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Colocalization of fluorescent markers in confocal microscope images of plant cells

Nature Protocols volume 3, pages 619–628 (2008)Cite this article

Abstract

This protocol describes the steps needed to perform quantitative statistical colocalization on two-color confocal images, specifically of plant cells. The procedure includes a calibration test to check the chromatic alignment of the confocal microscope. A software tool is provided to calculate the Pearson and Spearman correlation coefficients ('Pearson–Spearman correlation colocalization' ImageJ plug-in) across regions of interest within the image. Steps are included to help the user practice using the software. The result is a quantitative estimate of the amount of colocalization in the images. Manual masking takes about 1–15 min per image, depending on the detail required, and calculating the correlation coefficients is almost instantaneous. Examples of suitable dyes for such two-color colocalization include Oregon Green or Alexa Fluor 488 dyes in the green range (excited with 488-nm laser line) and Alexa Fluor 555 dye in the red range (excited with 543-nm laser line).

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Figure 1: Examples of image outputs from confocal colocalization experiments.
Figure 2: A medium-quality JPEG image, decomposed into intensity (left) and hue (right) channels.
Figure 3
Figure 5: An image with a blue channel mask and an ImageJ selection.
Figure 6: Two types of masks.
Figure 4
Figure 7
Figure 8: Composite image as a result of including Figure 7 as a mask in a red–green image.
Figure 9: Columns from left to right: green channel, red channel, combined image with overlaid mask in blue channel, resulting in scatter plot of intensities across the two channels.
Figure 10: Test image.

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References

  1. North, A.J. Seeing is believing? A beginner's guide to practical pitfalls in image acquisition. J. Cell Biol., 172, 9–18 (2006).

    Article  CAS  Google Scholar 

  2. Bolte, S. & Cordelières, F.P. A guided tour into subcellular colocalization in light microscopy. J. Microsc., 224, 213–232 (2006).

    Article  CAS  Google Scholar 

  3. Costes, S.V., Daelemans, D., Cho, E.H., Dobbin, Z. & Pavlakis, G. Automatic and quantitative measurement of protein–protein colocalization in live cells. Biophys. J., 86, 3993–4003 (2004).

    Article  CAS  Google Scholar 

  4. Manders, E.M.M., Stap, J., Brakenhoff, G.J., Van Driel, R. & Aten, J.A. 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 (1992).

    CAS  Google Scholar 

  5. Lachmanovich, E. et al. Co-localization analysis of complex formation among membrane proteins by computerized fluorescence microscopy: application to immunofluorescence co-patching studies. J. Microsc., 212, 122–131 (2003).

    Article  CAS  Google Scholar 

  6. Glynn, M.W. & McAllister, A.K. Immunocytochemistry and quantification of protein colocalization in cultured neurons. Nat. Protoc., 1, 1287–1296 (2006).

    Article  CAS  Google Scholar 

  7. Heintzmann, R. & Cremer, C. Axial tomographic confocal fluorescence microscopy. J. Microsc., 206, 7–23 (2002).

    Article  CAS  Google Scholar 

  8. Dharmasiri, S. et al. AXR4 is required for localization of the auxin influx facilitator AUX1. Science, 312, 1218–1220 (2006).

    Article  CAS  Google Scholar 

  9. Abramoff, M.D., Magelhaes, P.J. & Ram, S.J. Image processing with ImageJ. Biophotonics Int., 11, 36–42 (2004).

    Google Scholar 

  10. Zucker, R.M. & Price, O. Evaluation of confocal microscopy system performance. Cytometry, 44, 273–294 (2001).

    Article  CAS  Google Scholar 

  11. Manders, E.M.M. Chromatic shift in multicolour confocal microscopy. J. Microsc., 185, 321–328 (1997).

    Article  Google Scholar 

  12. Pawley, J. The 39 steps: a cautionary tale of quantitative 3-D fluorescence microscopy. Biotechniques, 28, 884–888 (2000).

    Article  CAS  Google Scholar 

  13. Pawley, J. Handbook of Confocal Microscopy 3rd edn (Springer Science, New York, 2006).

    Book  Google Scholar 

  14. Smallcombe, A. Multicolor imaging: the important question of co-localization. Biotechniques, 30, 1240–1245 (2001).

    Article  CAS  Google Scholar 

  15. Kass, M., Witkin, A. & Terzopolous, D. Snakes: active contour models. Int. J. Comp. Vis., 1, 321–331 (1988).

    Article  Google Scholar 

  16. Friml, J., Benkova, E., Mayer, U., Palme, K. & Muster, G. Automated whole mount localisation techniques for plant seedlings. Plant J., 34, 115–124 (2003).

    Article  CAS  Google Scholar 

  17. Sauer, M., Paciorek, T., Benkova, E. & Friml, J. Immunocytochemical techniques for whole-mount in-situ protein localization in plants. Nat. Protoc., 1, 98–103 (2006).

    Article  CAS  Google Scholar 

  18. Centonze, V. & Pawley, J. Tutorial on practical confocal microscopy and use of the confocal test specimen. In Handbook of Biological Confocal Microscopy 3rd edn. (ed. Pawley J.) 627–649 (Springer Science and Business Media, New York, 2006).

    Chapter  Google Scholar 

  19. Swarup, R. et al. Structure–function analysis of the presumptive Arabidopsis auxin permease AUX1. Plant Cell, 16, 3069–3083 (2004).

    Article  CAS  Google Scholar 

  20. Li, Q. et al. A Syntaxin 1, Gαo, and N-type calcium channel complex at a presynaptic nerve terminal: analyses by quantitative immunocolocalization. J. Neurosci., 24, 4070–4081 (2004).

    Article  CAS  Google Scholar 

  21. Chinga, G. & Syverud, K. Quantification of paper mass distributions within local picking areas. Nordic Pulp Paper Res. J., 22, 441–446 (2007).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We acknowledge EPSRC and BBSRC CISB programme funding to CPIB and funding from the Belgian Federal IUAPVI programme to M.J.B. and R.S. as part of the BARN consortium.

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Author notes

  1. Steven Mills

    Present address: Present address: Geospatial Research Centre (NZ) Ltd., Private Bag 4800, Christchurch 8140, New Zealand.,

Authors and Affiliations

  1. Centre for Plant Integrative Biology, Main Building, University of Nottingham, Sutton Bonington, LE12 5RD, UK

    Andrew P French, Steven Mills, Ranjan Swarup, Malcolm J Bennett & Tony P Pridmore

  2. School of Computer Science, University of Nottingham, Jubilee Campus, Wollaton Road, Nottingham, NG8 1BB, UK

    Steven Mills & Tony P Pridmore

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  1. Andrew P French
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Correspondence to Andrew P French.

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French, A., Mills, S., Swarup, R. et al. Colocalization of fluorescent markers in confocal microscope images of plant cells. Nat Protoc 3, 619–628 (2008). https://doi.org/10.1038/nprot.2008.31

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