A fluorescence correlation spectroscopy study of the diffusion of an organic dye in the gel phase and fluid phase of a single lipid vesicle

Abstract

The mobility of the organic dye DCM (4-dicyanomethylene-2-methyl-6-p-dimethyl aminostyryl-4H-pyran) in the gel and fluid phases of a lipid vesicle is studied by fluorescence correlation spectroscopy (FCS). Using FCS, translational diffusion of DCM is determined in the gel phase and fluid phase of a single lipid vesicle adhered to a glass surface. The size of a lipid vesicle (average diameter 100 nm) is smaller than the diffraction limited spot size (250 nm) of the microscope. Thus, the vesicle is confined within the laser focus. Three lipid vesicles (1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)) having different gel transition temperatures (-1, 23, and 41°C, respectively) were studied. The diffusion coefficient of the dye DCM in bulk water is 300 μ m²/s. In the lipid vesicle, the average D_t decreases markedly to ~5 μm²/s (60 times) in the gel phase (for DPPC at 20°C) and 40 μm2/s (~8 times) in the fluid phase (for DLPC at 20°C). This clearly demonstrates higher mobility in the fluid phase compared with the gel phase of a lipid. It is observed that the D_t values vary from lipid to lipid and there is a distribution of D_t values. The diffusion of the hydrophobic dye DCM (D_t~ 5 μ m²/s) in the DPPC vesicle is found to be 8 times smaller than that of a hydrophilic anioinic dye C343 (D_t~ 40 μ m²/s). This is attributed to different locations of the hydrophobic (DCM) and hydrophilic (C343) dyes.