A practical guide to evaluating colocalization in biological microscopy

Am J Physiol Cell Physiol. 2011 Apr;300(4):C723-42. doi: 10.1152/ajpcell.00462.2010. Epub 2011 Jan 5.


Fluorescence microscopy is one of the most powerful tools for elucidating the cellular functions of proteins and other molecules. In many cases, the function of a molecule can be inferred from its association with specific intracellular compartments or molecular complexes, which is typically determined by comparing the distribution of a fluorescently labeled version of the molecule with that of a second, complementarily labeled probe. Although arguably the most common application of fluorescence microscopy in biomedical research, studies evaluating the "colocalization" of two probes are seldom quantified, despite a diversity of image analysis tools that have been specifically developed for that purpose. Here we provide a guide to analyzing colocalization in cell biology studies, emphasizing practical application of quantitative tools that are now widely available in commercial and free image analysis software.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Biology / methods*
  • Fluorescent Dyes / metabolism*
  • Image Interpretation, Computer-Assisted / methods*
  • Immunohistochemistry / methods
  • Microscopy, Fluorescence / methods*
  • Software


  • Fluorescent Dyes