Imaging Fluorescence Correlation Spectroscopy Characterizes Membrane Dynamics and Organization

Abstract

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