July 25, 2025
Colocalization aims to quantify the degree of overlap between two or more channels in an image, for example representing subcellular fluorescence markers. This can be done using a number of different metrics, such as Pearson’s correlation coefficient or Manders’ overlap coefficient. A good overview of these approaches can be found in this review and this Microcourse Youtube video.
Fiji offers a number of plugins for colocalization analysis, however currently the best supported one is the JACoP plugin revamped by BIOP. It implements both Pearson’s and Manders’ coefficients, as well as Costes’ automated thresholding.
CellProfiler provides a MeasureColocalization module for colocalization analysis. The module can be used to calculate a wide range of metrics, such as Pearson’s correlation coefficient, Manders’ overlap coefficient, and Rank Weighted Colocalization coefficient. A helpful example/tutorial is provided on the CellProfiler Examples page.