Calibration and Limits of Camera-Based Fluorescence Correlation Spectroscopy: A Supported Lipid Bilayer Study

摘要: Camera-based fluorescence correlation spectroscopy (FCS) approaches allow the measurement of thousands contiguous points yielding excellent statistics and details sample structure. Imaging total internal reflection FCS (ITIR-FCS) provides these measurements on lipid membranes. Herein, we determine influence point spread function (PSF) optical system, laser power used, time resolution camera accuracy diffusion coefficient concentration measurements. We demonstrate that PSF can be accurately determined by ITIR-FCS varied over a wide range with limited whereas are sensitive to changes in parameters. One advantage is has performed only once for given setup, contrast confocal which calibrations have at least per day. Using optimized experimental conditions provide coefficients ten different membranes consisting one, two three constituents, measured 200000 individual functions. software binning thus inherent providing multiple observation areas single test law show how they complemented local information provided difference cross-correlation functions (ΔCCF). With determination optimization becomes calibration-free method. This allows us absolute bilayers compositions, were stable many bilayer preparations one year, using calibration.