To form continuous bilayers on NanoSIMS substrates, 100nm vesicles were incubated over plasma cleaned substrates for 1 minute before being washed extensively with MilliQ water. Continuous bilayers on substrates were found to be more susceptible to de-wetting during flash freezing, so substrates with continuous bilayers were removed from MilliQ water with the bilayer facing upside-down. This ensured that a drop of water remained in contact with the bilayer at all times before the substrate was flash-frozen in liquid nitrogen. Once frozen, substrates were subjected to low pressure using the same method as GUV patch samples.