Application of Fluorescence Correlation Spectroscopy (FCS) to Measure the Dynamics of Fluorescent Proteins in Living Cells

Fluorescence correlation spectroscopy (FCS) can add dynamic molecular information to images of live cells. For example, a confocal laser scanning microscope (CLSM) equipped with an accessory FCS unit provides the possibility to first image the spatial distribution of a fluorescent protein before probing its mobility within defined regions of interest. Whereas specific protein–protein interactions are preferably assayed with a dual-color approach, single-color FCS can still provide valuable information about the size of the diffusing entities and potential interactions with other, nonfluorescent, proteins or subcellular structures. Because number fluctuations are measured, the concentrations of freely diffusing complexes and their state of oligomerization are accessible.