Sources of Variability. In order to further explore sources of the observed GUV variability, GUVs with pure POPC composition ( 2 H31-POPC: 13 C18-POPC:POPC 20:20:60) were formed by either gentle hydration or electroformation. 30 GUV patches with this composition were analyzed for each method, and their absolute concentrations determined via external calibration curves. In Figure , the calculated 2 H31-POPC concentrations in the pure POPC samples are compared to the 2 H31-POPC concentrations in the previously discussed ternary mixture. Ternary GUV patches formed either by electroformation or gentle hydration displayed significantly more variability in 2 H31-POPC concentrations relative to pure POPC patches formed by the same method. This lower variability for the pure POPC GUVs can also be seen for the C18-labeled lipids (Figure ). These results suggest that more complex lipid compositions lead to considerably more compositional variability. It also suggests that the observed compositional variability is not only due to residual contamination of either the platinum electrodes used for electroformation or glass vials used for gentle hydration. If these surfaces had significant contamination, pure POPC patches would have compositional variability comparable to ternary patches. Comparison of 2 H31-POPC concentration variability between pure POPC patches formed via either electroformation or gentle hydration did not show a significant difference in compositional variability (Figure ). This suggests that the higher variability observed in GUV patches formed via gentle hydration (Figures and) is not due to higher residual contamination on glass vials relative to the platinum electrodes, but rather results from the complexity of the ternary mixture and the method of GUV formation. Additionally, the average 2 H -, 12 C -and 13 C -counts were compared between regions of interest with and without bilayer to gauge the level of contamination on the substrate surface (Figure ). Regions of interest containing bilayer showed considerably higher signal on all detectors than regions of interest on exposed substrate. Further analysis was also performed to correct the observed variabilities in ternary GUV patches for noise due to sample preparation and analysis (discussed in section 10 of the supporting information).