Diffusion processes in membranes containing coexisting domains investigated by Fluorescence Correlation Spectroscopy

Abstract

The aim of this study is to link domain and microdomain formation to diffusion behaviour in artificial planar membranes using Fluorescence Correlation Spectroscopy (FCS) and Monte Carlo (MC) simulations. In recent years it has been established that the membrane structure is not homogeneous but contains various domains e.g. so called rafts those including cholesterol. However, even in simple pure lipid systems in a gel and fluid coexistence phase macrodomains and microdomains (in which the lipid states differ) can be observed. The existence of domains is also predicted from statistical thermodynamics simulations, and has been observed by Confocal Fluorescence Microscopy (CFM) or Atomic Force Microscopy (AFM).The presence of gel and fluid heterogeneities can influence diffusion processes in membranes. Therefore diffusion processes in hydrated multilamellar membranes of mixtures of various phospholipids supported on a quartz coverslip were investigated by FCS. Fluorescent intensity fluctuations, analysed via a correlation function, give information on the translational diffusion coefficient, Dτ, and the diffusion time, τD, as well as on the mean number of dye molecules in the laser focus, <N>.The diffusion coefficient in a pure fluid phase was found to be approximately two order in magnitude faster than the diffusion coefficient in a pure gel phase. Decreasing temperature, decreasing the amount of a gel fraction, or a presence of salt e.g. NaCl lower its value. The shape of the correlation profiles corresponding to a pure gel or a pure fluid phase differs from this corresponding to the gel and fluid coexistence region. The FCS curves in a pure gel and in a pure fluid phase were fitted with a one component fit, whereas in a gel and fluid coexistence phase with a two diffusion coefficient fit assuming macroscopic gel and fluid domain coexistence. The following phenomena was observed: decreasing value of a fast Dτ and increasing value of a slow Dτ, which may hypothesise microdomain formation. Experimental profiles tried to be described with Monte Carlo simulations, which make use of thermodynamical properties of lipid mixtures derived from calorimetric measurements. The MC snapshots show existence of microscopic gel and fluid inheterogeneities in huge fluid and gel macrodomains. Since our experimental correlation profiles are consistent with theoretically predicted profiles it may confirm that there are microdomains, whose length is smaller than the focus diameter (<1 µm).This study means an important step in the better understanding of the lateral organisation of the lipid membrane, its dynamics and kinetics.