Colocalization analysis in fluorescence microscopy

Methods Mol Biol. 2013;931:97-109. doi: 10.1007/978-1-62703-056-4_5.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Membrane / metabolism
  • Fluorescent Dyes / chemistry*
  • Image Processing, Computer-Assisted*
  • Intracellular Space / metabolism
  • Microscopy, Fluorescence / methods
  • Microspheres
  • Single-Cell Analysis
  • Software


  • Fluorescent Dyes