A practical guide to evaluating colocalization in biological microscopy

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737 Scopus citations


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.

Original languageEnglish (US)
Pages (from-to)C723-C742
JournalAmerican Journal of Physiology - Cell Physiology
Issue number4
StatePublished - Apr 1 2011


  • Confocal microscopy
  • Digital image analysis
  • Fluorescence microscopy

ASJC Scopus subject areas

  • Cell Biology
  • Physiology

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