Colocalization Analysis in Fluorescence Microscopy

  • Jeremy Adler
  • Ingela Parmryd
Part of the Methods in Molecular Biology book series (MIMB, volume 931)


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 by area or with the M1 and M2 coefficients, and should be compared to random distributions. Correlation analysis should use the Pearson and Spearman coefficients, which need to be measured by replicate based noise corrected correlation to eliminate errors arising from differences in image quality. Ideally, both co-occurrence and correlation should be reported.

Key words

Colocalization Co-occurrence Correlation Pearson correlation coefficient RBNCC 



This work was supported by a grant from Signhild Engkvist’s Foundation. The authors declare that they are shareholders in the company, No More Noise.


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Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  1. 1.Department of Immunology, Genetics and PathologyUppsala UniversityUppsalaSweden
  2. 2.Department of Medical BiologyUppsala UniversityUppsalaSweden

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