Large unilamellar vesicles (LUVs) with increasing PBd22-PEO14 mole fraction were formed by extrusion (through 100 nm track-etched pores) and mixed with colloidal gold, which act as fiducial markers for tomogram alignment. From the tomogram, transmembrane line profiles of individual vesicles were collected in the same direction (inside to outside) to allow differentiation between the inner and outer membrane leaflets (data available in Supplementary Information Section 1). These line profiles were then averaged to find the electron intensity profile for a vesicle composition as shown in Figure . The electron intensity profiles were used to infer structural features in the bilayer, such as the membrane thickness from the full width half maximum (FWHM), and the hydrophobic core thickness using peak-to-peak distance.