized application. Our findings represent an important step toward developing cryo-EM into a robust method for investigating phase behavior of membranes at length scales relevant to lipid rafts.