Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Mechanistic investigation of mEos4b reveals a strategy to reduce track interruptions in sptPALM

Nature Methods volume 16, pages 707–710 (2019)Cite this article

Subjects

Abstract

Green-to-red photoconvertible fluorescent proteins repeatedly enter dark states, causing interrupted tracks in single-particle-tracking localization microscopy (sptPALM). We identified a long-lived dark state in photoconverted mEos4b that results from isomerization of the chromophore and efficiently absorbs cyan light. Addition of weak 488-nm light swiftly reverts this dark state to the fluorescent state. This strategy largely eliminates slow blinking and enables the recording of longer tracks in sptPALM with minimum effort.

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

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Learn more

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

Fig. 1: mEos4b mechanistic studies.
Fig. 2: Increase in mEos4b track length on 488-nm light illumination.

Data availability

Crystal structures of mEos4b in its green, red and long-lived dark state are available in the Protein Data Bank with accession codes 6GOY, 6GP0 and 6GP1, respectively. The main figures of the paper have associated raw data. Raw data for Supplementary Figures are available upon request. All unique biological materials used in this study and the data that support the findings are available from the corresponding authors upon reasonable request.

References

  1. Shen, H. et al. Chem. Rev. 117, 7331–7376 (2017).

    Article  CAS  Google Scholar 

  2. Acharya, A. et al. Chem. Rev. 117, 758–795 (2017).

    Article  CAS  Google Scholar 

  3. Annibale, P., Vanni, S., Scarselli, M., Rothlisberger, U. & Radenovic, A. Nat. Methods 8, 527–528 (2011).

    Article  CAS  Google Scholar 

  4. Paez-Segala, M. G. et al. Nat. Methods 12, 215–218 (2015).

    Article  CAS  Google Scholar 

  5. Lee, S.-H., Shin, J. Y., Lee, A. & Bustamante, C. Proc. Natl Acad. Sci. USA 109, 17436–17441 (2012).

    Article  CAS  Google Scholar 

  6. Berardozzi, R., Adam, V., Martins, A. & Bourgeois, D. J. Am. Chem. Soc. 138, 558–565 (2016).

    Article  CAS  Google Scholar 

  7. Avilov, S. et al. PLoS ONE 9, e98362 (2014).

    Article  Google Scholar 

  8. Chenouard, N. et al. Nat. Methods 11, 281–289 (2014).

    Article  CAS  Google Scholar 

  9. Annibale, P., Vanni, S., Scarselli, M., Rothlisberger, U. & Radenovic, A. PLoS ONE 6, e22678 (2011).

    Article  CAS  Google Scholar 

  10. Bourgeois, D. Int. J. Mol. Sci. 18, 1187 (2017).

    Article  Google Scholar 

  11. Bourgeois, D. & Adam, V. IUBMB Life 64, 482–491 (2012).

    Article  CAS  Google Scholar 

  12. Dedecker, P., De Schryver, F. C. & Hofkens, J. J. Am. Chem. Soc. 135, 2387–2402 (2013).

    Article  CAS  Google Scholar 

  13. Thédié, D., Berardozzi, R., Adam, V. & Bourgeois, D. J. Phys. Chem. Lett. 8, 4424–4430 (2017).

    Article  Google Scholar 

  14. Grimm, J. B. et al. Nat. Methods 13, 985–988 (2016).

    Article  CAS  Google Scholar 

  15. Saurabh, S., Perez, A. M., Comerci, C. J., Shapiro, L. & Moerner, W. E. J. Am. Chem. Soc. 138, 10398–10401 (2016).

    Article  CAS  Google Scholar 

  16. Tsunoyama, T. A. et al. Nat. Chem. Biol. 14, 497–506 (2018).

    Article  CAS  Google Scholar 

  17. Liu, H. et al. Proc. Natl Acad. Sci. USA 115, 343–348 (2018).

    Article  CAS  Google Scholar 

  18. Basu, S. et al. Nat. Commun. 9, 2520 (2018).

    Article  Google Scholar 

  19. Nguyen, H. L., Gruber, D., Mcgraw, T., Bulinski, J. C. & Sheetz, M. P. Biol. Bull. 194, 354–357 (2007).

    Article  Google Scholar 

  20. Adam, V. et al. Proc. Natl Acad. Sci. USA 105, 18343–18348 (2008).

    Article  CAS  Google Scholar 

  21. Fricke, F., Beaudouin, J., Eils, R. & Heilemann, M. Sci. Rep. 5, 14072 (2015).

    Article  Google Scholar 

  22. Ando, R., Hama, H., Yamamoto-Hino, M., Mizuno, H. & Miyawaki, A. Proc. Natl Acad. Sci. USA 99, 12651–12656 (2002).

    Article  CAS  Google Scholar 

  23. Sawano, A. Nucleic Acids Res. 28, e78 (2000).

    Article  CAS  Google Scholar 

  24. Duwé, S. et al. ACS Nano 9, 9528–9541 (2015).

    Article  Google Scholar 

  25. Ovesný, M., Křížek, P., Borkovec, J., Švindrych, Z. & Hagen, G. M. Bioinformatics 30, 2389–2390 (2014).

    Article  Google Scholar 

  26. Dedecker, P., Duwé, S., Neely, R. K. & Zhang, J. J. Biomed. Opt. 17, 126005–126008 (2012).

    Article  Google Scholar 

  27. English, B. P. et al. Nat. Methods 12, 838–840 (2015).

    Article  Google Scholar 

  28. Jaqaman, K. et al. Nat. Methods 5, 695–702 (2008).

    Article  CAS  Google Scholar 

  29. Byrdin, M. & Dominique, B. Spectrosc. Eur. 28, 14–17 (2016).

    CAS  Google Scholar 

  30. Kabsch, W. Acta Crystallogr. Sect. D 66, 125–132 (2010).

    Article  CAS  Google Scholar 

  31. McCoy, A. J. et al. J. Appl. Crystallogr. 40, 658–674 (2007).

    Article  CAS  Google Scholar 

  32. Nienhaus, K., Nienhaus, G. U., Wiedenmann, J. & Nar, H. Proc. Natl Acad. Sci. USA 102, 9156–9159 (2005).

    Article  CAS  Google Scholar 

  33. Adams, P. D. et al. Acta Crystallogr. D 66, 213–221 (2010).

    Article  CAS  Google Scholar 

  34. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Acta Crystallogr. D 66, 486–501 (2010).

    Article  CAS  Google Scholar 

  35. Moriarty, N. W., Grosse-Kunstleve, R. W. & Adams, P. D. Acta Crystallogr. D 65, 1074–1080 (2009).

    Article  CAS  Google Scholar 

  36. Burnley, B. T., Afonine, P. V., Adams, P. D. & Gros, P. eLife 1, e00311 (2012).

    Article  Google Scholar 

  37. Terwilliger, T. C. & Berendzen, J. Acta Crystallogr. D 51, 609–618 (1995).

    Article  CAS  Google Scholar 

  38. Ursby, T. & Bourgeois, D. Acta Crystallogr. A 53, 564–575 (1997).

    Article  Google Scholar 

  39. Henderson, R. & Moffat, J. K. Acta Crystallogr. B 27, 1414–1420 (2002).

    Article  Google Scholar 

  40. Coquelle, N. et al. Nat. Chem. 10, 31–37 (2018).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by the Agence Nationale de la Recherche (grant no. ANR-17-CE11-0047-01 to D.B.) and used the M4D imaging platform of the Grenoble Instruct-ERIC Center (ISBG: UMS 3518 CNRS-CEA-UGA-EMBL) with support from FRISBI (grant no. ANR-10-INBS-05-02) and GRAL (grant no. ANR-10-LABX-49-01) within the Grenoble Partnership for Structural Biology (PSB). E.D.Z. thanks the Research Foundation Flanders (FWO) for a doctoral fellowship and travel bursary. S.H. thanks the FWO for a postdoctoral fellowship. We acknowledge support from the European Research Council for ERC Starting Grant NanoCellActivity and the FWO for grant G0B8817N (both to P.D.). The authors thank the staff of beamline ID-23-1 from the ESRF, Grenoble, France. We acknowledge W. Vandenberg, I. Arnal and U. Endesfelder for insightful discussions. We thank N. Zala, J.P. Kleman, A. Conte-Daban and I. Govaerts for practical assistance. Part of the SPT analysis was conducted using resources provided by the VSC (Flemish Supercomputer Center), funded by the Research Foundation Flanders (FWO) and the Flemish Government, Department for Economy, Science and Innovation (EWI).

Author information

Authors and Affiliations

  1. Department of Chemistry, KU Leuven, Heverlee, Belgium

    Elke De Zitter, Viola Mönkemöller, Siewert Hugelier, Luc Van Meervelt & Peter Dedecker

  2. University Grenoble Alpes, CEA, CNRS, IBS, Grenoble, France

    Daniel Thédié, Joël Beaudouin, Virgile Adam, Martin Byrdin & Dominique Bourgeois

Authors
  1. Elke De Zitter
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Daniel Thédié
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Viola Mönkemöller
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Siewert Hugelier
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Joël Beaudouin
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Virgile Adam
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Martin Byrdin
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Luc Van Meervelt
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Peter Dedecker
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Dominique Bourgeois
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

D.B. and P.D. designed the project with E.D.Z. E.D.Z., V.A., M.B. and D.B. carried out mechanistic investigations. D.T. performed in vitro SM and ensemble experiments with contributions from E.D.Z. and D.B. V.M. initiated application to sptPALM. V.M., S.H. and P.D. performed and analyzed sptPALM experiments. S.H., V.M. and D.T. performed JF dye experiments and analysis. D.T. and J.B. performed fixed cell experiments. D.B. performed Monte Carlo simulations with assistance from S.H. L.V.M. assisted in crystallographic analysis. E.D.Z., D.T., P.D. and D.B. wrote the manuscript with input from all authors.

Corresponding authors

Correspondence to Peter Dedecker or Dominique Bourgeois.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Peer review information: Rita Strack was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Figs. 1–30, Supplementary Tables 1–4 and Supplementary Notes 1–9

Reporting Summary

Supplementary Video 1: MAP4-mEos4b single-particle tracking experiments in the absence of additional 488-nm light.

Clip of the raw data from a sptPALM experiment. The sptPALM experiments with mEos4b were performed under 561-nm (55 W cm–2, 40 ms) and 405-nm (about 0.8 mW cm–2) illumination. (Movie plays at half speed: 12.5 frames per second, field of view: 24 ×24 µm2). The movie is representative of n > 10 cells.

Supplementary Video 2: MAP4-mEos4b single-particle tracking experiments using additional 488-nm illumination.

Clip of the raw data from a sptPALM experiment. The sptPALM experiments with mEos4b were performed under 561-nm (55 W cm–2, 40 ms) with additional 488-nm illumination (4.80 W cm–2). (Movie plays at half speed: 12.5 frames per second, field of view: 24 × 24 µm2). The movie is representative of n > 10 cells.

Supplementary Video 3: PA-JF549 single-particle tracking experiments.

Clip of the raw data from a sptPALM experiment. The sptPALM experiment with HaloTag–MAP4 labeled with the HaloTag ligand PA-JF549 was performed under 561-nm (55 W cm–2, 40 ms) and 405-nm (about 1 mW cm–2) illumination. (Movie plays at half speed: 12.5 frames per second, field of view: 24 × 24 µm2). The movie is representative of n > 5 cells.

Supplementary Video 4: Trace of a long track of a MAP4-mEos4b molecule in a single-particle tracking experiments using additional 488-nm illumination.

Clip of the raw data from a sptPALM experiment showing one of the long tracks presented in Fig. Figure 2, and being progressively traced along the frames. (Movie: 12.5 frames s–1; field of view: 4 × 4 µm2). The movie is representative of n >> 100 tracks.

Source data

Rights and permissions

Reprints and Permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

De Zitter, E., Thédié, D., Mönkemöller, V. et al. Mechanistic investigation of mEos4b reveals a strategy to reduce track interruptions in sptPALM. Nat Methods 16, 707–710 (2019). https://doi.org/10.1038/s41592-019-0462-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41592-019-0462-3

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing