Although measuring the 2 H -/ 13 C -ratio is useful for comparing the variability in different methods while avoiding concerns regarding surface contamination and calibration accuracy (further discussed in section 4 of the supporting information), quantification of absolute concentration is useful for determining how much the mol % of a particular lipid varies from GUV to GUV. Therefore, external calibration curves, such as those shown in Figure , were used to relate quantitative ion ratios to labeled lipid concentration. This allowed for the concentration of each labeled species within one GUV patch to be determined. This analysis was performed for the GUV samples discussed in Figure .