SIMS can be used to measure isotopic ratios present in a sample with high precision . The Cameca NanoSIMS 50L functions by collisional sputtering of a freeze-dried bilayer containing isotopically labeled lipids with primary cesium ions (Cs + ). This process ejects secondary ions, which are then separated by a mass analyzer, allowing for up to seven individual species to be detected simultaneously. The high sensitivity and mass precision of the NanoSIMS 50L allows for compositional information of individual bilayers to be obtained . For the experiments reported here, GUVs formed via either gentle hydration or electroformation were exposed to NanoSIMS substrates (10 nm SiO2 coated Si) where they rupture to form supported bilayer patches whose area reflects the surface area of the parent GUV. This process is depicted in Figure . In parallel, continuous supported bilayers (SLBs) were formed by conventional small vesicle fusion. SIMS can then be used to examine the compositional variability of the resulting SLBs formed via different methods.