Critical evaluation of quantitative colocalization analysis in confocal fluorescence microscopy

  • Yong WuEmail author
  • Vadim Zinchuk
  • Olga Grossenbacher-Zinchuk
  • Enrico Stefani


Spatial colocalization of fluorescently labeled proteins can reveal valuable information about proteinprotein interactions. Compared to qualitative visual interpretation of dual color images, quantitative colocalization analysis (QCA) provides more objective evaluations to the degree of colocalization. However, the finite resolution power of microscopes and the spatial patterns of intracellular structures may compromise the reliability of many classical QCA methods. In this paper, we discuss the strength and weakness of some mostly used QCA methods. By studying their applications on computer-simulated images and biological images, we show that classical pixel intensity based QCA methods are often vulnerable to coincidental overlapping among resolution elements (resel) distributions and thus not suitable to images with high molecular density or with low resolution. Also, many QCA methods can mistakenly regard long range correlation as colocalization due to protein localization in intracellular structures. The newly developed protein-protein index (PPI) approach is able to reduce the influence from resel overlapping and spatial intracellular pattern compared to previous methods, significantly improving the reliability of QCA.

Key words

fluorescence microscopy confocal microscopy image analysis colocalization 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. [1]
    Adler, J., Parmryd, I. 2010. Quantifying colocalization by correlation: The Pearson correlation coefficient is superior to the Mander’s overlap coefficient. Cytometry A 77, 733–742.PubMedGoogle Scholar
  2. [2]
    Betzig, E., Patterson, G.H., Sougrat, R., Lindwasser, O.W., Olenych, S., Bonifacino, J.S., Davidson, M.W., Lippincott-Schwartz, J., Hess, H.F. 2006. Imaging intracellular fluorescent proteins at nanometer resolution. Science 313, 1642–1645.PubMedCrossRefGoogle Scholar
  3. [3]
    Bolte, S., Cordelieres, F.P. 2006. A guided tour into subcellular colocalization analysis in light microscopy. J Microsc 224, 213–232.PubMedCrossRefGoogle Scholar
  4. [4]
    Boutte, Y., Crosnier, M.T., Carraro, N., Traas, J., Satiat-Jeunemaitre, B. 2006. The plasma membrane recycling pathway and cell polarity in plants: Studies on PIN proteins. J Cell Sci 119, 1255–1265.PubMedCrossRefGoogle Scholar
  5. [5]
    Comeau, J.W.D., Costantino, S., Wiseman, P.W. 2006. A guide to accurate fluorescence microscopy colocalization measurements. Biophys J 91, 4611–4622.PubMedCrossRefGoogle Scholar
  6. [6]
    Comeau, J.W.D., Kolin, D.L., Wiseman, P.W. 2008. Accurate measurements of protein interactions in cells via improved spatial image cross-correlation spectroscopy. Mol Biosyst 4, 672–685.PubMedCrossRefGoogle Scholar
  7. [7]
    Costes, S.V., Daelemans, D., Cho, E.H., Dobbin, Z., Pavlakis, G., Lockett, S. 2004. Automatic and quantitative measurement of protein-protein colocalization in live cells. Biophys J 86, 3993–4003.PubMedCrossRefGoogle Scholar
  8. [8]
    Demandolx, D., Davoust, J. 1997. Multicolour analysis and local image correlation in confocal microscopy. J Microsc 185, 21–36.CrossRefGoogle Scholar
  9. [9]
    Dutartre, H., Davoust, J., Gorvel, J.P., Chavrier, P. 1996. Cytokinesis arrest and redistribution of actincytoskeleton regulatory components in cells expressing the Rho GTPase CDC42Hs. J Cell Sci 109, 367–377.PubMedGoogle Scholar
  10. [10]
    Fox, M.H., Arndt-Jovin, D.J., Jovin, T.M., Baumann, P.H., Robert-Nicoud, M. 1991. Spatial and temporal distribution of DNA replication sites localized by immunofluorescence and confocal microscopy in mouse fibroblasts. J Cell Sci 99, 247–253.PubMedGoogle Scholar
  11. [11]
    Gustafsson, M.G. 2000. Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy. J Microsc 198, 82–87.PubMedCrossRefGoogle Scholar
  12. [12]
    Hell, S.W. 2003. Toward fluorescence nanoscopy. Nat Biotechnol 21, 1347–1355.PubMedCrossRefGoogle Scholar
  13. [13]
    Lachmanovich, E., Shvartsman, D.E., Malka, Y., Botvin, C., Henis, Y.I., Weiss, A.M. 2003. Colocalization analysis of complex formation among membrane proteins by computerized fluorescence microscopy: Application to immunofluorescence copatching studies. J Microsc 212, 122–131.PubMedCrossRefGoogle Scholar
  14. [14]
    Li, Q., Lau, A., Morris, T.J., Guo, L., Fordyce, C.B., Stanley, E.F. 2004. A syntaxin 1, G alpha(o), and Ntype calcium channel complex at a presynaptic nerve terminal: Analysis by quantitative immunocolocalization. J Neurosci 24, 4070–4081.PubMedCrossRefGoogle Scholar
  15. [15]
    Manders, E.M., Stap, J., Brakenhoff, G.J., van Driel, R., Aten, J.A. 1992. Dynamics of three-dimensional replication patterns during the S-phase, analysed by double labelling of DNA and confocal microscopy. J Cell Sci 103, 857–862.PubMedGoogle Scholar
  16. [16]
    Manders, E.M.M., Verbeek, F.J., Aten, J.A. 1993. Measurement of colocalization of objects in dual-color confocal images. J Microsc 169, 375–382.CrossRefGoogle Scholar
  17. [17]
    Ropero, A.B., Eghbali, M., Minosyan, T.Y., Tang, G., Toro, L., Stefani, E. 2006. Heart estrogen receptor alpha: Distinct membrane and nuclear distribution patterns and regulation by estrogen. J Mol Cell Cardiol 41, 496–510.PubMedCrossRefGoogle Scholar
  18. [18]
    van Steensel, B., van Binnendijk, E.P., Hornsby, C.D., van der Voort, H.T., Krozowski, Z.S., de Kloet, E.R., van Driel, R. 1996. Partial colocalization of glucocorticoid and mineralocorticoid receptors in discrete compartments in nuclei of rat hippocampus neurons. J Cell Sci 109, 787–792.PubMedGoogle Scholar
  19. [19]
    Wu, Y., Eghbali, M., Ou, J., Lu, R., Toro, L., Stefani, E. 2010. Quantitative determination of spatial protein-protein correlations in fluorescence confocal microscopy. Biophys J 98, 493–504.PubMedCrossRefGoogle Scholar
  20. [20]
    Zinchuk, V., Grossenbacher-Zinchuk, O. 2009. Recent advances in quantitative colocalization analysis: Focus on neuroscience. Prog Histochem Cytochem 44, 125–172.PubMedCrossRefGoogle Scholar
  21. [21]
    Zinchuk, V., Zinchuk, O. 2008. Quantitative colocalization analysis of confocal fluorescence microscopy images. In: Current Protocols in Cell Biology. Vol 39. John Wiley & Sons, 4.19.1–4.19.16.Google Scholar

Copyright information

© International Association of Scientists in the Interdisciplinary Areas and Springer-Verlag Berlin Heidelberg 2012

Authors and Affiliations

  • Yong Wu
    • 1
    Email author
  • Vadim Zinchuk
    • 2
  • Olga Grossenbacher-Zinchuk
    • 3
  • Enrico Stefani
    • 1
  1. 1.Department of Anesthesiology, David Geffen School of MedicineUniversity of CaliforniaLos AngelesUSA
  2. 2.Department of Anatomy and Cell Biology, Faculty of MedicineKochi UniversityKochiJapan
  3. 3.Institute of AnatomyUniversity of FribourgFribourgSwitzerland

Personalised recommendations