Image correlation spectroscopy for measurements of particle densities and colocalization

Cells interact with their environment through receptor proteins expressed at their plasma membrane, and protein-protein interactions govern the transduction of signals across the membrane into the cell. Therefore, the ability to measure receptor densities and protein colocalization within the membrane of intact cells is of paramount importance. This unit describes a technique to extract these parameters from fluorescence microscopy images obtained using a commercial confocal laser scanning microscope (CLSM) and other similar types of microscopes. It is based on the analysis of spatial fluorescence intensity fluctuations in the images, which can then be related to particle density and aggregation state via calculation of a spatial autocorrelation function, or used to measure particle colocalization via calculation of a spatial cross-correlation function from dual-color images of proteins tagged with two different fluorophores and imaged in two detection channels. These parameters offer key insights on the interaction of the cell with its environment.