Abstract
The measurement of colocalization requires images of two fluorophores that are aligned, with no cross talk, and that the intensities remain within the response range of the microscope. Quantitation depends upon differentiating between the presence and absence of fluorescence, and measurements should be made within biologically relevant regions of interest. Co-occurrence can be measured simply by area or with the M1 and M2 coefficients, and should be compared to random distributions. Correlation analysis should use the Pearson and Spearman coefficients, which need to be measured by replicate based noise corrected correlation to eliminate errors arising from differences in image quality. Ideally, both co-occurrence and correlation should be reported.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Manders E, Verbeek FJ, Aten JA (1993) Measurement of co-localisation of objects in dual-colour confocal images. J Microsc 169:375–382
Adler J, Bergholm F, Pagakis SN, Parmryd I (2008) Noise and colocalization in fluorescence microscopy: solving a problem. Microsc Anal 22:7–10
Cromey DW (2010) Avoiding twisted pixels: ethical guidelines for the appropriate use and manipulation of scientific digital images. Sci Eng Ethics 16:639–667
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
Bolte S, Cordelieres FP (2006) A guided tour into subcellular colocalization analysis in light microscopy. J Microsc 224:213–232
Li Q, Lau A, Morris TJ, Guo L, Fordyce CB et al (2004) A syntaxin 1, Galpha(o), and N-type calcium channel complex at a presynaptic nerve terminal: analysis by quantitative immunocolocalization. J Neurosci 24:4070–4081
Dunn KW, Kamocka MM, McDonald JH (2011) A practical guide to evaluating colocalization in biological microscopy. Am J Physiol Cell Physiol 300:C723–C742
Costes SV, Daelemans D, Cho EH, Dobbin Z, Pavlakis G et al (2004) Automatic and quantitative measurement of protein-protein colocalization in live cells. Biophys J 86:3993–4003
Wong B (2011) Color blindness. Nat Methods 8:441
Adler J, Pagakis SN, Parmryd I (2008) Replicate-based noise corrected correlation for accurate measurements of colocalization. J Microsc 230:121–133
Barlow AL, Macleod A, Noppen S, Sanderson J, Guerin CJ (2010) Colocalization analysis in fluorescence micrographs: verification of a more accurate calculation of pearson’s correlation coefficient. Microsc Microanal 16:710–724
Bergholm F, Adler J, Parmryd I (2010) Analysis of bias in the apparent correlation coefficient between image pairs corrupted by severe noise. J Math Imaging Vis 37:204–219
Acknowledgements
This work was supported by a grant from Signhild Engkvist’s Foundation. The authors declare that they are shareholders in the company, No More Noise.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2012 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Adler, J., Parmryd, I. (2012). Colocalization Analysis in Fluorescence Microscopy. In: Taatjes, D., Roth, J. (eds) Cell Imaging Techniques. Methods in Molecular Biology, vol 931. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-056-4_5
Download citation
DOI: https://doi.org/10.1007/978-1-62703-056-4_5
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-055-7
Online ISBN: 978-1-62703-056-4
eBook Packages: Springer Protocols