Colocalization analysis is the most common technique used for quantitative analysis of fluorescence microscopy images. Several metrics have been developed for measuring the colocalization of two probes, including Pearson's correlation coefficient (PCC) and Manders' correlation coefficient (MCC). However, once measured, the meaning of these measurements can be unclear; interpreting PCC or MCC va...

One of the most routine uses of fluorescence microscopy is colocalization, i.e., the demonstration of a relationship between pairs of biological molecules. Frequently this is presented simplistically by the use of overlays of red and green images, with areas of yellow indicating colocalization of the molecules. Colocalization data are rarely quantified and can be misleading. Our results from bo...

The measurement of colocalization requires images of two fluorophores that are aligned, with no cross talk, and that the intensities remain within the response range of the microscope. Quantitation depends upon differentiating between the presence and absence of fluorescence, and measurements should be made within biologically relevant regions of interest. Co-occurrence can be measured simply b...