Atomic Recombination in Nanosims as a Method to Measure Nanometer-Scale Intermolecular Distances in Lipid Bilayers

Abstract

The nanometer scale organization of the eukaryotic plasma membrane, often described in terms of the lipid raft hypothesis, is presumed to be critical for signaling, viral budding, and other membrane phenomena. Here, we use secondary ion mass spectrometry (SIMS) to probe the nanometer scale structure of supported lipid bilayers (SLBs) by taking advantage of the intermolecular recombination of atomic ions into diatomic species that occurs in dynamic SIMS. In this experiment, one can measure mean distances between molecules on a length scale far below the lateral resolution of the NanoSIMS 50L instrument, which combines high spatial resolution and high mass resolution for chemical imaging. As an example, we show that in lipid bilayers, the efficiency of atomic recombination to form secondary 13C15N- ions depends on the distance between 13C and 15N atoms installed on headgroups of different lipid molecules. Specifically, with site-specific isotopic labeling of lipid headgroups, we determine the dependence of recombination efficiency on the average distance between 13C- and 15N-labeled phospholipids in supported monolayers and SLBs. We refer to this method of measuring nanometer-scale distances between isotopically-labeled molecules as a chemical ruler, similar in concept to FRET but different in physical phenomenon. High precision isotope ratio analysis with the NanoSIMS 50L makes this a potentially unique way to study proximity between membrane-associated components on the sub-5 nm length scale without the use of fluorescent dyes that perturb bilayer structure. We then apply this chemical ruler to study the structure of SLBs of lipid compositions commonly believed to contain nanometer-scale liquid-ordered domains that often serve as models for lipid rafts.