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Colocalization in microscopy refers to different data analysis methods to characterize the degree of overlap between two channels in an image (conventionally called R and G channels, or red and green channels, independently of the WaveLength they have actually registered). A typical application in fluorescence microscopy is to study the presence of two labeled targets in the same region of a cell. This region to be analyzed can be further defined using region of interest (ROI) tools within Huygens Object Analyzer, Colocalization Analyzer, and Object Tracker.

For an accessible introduction see Colocalization Coefficients and Colocalization Basics.

Mind that Blur And Noise affect Colocalization.

See Colocalization Analyzer and Colocalization Theory for more details.

Object colocalization

Notice that the Object Analyzer in the Huygens Software also provides colocalization measurements at the object level. The Colocalization Analyzer works more at the level of the whole image, despite some local statistics of the colocalizing regions can be easily retrieved.

Both analyzers work, in this sense, in complementary ways.

• The Object Analyzer allows you to define objects (see Object Segmentation) and see how much they overlap, in volume or intensity. Objects defined like this can overlap with other objects, or not. You can filter out objects that do not colocalize at all.
• The Colocalization Analyzer explores the whole image to search for colocalizing regions based on the usual colocalization coefficients. These regions are then segmented and treated as objects to analyze. These objects are therefore always volumes of intersection.