Viscosity-sensitive membrane dyes as tools to estimate the crystalline structure of lipid bilayers

Lipid membranes are crucial for cellular integrity and regulation, and tight control of their structural and mechanical properties is vital to ensure that they function properly. Fluorescent probes sensitive to the mem-brane’s microenvironment are useful for investigating lipid membrane properties, however, there is currently a lack of quantitative correlation between the exact parameters of lipid organization and a readout from these dyes. Here, we investigate this relationship for ‘molecular rotors’, or microviscosity sensors, by simultaneously measuring their fluorescence lifetime to determine the membrane viscosity, while using the X-Ray diffraction the determine the membrane’s structural properties. Our results reveal a phase-dependent correlation be-tween the membrane’s structural parameters and mechanical properties measured by a BODIPY-based mo-lecular rotor, giving excellent predictive power for the structural descriptors of the lipid bilayer. We also demonstrate that differences in membrane thickness between different lipid phases is not a prerequisite for formation of lipid microdomains and that this requirement can be disrupted by the presence of line-active molecules. Our results underpin the use of membrane-sensitive dyes as reporters of the structure of lipid membranes.