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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: [email protected]
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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

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