Abstract Lateral lipid heterogeneity (i.e., raft formation) in biomembranes plays a functional role in living cells. Three-component mixtures of low- and high-melting lipids plus cholesterol offer a simplified experimental model for raft domains in which a liquid-disordered (Ld) phase coexists with a liquid-ordered (Lo) phase. Using such models, we recently showed that cryogenic electron microscopy (cryo-EM) can detect phase separation in lipid vesicles based on differences in bilayer thickness. However, the considerable noise within cryo-EM data poses a significant challenge for accurately determining the membrane phase state at high spatial resolution. To this end, we have developed an image processing pipeline that utilizes machine learning (ML) to predict the bilayer phase in projection images of lipid vesicles. Importantly, the ML method exploits differences in both the thickness and molecular density of Lo compared to Ld, which leads to improved phase identification. To assess accuracy, we used artificial images of phase-separated lipid vesicles generated from all-atom molecular dynamics simulations of Lo and Ld phases. Synthetic ground truth datasets mimicking a series of compositions along a tieline of Ld+Lo coexistence were created and then analyzed with various ML models. For all tieline compositions, we find that the ML approach can correctly identify the bilayer phase at 5 nm lateral resolution with &gt; 90% accuracy, thus providing a means to isolate the intensity profiles of coexisting Ld and Lo phases, as well as accurately determine domain size distributions, number of domains, and phase area fractions. The method described here provides a framework for characterizing nanoscopic lateral heterogeneities in membranes and paves the way for a more detailed understanding of raft properties in biological contexts. Significance Lipid rafts are important for cell function, but in most cases cannot be detected with conventional optical microscopy because of their extremely small size. Cryogenic electron microscopy (cryo-EM), because of its much greater spatial resolution, is capable of imaging domains as small as 5-10 nm. In this report, we show how machine learning techniques can be used to automatically and accurately identify raft-like domains in simulated cryo-EM images, a powerful approach that could ultimately lead to a better understanding of raft properties.

Cryo-EM images of phase separated lipid bilayer vesicles analyzed with a machine learning approach