Abstract
Scanning-fluctuation correlation spectroscopy was used to detect subresolution organizational fluctuations in the lipid liquid-crystalline phase for single lipid model systems. We used the fluorescent probe Laurdan which is sensitive to the amount of water in the membrane to show that there is a spatial heterogeneity on the scale of few pixels (the size of the pixel is 50 nm). We calculated the pixel variance of the GP function and we found that the variance has a peak at the phase transition for 3 different samples made of pure lipids. The pixel variance has an abrupt change at the phase transition of the membrane and then it slowly decreases at higher temperature. The relatively large variance of the GP indicates that the liquid phase of the membrane is quite heterogeneous even several degrees higher than the phase transition temperature. We interpreted this result as evidence of an underlying microscale structure of the membrane in which water is not uniformly distributed at the micron scale. Imaging of these microstructures shows that the pixels with different GP tend to concentrate in specific domains in the membrane. In the case of single lipid membrane, the statistical and fluctuation analysis of the GP data shows that even such simple lipid systems are capable of generating and maintaining stable structural and organizational heterogeneities.
Highlights
Lipid molecular interactions have been the subject of extensive biophysical investigation due to their relevance in the understanding of functioning and dynamics of biological membranes
Recent investigations using fluorescence based techniques such as single particle tracking, confocal and STED fluorescence correlation spectroscopy, foster resonance energy transfer have revealed that lipid phase domains in biological membranes are small and transient in nature [5,6,7,8,9]
A broadening of the generalized polarization (GP) distribution is observed as the system temperature is lowered towards the main lipid phase transition temperature independently of the specific lipid species under investigation as shown in Fig. 1 where the variation of GP pixel standard deviation is plotted against the normalized temperature for three different phosphocholines: DPPC, DMPC and DEPC (16:0 16:0; 14:0 14:0; 13:0 13:0-PC respectively)
Summary
Lipid molecular interactions have been the subject of extensive biophysical investigation due to their relevance in the understanding of functioning and dynamics of biological membranes. Recent investigations using fluorescence based techniques such as single particle tracking, confocal and STED fluorescence correlation spectroscopy, foster resonance energy transfer have revealed that lipid phase domains in biological membranes are small and transient in nature [5,6,7,8,9] For this reason high spatial and temporal resolution is needed for these studies. Among the available model membrane systems, giant unilamellar vesicles (GUVs) are extremely useful for light microscopy experiments due to their cell-like geometry and size which allows direct visualization of extended membrane portions at submicron resolution under the microscope These systems provide a free standing bilayer eliminating possible artifacts due to the interaction with the substrate observed in supported bilayers, which can hinder investigation of processes such as lipid phase transition and lipid diffusion [10]. In the case of binary mixtures displaying phase separation, the analysis of the fluctuations of Laurdan GP along a line spanning different domains revealed the spatial correlations of the fluctuations and the movement of the borders between domains [43]
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