Image correlation spectroscopy. II. Optimization for ultrasensitive detection of preexisting platelet-derived growth factor-beta receptor oligomers on intact cells

Previously we introduced image correlation spectroscopy (ICS) as an imaging analog of fluorescence correlation spectroscopy (FCS). Implementation of I CS with image collection via a standard fluorescence confocal microscope an d computer-based autocorrelation analysis was shown to facilitate measureme nts of absolute number densities and determination of changes in aggregatio n state for fluorescently labeled macromolecules. In the present work we il lustrate how to use ICS to quantify the aggregation state of immunolabeled plasma membrane receptors in an intact cellular milieu, taking into account background fluorescence. We introduce methods that enable us to completely remove white noise contributions from autocorrelation measurements for ind ividual images and illustrate how to perform background corrections for aut ofluorescence and nonspecific fluorescence on cell population means obtaine d via ICS. The utilization of photon counting confocal imaging with ICS ana lysis in combination with the background correction techniques outlined ena bled us to achieve very low detection limits with standard immunolabeling m ethods on normal, nontransformed human fibroblasts (AG1523) expressing rela tively low numbers of platelet-derived growth factor-beta (PDGF-beta) recep tors. Specifically, we determined that the PDGF-beta receptors were preaggr egated as tetramers on average with a mean surface density of 2.3 clusters mu m(-2) after immunolabeling at 4 degrees C. These measurements, which sho w preclustering of PDGF-beta receptors on the surface of normal human fibro blasts, contradict a fundamental assumption of the ligand-induced dimerizat ion model for signal transduction and provide support for an alternative mo del that posits signal transduction from within preexisting receptor aggreg ates.