Dual-color fluorescence cross-correlation spectroscopy (FCCS) allows for the determination of molecular mobility and concentrations and for the quantitative analysis of molecular interactions such as binding or cleavage at very low concentrations. This protocol discusses considerations for preparing a biological system for FCCS experiments and offers practical advice for performing FCCS on a commercially available setup. Although FCCS is closely related to two-color confocal microscopy, critical adjustments and test measurements are necessary to establish successful FCCS measurements, which are described in a step-by-step manner. Moreover, we discuss control experiments for a negative cross-correlation artifact, arising from a lack of detection volume overlap, and a positive artifact, arising from cross-talk. FCCS has been applied to follow molecular interactions in solutions, on membranes and in cells and to analyze dynamic colocalization during intracellular transport. It is a technique that is expected to see new applications in various fields of biochemical and cell biological research.
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