Measuring colocalization by dual color single molecule imaging. Thresholds, error rates, and sensitivity

Dual color single molecule tracking is a state-of-the-art fluorescence microscopy technique to study molecular associations. Featuring high spatial and temporal resolution, the method allows for catching associations with extremely high reliability. In this review, we describe robust data analysis approaches to identify and characterize molecular associations from dual color fluorescence images and show how colocalization can be statistically discriminated from accidental colocalizations. We discriminate the case of static analysis where colocalizations are revealed from individual dual color fluorescence images, and the case of dynamic analysis where colocalizations are tracked in time.