The lateral distribution of cell-surface proteins and their co-localization in membrane microdomains can be studied by digital imaging microscopy with a resolution of 200-300 nm, as set by Abbe's law . The application of confocal laser scanning microscopy or multiphoton excitation  greatly improves image sharpness and contrast by excluding photons arising from out-of-focus planes. This resolution is not sufficient to directly indicate molecular associations; however, it allows the observation of overlap/segregation between different protein clusters or lipid microdomains. Quantitative analysis of the size of clusters/microdomains can be derived from the spatial autocorrelation function of the fluorescence intensity distribution . Using this approach, cluster sizes of predominantly raft-localized proteins IL-2Ra, HLA I and II, as well as GPI-anchored proteins CD48, were determined to be ~600-700 nm, which coincided well with cluster sizes determined from electron microscopic analysis of immunogold-labeled samples. These domain sizes correlated well with the mean barrier-free path measured for the MHC I glycoprotein and its truncation mutants , as well as dimensions of confinement zones derived from single particle tracking in which E-cadherin or epidermal growth factor receptor (EGFR) molecules could freely diffuse . Disruption of lipid rafts by cholesterol extraction blurred the boundaries of protein clusters and extended their diameter to ~1000 nm .
The extent of overlap between clusters of different membrane proteins and lipid microdomains can be characterized by the cross-correlation coefficient of the pixel intensities of individual fluorescence distributions [11,39,40]. For a pair of images x and y, the cross-correlation coefficient is calculated as:
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