High-order autocorrelation with imaging fluorescence correlation spectroscopy: Application to IgE on supported planar membranes

The use of high-order autocorrelation with imaging fluorescence correlation spectroscopy is described. Fluorescently labeled, antitrinitrophenyl IgE antibodies were specifically bound to substrate-supported planar membranes composed of trinitrophenylaminocaproyldipalmitoylphosphatidylethanolamine and dipalmitoylphosphatidylcholine. The IgE-coated membranes were illuminated with a laser beam that was totally internally reflected at the substrate/solution interface. The evanescently excited fluorescence arising from the membrane-bound IgE was measured with a CCD camera. The images were corrected for background and for the elliptically Gaussian spatial dependence of the evanescent excitation intensity. A series of high-order pixel-to-pixel spatial fluorescence fluctuation autocorrelation functions was calculated from the images, The autocorrelation functions generated multiple independent parameters which were used to characterize the nonuniform spatial distributions of the membrane-bound IgE. These parameters varied with the IgE density and also changed significantly when the IgE-coated membranes were further treated with unlabeled, polyclonal anti-IgE. The high-order autocorrelation functions calculated from images of planar membranes containing fluorescently labeled lipids rather than bound, labeled IgE demonstrated that the spatial nonuniformities were prominent only in the presence of IgE. Images of fluorescent beads were used to demonstrate the principles and the methods.