To further examine potential sources of variability, lipid films from the master stock containing 13 C18-DSPC and 2 H31-POPC were dried down directly on NanoSIMS substrates and imaged. No significant separation within the 50nm lateral resolution of the NanoSIMS was observed between 13 C18-DSPC and 2 H31-POPC within the film (Figure ). However, atomic recombination experiments demonstrated that there is nanoscale separation between POPC and DSPC (Figure ). Although this does not demonstrate that this nanoscale separation is responsible for the observed GUV compositional variability, it does suggest that there are preferential interactions within lipid films. These interactions may contribute to lipid sorting while GUVs are being formed. Additional experiments also suggest that the size of the GUV patch analyzed is not correlated with the either 13 C18-DSPC or 2 H31-POPC concentrations (section 12 of the supporting information). Furthermore, additional data suggest that two different GUVs typically do not rupture to form one patch (section 13 of the supporting information). This suggests that most patches are the product of a single GUV and that minimal compositional averaging between GUVs occurs.