Understanding interactions of calcium with lipid membranes at the molecular level is of great importance in light of their involvement in calcium signaling, association of proteins with cellular membranes, and membrane fusion. We quantify these interactions in detail by employing a combination of spectroscopic methods with atomistic molecular dynamics simulations. Namely, time-resolved fluorescent spectroscopy of lipid vesicles and vibrational sum frequency spectroscopy of lipid monolayers are used to characterize local binding sites of calcium in zwitterionic and anionic model lipid assemblies, while dynamic light scattering and zeta potential measurements are employed for macroscopic characterization of lipid vesicles in calcium-containing environments. To gain additional atomic-level information, the experiments are complemented by molecular simulations that utilize an accurate force field for calcium ions with scaled charges effectively accounting for electronic polarization effects. We demonstrate that lipid membranes have substantial calcium-binding capacity, with several types of binding sites present. Significantly, the binding mode depends on calcium concentration with important implications for calcium buffering, synaptic plasticity, and protein-membrane association.

Confocal fluorescence correlation spectroscopy (FCS) allows for the determination of lateral diffusion coefficients and surface densities in planar phospholipid systems. The determination of the vertical (z-) position of the laser focus relative to the phospholipid surface plane is of crucial importance for the accuracy of the confocal FCS experiment. In this work we determine for the first time this vertical (z-) position of the laser focus by a so-called “Z-scan”, which is based on the determination of diffusion times and particle numbers in 0.2 μm steps along the vertical (z-) axis. Experiments on supported phospholipid bilayers composed of dioleoylphosphatidylcholine (DOPC) and small amounts of Rhodamine Red-X 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt (Rhodamine Red-X DHPE) adsorbed onto atomically flat mica and borosilicate glass demonstrate that results obtained by the Z-scan approach are significantly more precise than those results obtained when the fluorescence intensity maximum is used as an indicator in the determination of the vertical (z-) position of the sample. In addition to this basic contribution for the investigation of planar bilayer systems by confocal FCS, the lateral diffusion coefficients of Rhodamine Red-X DHPE in supported phospholipid bilayers composed of DOPC and cholesterol as well as in DOPC or dipalmitoylphosphatidylcholine (DPPC) monolayers adsorbed at a liquid−liquid interface were determined.

Understanding of cellular regulatory pathways that involve lipid membranes requires the detailed knowledge of their physical state and structure. However, mapping the viscosity and diffusion in the membranes of complex composition is currently a non-trivial technical challenge. We report fluorescence lifetime spectroscopy and imaging (FLIM) of a meso-substituted BODIPY molecular rotor localised in the leaflet of model membranes of various lipid compositions. We prepare large and giant unilamellar vesicles (LUVs and GUVs) containing phosphatidylcholine (PC) lipids and demonstrate that recording the fluorescence lifetime of the rotor allows us to directly detect the viscosity of the membrane leaflet and to monitor the influence of cholesterol on membrane viscosity in binary and ternary lipid mixtures. In phase-separated 1,2-dioleoyl-sn-glycero-3-phosphocholine-cholesterol-sphingomyelin GUVs we visualise individual liquid ordered (Lo) and liquid disordered (Ld) domains using FLIM and assign specific microscopic viscosities to each domain. Our study showcases the power of FLIM with molecular rotors to image microviscosity of heterogeneous microenvironments in complex biological systems, including membrane-localised lipid rafts.

The lateral diffusion coefficients of a BODIPY tail-labeled lipid in two model systems, namely, free-standing giant unilamellar vesicles (GUVs) and supported phospholipid bilayers (SPBs), were determined by fluorescence correlation spectroscopy (FCS) using the Z-scan approach. For the first time, the performed measurements on 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) bilayers maintain exactly the same experimental conditions for both systems, which allows for a quantitative comparison of lipid diffusion in these two commonly used model membranes. The results obtained revealed that the lipid mobility in free-standing bilayers (D=7.8+/-0.8 microm2 s-1) is significantly higher than in the bilayer created on the solid support (mica) (D=3.1+/-0.3 microm2 s-1).