Thickness measurements of in vitro lipid bilayers match closely between cryo-EM and cryo-ET. (A) Representative untilted 2D cryo-EM micrograph of in vitro–extruded vesicles with varying radii. (B) Thickness of vesicle bilayers, measured as previously described (Sharma et al., 2024), revealed in untilted micrographs, shown as color on the micrograph, reveals the thickness variation in the micrograph. The scale bar is 150 nm. (C) Histogram of thicknesses across contours in the micrographs showing the median thickness of 3.56 nm. (D) Representative slice of a reconstructed tomogram of in vitro vesicles acquired from the same grid. The scale bar is 150 nm. (E) Surface map shows the variation of thickness measured by the Surface Morphometrics pipeline. (F) Histogram of thickness across triangles in the tomograms shows the median thickness of 3.52 nm, <2% different than the in vitro micrographs, despite larger defocus and reduced signal-to-noise leading to increased variance across triangles. n = 3 tomograms, 47 vesicles. (G) 2D histogram showing no significant correlation between curvature and thickness in in vitro vesicles. (H) Average thickness per vesicle is plotted against vesicle radius, with a linear regression line in red, showing no significant correlation between vesicle radius and thickness. R² = 0.096.