A novel leaflet-selective fluorescence labeling technique reveals differences between inner and outer leaflets at high bilayer curvature

► We introduce a method to label the inner or outer leaflet of lipid vesicles. ► In SUV, the outer leaflet headgroups are less tightly packed and more hydrated. ► In the inner leaflet, headgroups are more tightly packed and less hydrated. ► The differences between the two leaflets vanish when vesicles size is increased. ► The differences are also small when lipids with short acyl chains are used.