Cell membranes possess a complex three-dimensional architecture, including nonrandom lipid lateral organization within the plane of a bilayer leaflet, and compositional asymmetry between the two leaflets. As a result, delineating the membrane structure–function relationship has been a highly challenging task. Even in simplified model systems, the interactions between bilayer leaflets are poorly understood, due in part to the difficulty of preparing asymmetric model membranes that are free from the effects of residual organic solvent or osmotic stress. To address these problems, we have modified a technique for preparing asymmetric large unilamellar vesicles (aLUVs) via cyclodextrin-mediated lipid exchange in order to produce tensionless, solvent-free aLUVs suitable for a range of biophysical studies. Leaflet composition and structure were characterized using isotopic labeling strategies, which allowed us to avoid the use of bulky labels. NMR and gas chromatography provided precise quantification of the extent of lipid exchange and bilayer asymmetry, while small-angle neutron scattering (SANS) was used to resolve bilayer structural features with subnanometer resolution. Isotopically asymmetric POPC vesicles were found to have the same bilayer thickness and area per lipid as symmetric POPC vesicles, demonstrating that the modified exchange protocol preserves native bilayer structure. Partial exchange of DPPC into the outer leaflet of POPC vesicles produced chemically asymmetric vesicles with a gel/fluid phase-separated outer leaflet and a uniform, POPC-rich inner leaflet. SANS was able to separately resolve the thicknesses and areas per lipid of coexisting domains, revealing reduced lipid packing density of the outer leaflet DPPC-rich phase compared to typical gel phases. Our finding that a disordered inner leaflet can partially fluidize ordered outer leaflet domains indicates some degree of interleaflet coupling, and invites speculation on a role for bilayer asymmetry in modulating membrane lateral organization.

Focused electron beam induced deposition (FEBID) is an important synthesis method as it is an extremely flexible tool for fabricating functional (3D) structures with nanometer spatial resolution. However, FEBID has historically suffered from carbon impurities up to 90 at %, which significantly limits the intended functionalities. In this study we demonstrate that MeCpPtIVMe3 deposits can be fully purified by an electron-beam assisted approach using H2O vapor at room temperature, which eliminates sample and/or gas heating and complicated gas delivery systems, respectively. We demonstrate that local pressures of 10 Pa results in an electron-limited regime, thus enabling high purification rates of better than 5 min·nA–1·μm–2 (30 C·cm–2) for initially 150 nm thick deposits. Furthermore, TEM measurements suggest the purification process for the highly compact deposits occurs via a bottom-up process.

Despite the ubiquity of transbilayer asymmetry in natural cell membranes, the vast majority of existing research has utilized chemically well-defined symmetric liposomes, where the inner and outer bilayer leaflets have the same composition. Here, we review various aspects of asymmetry in nature and in model systems in anticipation for the next phase of model membrane studies.