Imaging Fluorescence Correlation Spectroscopy Characterizes Membrane Dynamics and Organization

摘要: Imaging Total Internal Reflection Fluorescence Correlation Spectroscopy (ITIR-FCS), a camera based FCS approach, characterizes membrane dynamics by measuring the diffusion coefficient at every point on patch and accesses organization using so-called ΔCCF approach in which cross-correlation of neighboring pixels is used to characterize heterogeneity. Here we discuss various technical aspects ITIR-FCS influence time resolution, measurement length, laser power reliability results. We show that can unambiguously determine spread function system this essential obtain proper absolute values for coefficients concentrations. apply first range supported lipid bilayers with increasing complexity from single binary ternary mixtures consisting different phospholipids, sphingomyelin, cholesterol. yields very consistent over dozens bilayer preparations. complement bottom-up whereby an artificial increased step-by-step cellular plasma membrane, top-down live cell membranes their natural state altered composition biochemical approaches. Since be binned per software after recording, it ideal testing laws, dependence size observation area, demonstrate existence domains membranes.