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 (cryoEM) can be used to directly image coexisting nanoscopic domains in synthetic and bio-derived membranes without extrinsic probes. Analyzing a series of single-component liposomes composed of synthetic lipids of varying lengths, we demonstrate that cryoEM can distinguish bilayer thickness differences as small as 0.5 angstrom, comparable to the resolution of small-angle scattering methods. Simulated images from computational models reveal that features in cryoEM 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, these methods reveal 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.

Direct label-free imaging of nanodomains in biomimetic and biological membranes by cryogenic electron microscopy