As shown in Figure and C, GUV patches produced via electroformation showed consistently less variability in both 13 C18-DSPC and 2 H31-POPC concentration than those formed via gentle hydration. To determine the lower bound on variability (i.e., how much of the variation in concentration measurements is due to instrument noise and surface contamination) a monolayer with overall composition 2 H31-POPC: 13 C18-POPC:POPC 20:20:60 (pure POPC composition) was formed on a NanoSIMS substrate and compared to both GUV samples. Since a monolayer should be compositionally homogenous across the substrate surface, this measurement can be used to approximate how much of the variability is due to the experimental method. As seen in Figure , pure POPC monolayers demonstrated consistently less variability in 2 H31-POPC concentrations than that of GUVs formed via electroformation or gentle hydration. However, these monolayers cannot be compared via the 2 H -/ 13 C -ratio as was done in Figure , because the correlation between 2 H31-POPC and 13 C18-POPC concentrations in pure POPC is dramatically different from the correlation between 2 H31-POPC and 13 C18-DSPC in ternary mixtures (Figure ). Additionally, ternary monolayers formed from the ternary master stock containing 2 H31-POPC and 13 C18-DSPC display macroscale separation (Figure ) and are therefore not comparable to the GUV samples, as is further discussed in section 5 of the supporting information. Therefore, monolayers can only be compared to GUV samples via absolute concentrations. Additional analysis suggests that the size of the region selected for quantification within the 25x25 μm analysis region does not significantly impact the distributions shown in Figures and (further discussed in section 6 of the supporting information). Therefore, the intrinsic signal to noise of a single patch is unlikely to substantially impact the GUV observed for different formation methods.