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Colocalization Analysis in Fluorescence Micrographs: Verification of a More Accurate Calculation of Pearson's Correlation Coefficient

Published online by Cambridge University Press:  15 October 2010

Andrew L. Barlow ,
Alasdair MacLeod ,
Samuel Noppen ,
Jeremy Sanderson  and
Christopher J. Guérin
Andrew L. Barlow
Affiliation:
PerkinElmer, Viscount Centre II, Millburn Hill Road, Warwick University Science Park, Coventry CV4 7HS, UK
Alasdair MacLeod
Affiliation:
PerkinElmer, Viscount Centre II, Millburn Hill Road, Warwick University Science Park, Coventry CV4 7HS, UK
Samuel Noppen
Affiliation:
Microscopy Core Facility, Department for Molecular Biomedical Research, Flanders Institute of Biotechnology (VIB), and University of Ghent, Fiers-Schell-Van Montagu Building, Technologiepark 927, B-9052 Ghent, Belgium
Jeremy Sanderson
Affiliation:
Microscopy Core Facility, Department for Molecular Biomedical Research, Flanders Institute of Biotechnology (VIB), and University of Ghent, Fiers-Schell-Van Montagu Building, Technologiepark 927, B-9052 Ghent, Belgium
Christopher J. Guérin*
Affiliation:
Microscopy Core Facility, Department for Molecular Biomedical Research, Flanders Institute of Biotechnology (VIB), and University of Ghent, Fiers-Schell-Van Montagu Building, Technologiepark 927, B-9052 Ghent, Belgium
*
Corresponding author. E-mail: chris.guerin@dmbr.vib-ugent.be
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Abstract

One of the most routine uses of fluorescence microscopy is colocalization, i.e., the demonstration of a relationship between pairs of biological molecules. Frequently this is presented simplistically by the use of overlays of red and green images, with areas of yellow indicating colocalization of the molecules. Colocalization data are rarely quantified and can be misleading. Our results from both synthetic and biological datasets demonstrate that the generation of Pearson's correlation coefficient between pairs of images can overestimate positive correlation and fail to demonstrate negative correlation. We have demonstrated that the calculation of a thresholded Pearson's correlation coefficient using only intensity values over a determined threshold in both channels produces numerical values that more accurately describe both synthetic datasets and biological examples. Its use will bring clarity and accuracy to colocalization studies using fluorescent microscopy.

Keywords

colocalizationfluorescence microscopyPearson's correlationalgorithmquantitation
Type
Fluorescence and Confocal Microscopies
Information
Microscopy and Microanalysis , Volume 16 , Issue 6 , December 2010 , pp. 710 - 724
Copyright
Copyright © Microscopy Society of America 2010

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References

REFERENCES

Adler, J., Pagakis, S.N. & Parmryd, I. (2008). Replicate based noise corrected correlation for accurate measurements of colocalization. J Microsc 230, 121–133.Google Scholar
Adler, J. & Parmryd, I. (2007). Letter to the Editor. J Microsc 227, 83.Google Scholar
Agnati, L.F., Fuxe, K., Torvinen, M., Genedani, S., Franco, R., Watson, S., Nussdorfer, G.G., Leo, G. & Guidolin, D. (2005). New methods to evaluate colocalization of fluorophores in immunocytochemical preparations as exemplified by a study on A2A and D2 receptors in Chinese hamster ovary cells. J Histochem Cytochem 53, 941–953.Google Scholar
Bolte, S. & Cordelières, F.P. (2006). A guided tour into sub-cellular colocalization analysis in light microscopy. J Microsc 224, 213–232.Google Scholar
Bolte, S. & Cordelières, F.P. (2007). Reply to Letter to the Editor. J Microsc 227, 84–85.Google Scholar
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.CrossRefGoogle ScholarPubMed
French, A.P., Mills, S., Swarup, R., Bennett, M.J. & Pridmore, T.P. (2008). Colocalization of fluorescent markers in confocal microscope images of plant cells. Nat Protoc 3(4), 619–628.CrossRefGoogle ScholarPubMed
Landmann, L. & Marbet, P. (2004). Colocalization analysis yields superior results after image restoration. Microsc Res Techniq 64, 103–112.CrossRefGoogle ScholarPubMed
Li, Q., Lau, A., Morris, T.J., Guo, L., Fordyce, C.B. & Stanley, E.F. (2004). A syntaxin 1, Gαo, and N-type calcium channel complex at a presynaptic nerve terminal: Analysis by quantitative immunolocalization. J Neurosc 24, 4070–4081.Google Scholar
Manders, E.M.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.Google Scholar
Manders, E.M.M., Verbeek, F.J. & Aten, J.A. (1993). Measurement of co-localisation of objects in dual-colour confocal images. J Microsc 169, 375–382.CrossRefGoogle Scholar
North, A.J. (2006). Seeing is believing? A beginners' guide to practical pitfalls in image acquisition. J Cell Biol 172, 9–18.CrossRefGoogle ScholarPubMed
Rasband, W.S. (1997–2010). ImageJ. Bethesda, MD: U.S. National Institutes of Health; available at http://rsb.info.nih.gov/ij/.Google Scholar
Scriven, D.R.L., Lynch, R.M. & Moore, E.D.W. (2008). Image acquisition for colocalization using optical microscopy. Am J Physiol Cell Physiol 294, C1119–C1122.Google Scholar
van Steensel, B., Van Binnendijk, E.P., Hornsby, C.D., Van der Voort, H.T.M., 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.CrossRefGoogle ScholarPubMed
Zinchuk, V., Zinchuk, O. & Okada, T. (2007). Quantitative colocalization analysis of multicolor confocal immunofluorescence microscopy images: Pushing pixels to explore biological phenomena. Acta Histochem Cytochem 40, 101–111.CrossRefGoogle ScholarPubMed
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