The pressure-dependent diffusion and partitioning of single lipid fluorophores in DMPC and DPPC monolayers were investigated with the use of a custom-made monolayer trough mounted on a combined fluorescence correlation spectroscopy (FCS) and wide-field microscopy setup. It is shown that lipid diffusion, which is essential for the function of biological membranes, is heavily influenced by the lateral pressure and phase of the lipid structure. Both of these may change dynamically during, e.g., protein adsorption and desorption processes. Using FCS, we measured lipid diffusion coefficients over a wide range of lateral pressures in DMPC monolayers and fitted them to a free-area model as well as the direct experimental observable mean molecular area. FCS measurements on DPPC monolayers were also performed below the onset of the phase transition (Π < 5 mN/m). At higher pressures, FCS was not applicable for measuring diffusion coefficients in DPPC monolayers. Single-molecule fluorescence microscopy and differential scanning calorimetry clearly showed that this was due to heterogeneous partitioning of the lipid fluorophores in condensed phases. The results were compared with dye partitioning in giant lipid vesicles. These findings are significant in relation to the application of lipid fluorophores to study diffusion in both model systems and biological systems. © 2009 by the Biophysical Society.
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