Cryogenic transmission electron microscopy was used to measure the thicknesses of the hydrocarbon regions of the membranes and confirm the unilamellar nature of the vesicles. Small unilamellar vesicles of GDGTs were vitrified on holey carbon grids using standard techniques and imaged by cryo-EM. The overwhelming majority of the vesicles were unilamellar displaying uni-vesicular or multi-vesicular (or "vesicle-in-vesicle") architectures (Figure , Figure ). In addition, some sponge-like morphologies comprising of an interconnected network of membranes were observed in case of P2 and PG1Glc which were excluded from bilayer profile analysis (Figure ). We measured the thickness of each membrane by segmenting vesicles into small regions and aligning and averaging the segments to derive 2D class averages (Figure ). The linear profile of each membrane was measured across the center of the aligned and averaged images to obtain corresponding electron density profiles (Figure ). In agreement with the SAXS results, we found that the membrane thicknesses for all GDGT lipids were similar, with PG2 slightly thinner than the rest. Congruent with previous work, measured membrane thicknesses were approximately 8 Å thinner than those measured by SAXS (Figure ). We note that the membrane profiles have a different shape than those obtained from SAXS because they represent the projection of the spherical density rather than the EDP normal to the plane of the bilayer. Together, the SAXS and cryo-EM data demonstrate that the thicknesses of GDGT membranes is principally determined by their hydrocarbon chains rather than their headgroups. . C. Representative cryogenic electron microscopy images of unilamellar (and multi-compartment) vesicles of P2 prepared in Tris buffer (50 mM, pH 7.5). Scale bar represents 50 nm. The adjoining images show the 2D class averages for each lipid. D. EDPs of GDGT membranes calculated from cryo-EM images. The peak-to-peak distances correspond to the thicknesses of the membranes. The centers of the 1 st peaks of all EDPs were arbitrarily set to '0'.