NanoSIMS Analysis. Analysis was performed on the Cameca NanoSIMS 50L at Stanford University. Correlative fluorescence imaging helped facilitate the selection of bilayer patches such that debris on the substrate surface was avoided. Bilayer patches were imaged with a 2pA 133 Cs + primary beam. Ten 25x25 micron scans (256x256 pixels, 1ms dwell time) were collected, which is enough to remove all of the deposited material on the surface. Secondary ion detectors were set to 2 H - , C -, C -, 12 C2H -, 12 C2 2 H -, 13 C2H -, and 13 C2 2 H -for samples containing 2 H-or C-labeled lipids of cholesterol. If 15 N- labeled POPC was contained within SLB patches, secondary ion detectors were set to 2 H -, 12 C -, 13 C -, 12 C2H -, 12 C 14 N -, 13 C 15 N -, and 13 C2 2 H -. Standard samples (the calibration curves described below) were regularly analyzed to ensure that isotope ratios were reproducible from session to session.