Image Correlation Spectroscopy. II. Optimization for Ultrasensitive Detection of Preexisting Platelet-Derived Growth Factor- β Receptor Oligomers on Intact Cells

Abstract

Previously we introduced image correlation spectroscopy (ICS) as an imaging analog of fluorescence correlation spectroscopy (FCS). Implementation of ICS with image collection via a standard fluorescence confocal microscope and computer-based autocorrelation analysis was shown to facilitate measurements of absolute number densities and determination of changes in aggregation state for fluorescently labeled macromolecules. In the present work we illustrate 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 individual images and illustrate how to perform background corrections for autofluorescence and nonspecific fluorescence on cell population means obtained via ICS. The utilization of photon counting confocal imaging with ICS analysis in combination with the background correction techniques outlined enabled us to achieve very low detection limits with standard immunolabeling methods on normal, nontransformed human fibroblasts (AG1523) expressing relatively low numbers of platelet-derived growth factor- β (PDGF- β) receptors. Specifically, we determined that the PDGF- β receptors were preaggregated as tetramers on average with a mean surface density of 2.3 clusters μm −2 after immunolabeling at 4°C. These measurements, which show preclustering of PDGF- β receptors on the surface of normal human fibroblasts, contradict a fundamental assumption of the ligand-induced dimerization model for signal transduction and provide support for an alternative model that posits signal transduction from within preexisting receptor aggregates.