Comparing GUV Preparation Methods via Double-Labeling. First, we assessed the variability in relative concentrations of labeled lipids for different GUV preparation methods. Although direct concentration quantification via external calibration curves can give estimates of the concentration of a labeled species, this method is subject to inaccuracies. These experiments can be skewed by surface contamination and are heavily reliant on the accuracy of the calibration standards. In order to avoid these issues, and inspired by prior work , initial experiments examined the relative change in ion counts from patch to patch resulting from two lipids with different isotopic labels. This was done with a POPC:DSPC:CHOL 40:40:20 mixture. This mixture was chosen as GUV patches with this composition do not display macroscopic phase separation between lipid components within the 50 nm lateral resolution of the NanoSIMS primary ion beam (Figure ; there is nanoscale separation present in these SLB patches which can be detected by ion-recombination ). This mixture is also well-studied in GUVs and GUV patches .