Quantifying spatial correlations of fluorescent markers using enhanced background reduction with protein proximity index and correlation coefficient estimations (original) (raw)

Nature Protocols volume 6, pages 1554–1567 (2011)Cite this article

Subjects

Abstract

Interactions of proteins are examined by detecting their overlap using fluorescent markers. The observed overlap is then quantified to serve as a measure of spatial correlation. A major drawback of this approach is that it can produce false values because of the properties of the image background. To remedy this, we provide a protocol to reduce the contribution of image background and then apply a protein proximity index (PPI) and correlation coefficient to estimate colocalization. Background heterogeneity is reduced by the median filtering procedure, comprising two steps, to reduce random noise and background, respectively. Alternatively, background can be reduced by advanced thresholding. PPI provides separate values for each channel to characterize the contribution of each protein, whereas correlation coefficient determines the overall colocalization. The protocol is demonstrated using computer-simulated and real biological images. It minimizes human bias and can be universally applied to various cell types in which there is a need to understand protein-protein interactions. Background reductions require 3–5 min per image. Quantifications take <1 min. The entire procedure takes approximately 15–30 min.

This is a preview of subscription content, access via your institution

Access options

Subscribe to this journal

Receive 12 print issues and online access

$259.00 per year

only $21.58 per issue

Buy this article

Prices may be subject to local taxes which are calculated during checkout

Additional access options:

Similar content being viewed by others

References

  1. Kuriyan, J. & Eisenberg, D. The origin of protein interactions and allostery in colocalization. Nature 450, 983–990 (2007).
    Article CAS Google Scholar
  2. Konig, P. et al. FRET-CLSM and double-labeling indirect immunofluorescence to detect close association of proteins in tissue sections. Lab. Invest. 86, 853–864 (2006).
    Article Google Scholar
  3. Smallcombe, A. Multicolor imaging: the important question of co-localization. BioTechniques 30, 1240–1242, 1244–1246 (2001).
    Article CAS Google Scholar
  4. Zinchuk, V. & Grossenbacher-Zinchuk, O. Recent advances in quantitative colocalization analysis: focus on neuroscience. Prog. Histochem. Cytochem. 44, 125–172 (2009).
    Article CAS Google Scholar
  5. Manders, E.M.M., Verbeek, F.J. & Aten, J.A. Measurement of co-localization of objects in dual-colour confocal images. J. Microsc. 169, 375–382 (1993).
    Article Google Scholar
  6. Bolte, S. & Cordelieres, F.P. A guided tour into subcellular colocalization analysis in light microscopy. J. Microsc. 224, 213–232 (2006).
    Article CAS Google Scholar
  7. Zinchuk, V. & Zinchuk, O. Quantitative colocalization analysis of confocal fluorescence microscopy images. Curr. Protoc. Cell Biol. Chapter 4 Unit 4 19 (2008).
  8. French, A.P., Mills, S., Swarup, R., Bennett, M.J. & Pridmore, T.P. Colocalization of fluorescent markers in confocal microscope images of plant cells. Nat. Protoc. 3, 619–628 (2008).
    Article CAS Google Scholar
  9. Waters, J.C. Accuracy and precision in quantitative fluorescence microscopy. J. Cell Biol. 185, 1135–1148 (2009).
    Article CAS Google Scholar
  10. Ono, M. et al. Quantitative comparison of anti-fading media for confocal laser scanning microscopy. J. Histochem. Cytochem. 49, 305–312 (2001).
    Article CAS Google Scholar
  11. Landmann, L. & Marbet, P. Colocalization analysis yields superior results after image restoration. Microsc. Res. Tech. 64, 103–112 (2004).
    Article Google Scholar
  12. Zinchuk, V., Zinchuk, O., Akimaru, K., Moriya, F. & Okada, T. Ethanol consumption alters expression and colocalization of bile salt export pump and multidrug resistance protein 2 in the rat. Histochem. Cell Biol. 127, 503–512 (2007).
    Article CAS Google Scholar
  13. Shaw, P. & Rawlins, D.J. The point spread function of confocal microscope: its measurement and use in deconvolution. J. Microsc. 163, 151–165 (1991).
    Article Google Scholar
  14. Wu, Y. et al. Quantitative determination of spatial protein-protein correlations in fluorescence confocal microscopy. Biophys. J. 98, 493–504 (2010).
    Article CAS Google Scholar
  15. Lippincott-Schwartz, J. & Manley, S. Putting super-resolution fluorescence microscopy to work. Nat. Methods 6, 21–23 (2009).
    Article CAS Google Scholar
  16. Haugland, R.P. The Handbook: A Guide to Fluorescent Probes and Labeling Technologies 10th edn (Invitrogen, Inc., 2005).
  17. Schneider Gasser, E.M. et al. Immunofluorescence in brain sections: simultaneous detection of presynaptic and postsynaptic proteins in identified neurons. Nat. Protoc. 1, 1887–1897 (2006).
    Article CAS Google Scholar
  18. Adler, J. & Parmryd, I. Quantifying colocalization by correlation: the Pearson correlation coefficient is superior to the Mander's overlap coefficient. Cytometry A 77, 733–742 (2010).
    Article Google Scholar

Download references

Acknowledgements

This work was partially supported by National Institutes of Health grant no. HL088640 and the American Heart Association Postdoctoral Fellowship no. 10POST4230081. We thank M. Celio (Fribourg University) for help in organizing the study.

Author information

Authors and Affiliations

  1. Department of Anatomy and Cell Biology, Kochi University, Faculty of Medicine, Kochi, Japan
    Vadim Zinchuk
  2. Department of Anesthesiology, Division of Molecular Medicine, David Geffen School of Medicine, University of California at Los Angeles, Los Angeles, California, USA
    Yong Wu & Enrico Stefani
  3. Unit of Anatomy, Faculty of Medicine, University of Fribourg, Fribourg, Switzerland
    Olga Grossenbacher-Zinchuk

Authors

  1. Vadim Zinchuk
    You can also search for this author inPubMed Google Scholar
  2. Yong Wu
    You can also search for this author inPubMed Google Scholar
  3. Olga Grossenbacher-Zinchuk
    You can also search for this author inPubMed Google Scholar
  4. Enrico Stefani
    You can also search for this author inPubMed Google Scholar

Contributions

V.Z. conceived and organized the study, designed and conducted experiments and wrote the paper; Y.W. conducted experiments and contributed to writing, O.G.-Z. designed and conducted experiments and contributed to writing; and E.S. helped to organize the study.

Corresponding author

Correspondence toVadim Zinchuk.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Rights and permissions

About this article

Cite this article

Zinchuk, V., Wu, Y., Grossenbacher-Zinchuk, O. et al. Quantifying spatial correlations of fluorescent markers using enhanced background reduction with protein proximity index and correlation coefficient estimations.Nat Protoc 6, 1554–1567 (2011). https://doi.org/10.1038/nprot.2011.384

Download citation