NanoSIMS sample preparation. GUVs were ruptured onto silicon substrates to form SLB patches after being allowed to briefly cool to room temperature. Silicon substrates were plasma cleaned for 10 minutes after which they were submerged in phosphate buffer (240 mM NaCl, 10 mM NaH2PO4 at pH 7.4). Then GUVs were deposited over the submerged substrates and allowed to incubate until approximately 10-15% of the surface was covered in SLB patches. GUV deposition was observed with a Nikon Eclipse 80i epifluorescence microscope equipped with an Andor Clara camera. The substrate and bilayers were then extensively washed with MilliQ water. Although GUVs, particularly those formed via gentle hydration, can be multivesicular, upon GUV rupture to form an SLB patch, the internal vesicles are liberated and rinsed away. Cleaned substrates were flash frozen in liquid nitrogen and then subjected to low pressure for at least 12 hours to sublimate any vitreous ice.