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Colocalization Basics

Colocalization is defined as the presence of two or more color types of fluorescently-labeled molecules at the same location. Physically, this means that the colors emitted by them occupy the same pixel in the image. Biologically, this means that two or more different molecules attach to the same structure in the cell. In the context of digital imaging, this is described as the spatial overlap of two or more dyes in a multichannel image.


Although colocalization is relative, the information on the appearance of the distinct molecules at the same location can be of particular significance to researchers. If the question is "Does protein A overlap/colocalize with protein B and if so to which degree?", quantitative colocalization provides the answer.


It is important to follow several guidelines to have your colocalization images suitable for quantification. Ignoring them can undermine the reliability of quantitative colocalization analysis or even make your confocal images unusable for it.


CoLocalizer Pro app estimates colocalization by calculating values representing the proportion of colocalized pixels in dual-color fluorescence microscopy images. These values are called colocalization coefficients.


Table for interpretation of the results of colocalization coefficients calculations shows how to understand your data.

Coefficients calculated by CoLocalizer and their significance

Pearson's correlation coefficient Rr is one of the standard measures in pattern recognition. It is used for describing the correlation of the intensity distributions between channels. It takes into consideration the only similarity between shapes while ignoring the intensities of signals:

Rr Coefficient Algorithm

Manders’ overlap coefficient R indicates an overlap of the signals. This coefficient is not sensitive to the limitations of typical fluorescence imaging, such as efficiency of hybridization, sample photobleaching, and camera quantum efficiency:

R Coefficient Algorithm

Overlap coefficients k1 and k2 split the value of colocalization into two separate parameters. k1 and k2 coefficients depend on the sum of the products of the intensities of two channels. Thus, they are sensitive to the differences in the intensities of signals:

K1 Coefficient Algorithm
K2 Coefficient Algorithm

Colocalization coefficients m1 and m2 describe the contribution of each channel to the image ROI. They are not sensitive to the intensities of signals and can be used when the numbers of objects are not equal:

m1 Coefficient Algorithm
m2 Coefficient Algorithm

Colocalization coefficients M1 and M2 describe the contribution of each channel to the scattergram ROI. They are not sensitive to the intensities of signals and can be used when the numbers of objects are not equal:

M1 Coefficient Algorithm
M2 Coefficient Algorithm

Studies performed with the help of CoLocalizer 



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