Skip to main content
SpringerLink
Log in

Bright cyan fluorescent protein variants identified by fluorescence lifetime screening

  • Brief Communication
  • Published:

From Nature Methods

View current issue Submit your manuscript

Abstract

Optimization of autofluorescent proteins by intensity-based screening of bacteria does not necessarily identify the brightest variant for eukaryotes. We report a strategy to screen excited state lifetimes, which identified cyan fluorescent proteins with long fluorescence lifetimes (>3.7 ns) and high quantum yields (>0.8). One variant, mTurquoise, was 1.5-fold brighter than mCerulean in mammalian cells and decayed mono-exponentially, making it an excellent fluorescence resonance energy transfer (FRET) donor.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Figure 1: Fluorescence lifetime screening and spectral properties of cyan fluorescent protein variants.
Figure 2: Three-component lifetime unmixing of cyan fluorescent protein variants from a single (frequency) FLIM experiment.

Similar content being viewed by others

References

  1. Heim, R. & Tsien, R.Y. Curr. Biol. 6, 178–182 (1996).

    Article  CAS  Google Scholar 

  2. Chudakov, D.M., Lukyanov, S. & Lukyanov, K.A. Trends Biotechnol. 23, 605–613 (2005).

    Article  CAS  Google Scholar 

  3. Miyawaki, A. Dev. Cell 4, 295–305 (2003).

    Article  CAS  Google Scholar 

  4. Kremers, G.J., Goedhart, J., van Munster, E.B. & Gadella, T.W. Jr. Biochemistry 45, 6570–6580 (2006).

    Article  CAS  Google Scholar 

  5. Rizzo, M.A., Springer, G.H., Granada, B. & Piston, D.W. Nat. Biotechnol. 22, 445–449 (2004).

    Article  CAS  Google Scholar 

  6. van Munster, E.B. & Gadella, T.W. Adv. Biochem. Eng. Biotechnol. 95, 143–175 (2005).

    PubMed  Google Scholar 

  7. Merzlyak, E.M. et al. Nat. Methods 4, 555–557 (2007).

    Article  CAS  Google Scholar 

  8. Mena, M.A., Treynor, T.P., Mayo, S.L. & Daugherty, P.S. Nat. Biotechnol. 24, 1569–1571 (2006).

    Article  CAS  Google Scholar 

  9. Nguyen, A.W. & Daugherty, P.S. Nat. Biotechnol. 23, 355–360 (2005).

    Article  CAS  Google Scholar 

  10. Chen, Y., Muller, J.D., So, P.T. & Gratton, E. Biophys. J. 77, 553–567 (1999).

    Article  CAS  Google Scholar 

  11. Szymczak, A.L. et al. Nat. Biotechnol. 22, 589–594 (2004).

    Article  CAS  Google Scholar 

  12. Digman, M.A., Caiolfa, V.R., Zamai, M. & Gratton, E. Biophys. J. 94, L14–L16 (2008).

    Article  CAS  Google Scholar 

  13. Pepperkok, R., Squire, A., Geley, S. & Bastiaens, P.I. Curr. Biol. 9, 269–272 (1999).

    Article  CAS  Google Scholar 

  14. Verveer, P.J., Wouters, F.S., Reynolds, A.R. & Bastiaens, P.I. Science 290, 1567–1570 (2000).

    Article  CAS  Google Scholar 

  15. Yasuda, R. et al. Nat. Neurosci. 9, 283–291 (2006).

    Article  CAS  Google Scholar 

  16. Riedl, J. et al. Nat. Methods 5, 605–607 (2008).

    Article  CAS  Google Scholar 

  17. Vermeer, J.E., Van Munster, E.B., Vischer, N.O. & Gadella, T.W. Jr. J. Microsc. 214, 190–200 (2004).

    Article  CAS  Google Scholar 

  18. Bacia, K. & Schwille, P. Methods 29, 74–85 (2003).

    Article  CAS  Google Scholar 

  19. Perroud, T.D., Huang, B. & Zare, R.N. ChemPhysChem 6, 905–912 (2005).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank I. Elzenaar and M. Adjobo-Hermans for pilot experiments on the 2A co-expression assay and the members of our laboratory for encouraging discussions, J.E.M. Vermeer (University of Amsterdam) for providing plasmid encoding peroxi-YFP with a C-terminal SKL peroxisomal targeting sequence and E.L. Snapp (Albert Einstein College of Medicine) for providing plasmid encoding ER-tdTomato. Part of this work is supported by the EU integrated project on 'Molecular Imaging' (LSHG-CT-2003-503259).

Author information

Author notes

  1. Joachim Goedhart and Laura van Weeren: These authors contributed equally to this work.

Authors and Affiliations

  1. Swammerdam Institute for Life Sciences, Section of Molecular Cytology, Centre for Advanced Microscopy, University of Amsterdam, Amsterdam, The Netherlands

    Joachim Goedhart, Laura van Weeren, Mark A Hink, Norbert O E Vischer & Theodorus W J Gadella Jr

  2. Division of Cell Biology, The Netherlands Cancer Institute, Amsterdam, The Netherlands

    Kees Jalink

Authors
  1. Joachim Goedhart
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Laura van Weeren
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Mark A Hink
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Norbert O E Vischer
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Theodorus W J Gadella Jr
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

J.G. and T.W.J.G. designed research; J.G., L.v.W., M.A.H., K.J. and T.W.J.G. conducted experiments; J.G., L.v.W., M.A.H., N.O.E.V. and T.W.J.G. analyzed data; and J.G. and T.W.J.G. wrote the paper.

Corresponding author

Correspondence to Theodorus W J Gadella Jr.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6, Supplementary Table 1, Supplementary Note and Supplementary Data (PDF 4861 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Goedhart, J., van Weeren, L., Hink, M. et al. Bright cyan fluorescent protein variants identified by fluorescence lifetime screening. Nat Methods 7, 137–139 (2010). https://doi.org/10.1038/nmeth.1415

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nmeth.1415

  • Springer Nature America, Inc.

This article is cited by

Search

Navigation