Quantitative analysis of the factors that affect the determination of colocalization coefficients in dual-color confocal images

Colocalization analysis is a subject of great interest that allows us to determine those locations at which two or more types of molecules or structures are found simultaneously. In confocal dual-color images, in order to consider a given position as colocalized, the following two conditions must be satisfied: 1) the colors emitted by the molecules labeled with different fluorophores must occupy the same pixel in the image and 2) the intensities of each component of the image must be within a certain range. Since it is not straightforward to assess these two conditions without ambiguity, this can lead to either considering false colocalizations as positive, or, conversely, leaving out positive locations. In fact, at present, there is not a general procedure to determine the area of colocalization that contains those pixels that can be considered as really colocalized (i.e., the pixels for which the above conditions are fulfilled). As a result, it is the user who must decide on the appropriate selection which introduces personal bias. These issues and the guides for a computational procedure independent of the user that allows us to quantify the degree of colocalization in a dual-color system are discussed.