Fluorescence signatures for interleaflet coupling - missing link in monolayer-bilayer correspondence

Model membrane systems, including Langmuir monolayers, supported lipid bilayers (SLBs), and vesicles, are widely used to investigate the structure and dynamics of biological membranes and membrane-associated processes. It is widely assumed that lipid monolayers compressed to approximately 30 mN/m provide a physicochemical environment comparable to that of lipid bilayers. However, the validity of this correspondence remains debated. Here, we systematically test monolayer-bilayer equivalence using the fluorescence spectral responses of three molecular probes, DOPE-Atto633, Laurdan, and NR12S. Their spectra were recorded in DOPC and DPPC monolayers over a broad range of surface pressures, with and without cholesterol, and compared with corresponding bilayer systems formed as SLBs and small unilamellar vesicles (SUVs). Our results reveal pronounced differences between monolayer and bilayer environments depending on the lipid composition, cholesterol content, and the molecular probe used. For pure DOPC and DPPC, Laurdan revealed close monolayer-bilayer correspondence at approximately 30 mN/m, whereas NR12S required substantially higher surface pressures to reproduce bilayer-like spectral responses. Cholesterol further altered this correspondence, and for some compositions full spectral correspondence was not reached even at the highest surface pressures investigated. These findings challenge the general applicability of the established monolayer-bilayer correspondence and demonstrate that a single surface-pressure criterion cannot reliably reproduce the physicochemical properties of bilayer membranes across different lipid systems. They also underpin the importance of second leaflet and interleaflet coupling in shaping membrane structure and packing. More broadly, our findings emphasise the need to critically consider appropriate model systems when investigating biological membranes and their functions.