Using FIJI, lines (5 pixels thickness) were always drawn from inside the lumen to the external buffer regions to differentiate the inner and outer leaflets in the final electron intensity profile. Per vesicle, at least 15 lines were drawn across the bilayer at various slices in a tomogram. The line profiles were adjusted on OriginPro to overlap at the central hydrophobic core, baseline corrected, averaged and normalised to give an electron intensity profile for each vesicle composition.