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.

Formation of supported membranes by exposure of solid surfaces to phospholipid vesicles is a much-used technique in membrane research. Freshly cleaved mica, because of its superior flatness, is a preferred support, and we used ellipsometry to study membrane formation kinetics on mica. Neutral dioleoyl-phosphatidylcholine (DOPC) and negatively charged dioleoyl-phosphatidylserine/dioleoyl-phosphatidylcholine (20% DOPS/80% DOPC) vesicles were prepared by sonication. Results were compared with membrane formation on silica and glass, and the influence of stirring, buffer, and calcium was assessed. Without calcium, DOPC vesicles had a low affinity (Kd approximately 30 microM) for mica, and DOPS/DOPC vesicles hardly adsorbed. Addition of calcium promptly caused condensation of the adhering vesicles, with either loss of excess lipid or rapid additional lipid adsorption up to full surface coverage. Vesicle-mica interactions dominate the adsorption process, but vesicle-vesicle interactions also seem to be required for the condensation process. Membranes on mica proved unstable in Tris-HCl buffer. For glass, transport-limited adsorption of DOPC and DOPS/DOPC vesicles with immediate condensation into bilayers was observed, with and without calcium. For silica, vesicle adsorption was also rapid, even in the absence of calcium, but the transition to condensed layers required a critical surface coverage of about 50% of bilayer mass, indicating vesicle-vesicle interaction. For all three surfaces, additional adsorption of DOPC (but not DOPS/DOPC) vesicles to condensed membranes was observed. DOPC membranes on mica were rapidly degraded by phospholipase A2 (PLA2), which pleads against the role of membrane defects as initial PLA2 targets. During degradation, layer thickness remained unchanged while layer density decreased, in accordance with recent atomic force microscopy measurements of gel-phase phospholipid degradation by PLA2.

Simul 5 Complex is a one-dimensional dynamic simulation software designed for electrophoresis, and it is based on a numerical solution of the governing equations, which include electromigration, diffusion and acid-base equilibria. A new mathematical model has been derived and implemented that extends the simulation capabilities of the program by complexation equilibria. The simulation can be set up with any number of constituents (analytes), which are complexed by one complex-forming agent (ligand). The complexation stoichiometry is 1:1, which is typical for systems containing cyclodextrins as the ligand. Both the analytes and the ligand can have multiple dissociation states. Simul 5 Complex with the complexation mode runs under Windows and can be freely downloaded from our web page http://natur.cuni.cz/gas. The article has two separate parts. Here, the mathematical model is derived and tested by simulating the published results obtained by several methods used for the determination of complexation equilibrium constants: affinity capillary electrophoresis, vacancy affinity capillary electrophoresis, Hummel-Dreyer method, vacancy peak method, frontal analysis, and frontal analysis continuous capillary electrophoresis. In the second part of the paper, the agreement of the simulated and the experimental data is shown and discussed.