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.

A guide to choosing fluorescent proteins

Abstract

The recent explosion in the diversity of available fluorescent proteins (FPs)1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16 promises a wide variety of new tools for biological imaging. With no unified standard for assessing these tools, however, a researcher is faced with difficult questions. Which FPs are best for general use? Which are the brightest? What additional factors determine which are best for a given experiment? Although in many cases, a trial-and-error approach may still be necessary in determining the answers to these questions, a unified characterization of the best available FPs provides a useful guide in narrowing down the options.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

References

  1. 1

    Nagai, T. et al. A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications. Nat. Biotechnol. 20, 87–90 (2002).

    CAS  Article  Google Scholar 

  2. 2

    Nguyen, A.W. & Daugherty, P.S. Evolutionary optimization of fluorescent proteins for intracellular FRET. Nat. Biotechnol. 23, 355–360 (2005).

    CAS  Article  Google Scholar 

  3. 3

    Rizzo, M.A., Springer, G.H., Granada, B. & Piston, D.W. An improved cyan fluorescent protein variant useful for FRET. Nat. Biotechnol. 22, 445–449 (2004).

    CAS  Article  Google Scholar 

  4. 4

    Shaner, N.C. et al. Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat. Biotechnol. 22, 1567–1572 (2004).

    CAS  Article  Google Scholar 

  5. 5

    Wang, L., Jackson, W.C., Steinbach, P.A. & Tsien, R.Y. Evolution of new nonantibody proteins via iterative somatic hypermutation. Proc. Natl. Acad. Sci. USA 101, 16745–16749 (2004).

    CAS  Article  Google Scholar 

  6. 6

    Zapata-Hommer, O. & Griesbeck, O. Efficiently folding and circularly permuted variants of the Sapphire mutant of GFP. BMC Biotechnol. 3, 5 (2003).

    Article  Google Scholar 

  7. 7

    Ando, R., Hama, H., Yamamoto-Hino, M., Mizuno, H. & Miyawaki, A. An optical marker based on the UV-induced green-to-red photoconversion of a fluorescent protein. Proc. Natl. Acad. Sci. USA 99, 12651–12656 (2002).

    CAS  Article  Google Scholar 

  8. 8

    Chudakov, D.M. et al. Photoswitchable cyan fluorescent protein for protein tracking. Nat. Biotechnol. 22, 1435–1439 (2004).

    CAS  Article  Google Scholar 

  9. 9

    Chudakov, D.M. et al. Kindling fluorescent proteins for precise in vivo photolabeling. Nat. Biotechnol. 21, 191–194 (2003).

    CAS  Article  Google Scholar 

  10. 10

    Karasawa, S., Araki, T., Nagai, T., Mizuno, H. & Miyawaki, A. Cyan-emitting and orange-emitting fluorescent proteins as a donor/acceptor pair for fluorescence resonance energy transfer. Biochem. J. 381, 307–312 (2004).

    CAS  Article  Google Scholar 

  11. 11

    Matz, M.V. et al. Fluorescent proteins from nonbioluminescent Anthozoa species. Nat. Biotechnol. 17, 969–973 (1999).

    CAS  Article  Google Scholar 

  12. 12

    Wiedenmann, J. et al. EosFP, a fluorescent marker protein with UV-inducible green-to-red fluorescence conversion. Proc. Natl. Acad. Sci. USA 101, 15905–15910 (2004).

    CAS  Article  Google Scholar 

  13. 13

    Verkhusha, V.V. & Sorkin, A. Conversion of the monomeric red fluorescent protein into a photoactivatable probe. Chem. Biol. 12, 279–285 (2005).

    CAS  Article  Google Scholar 

  14. 14

    Tsutsui, H., Karasawa, S., Shimizu, H., Nukina, N. & Miyawaki, A. Semi-rational engineering of a coral fluorescent protein into an efficient highlighter. EMBO Rep. 6, 233–238 (2005).

    CAS  Article  Google Scholar 

  15. 15

    Patterson, G.H. & Lippincott-Schwartz, J. Selective photolabeling of proteins using photoactivatable GFP. Methods 32, 445–450 (2004).

    CAS  Article  Google Scholar 

  16. 16

    Griesbeck, O., Baird, G.S., Campbell, R.E., Zacharias, D.A. & Tsien, R.Y. Reducing the environmental sensitivity of yellow fluorescent protein. Mechanism and applications. J. Biol. Chem. 276, 29188–29194 (2001).

    CAS  Article  Google Scholar 

  17. 17

    Zhang, J., Campbell, R.E., Ting, A.Y. & Tsien, R.Y. Creating new fluorescent probes for cell biology. Nat. Rev. Mol. Cell Biol. 3, 906–918 (2002).

    CAS  Article  Google Scholar 

  18. 18

    Tsien, R.Y. The green fluorescent protein. Annu. Rev. Biochem. 67, 509–544 (1998).

    CAS  Article  Google Scholar 

  19. 19

    Gross, L.A., Baird, G.S., Hoffman, R.C., Baldridge, K.K. & Tsien, R.Y. The structure of the chromophore within DsRed, a red fluorescent protein from coral. Proc. Natl. Acad. Sci. USA 97, 11990–11995 (2000).

    CAS  Article  Google Scholar 

  20. 20

    Hansen, M.C., Palmer, R.J., Jr, Udsen, C., White, D.C. & Molin, S. Assessment of GFP fluorescence in cells of Streptococcus gordonii under conditions of low pH and low oxygen concentration. Microbiology 147, 1383–1391 (2001).

    CAS  Article  Google Scholar 

  21. 21

    Zhang, C., Xing, X.H. & Lou, K. Rapid detection of a gfp-marked Enterobacter aerogenes under anaerobic conditions by aerobic fluorescence recovery. FEMS Microbiol. Lett. 249, 211–218 (2005).

    CAS  Article  Google Scholar 

  22. 22

    Verkhusha, V.V. & Lukyanov, K.A. The molecular properties and applications of Anthozoa fluorescent proteins and chromoproteins. Nat. Biotechnol. 22, 289–296 (2004).

    CAS  Article  Google Scholar 

  23. 23

    Zacharias, D.A., Violin, J.D., Newton, A.C. & Tsien, R.Y. Partitioning of lipid-modified monomeric GFPs into membrane microdomains of live cells. Science 296, 913–916 (2002).

    CAS  Article  Google Scholar 

  24. 24

    Long, J.Z., Lackan, C.S. & Hadjantonakis, A.K. Genetic and spectrally distinct in vivo imaging: embryonic stem cells and mice with widespread expression of a monomeric red fluorescent protein. BMC Biotechnol. 5, 20 (2005).

    Article  Google Scholar 

  25. 25

    Zhu, H. et al. Ubiquitous expression of mRFP1 in transgenic mice. Genesis 42, 86–90 (2005).

    CAS  Article  Google Scholar 

  26. 26

    Miesenbock, G., De Angelis, D.A. & Rothman, J.E. Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins. Nature 394, 192–195 (1998).

    CAS  Article  Google Scholar 

  27. 27

    Matsuyama, S., Llopis, J., Deveraux, Q.L., Tsien, R.Y. & Reed, J.C. Changes in intramitochondrial and cytosolic pH: early events that modulate caspase activation during apoptosis. Nat. Cell Biol. 2, 318–325 (2000).

    CAS  Article  Google Scholar 

  28. 28

    Hiraoka, Y., Shimi, T. & Haraguchi, T. Multispectral imaging fluorescence microscopy for living cells. Cell Struct. Funct. 27, 367–374 (2002).

    Article  Google Scholar 

  29. 29

    Habuchi, S. et al. Reversible single-molecule photoswitching in the GFP-like fluorescent protein Dronpa. Proc. Natl. Acad. Sci. USA 102, 9511–9516 (2005).

    CAS  Article  Google Scholar 

  30. 30

    Lukyanov, K.A., Chudakov, D.M., Lukyanov, S. & Verkhusha, V.V. Innovation: Photoactivatable fluorescent proteins. Nat. Rev. Mol. Cell Biol. (2005); advance online publication, 15 September 2005 (10.1038/nrm1741).

  31. 31

    Shkrob, M.A. et al. Far-red fluorescent proteins evolved from a blue chromoprotein from Actinia equina. Biochem. J. (2005); advance online publication,15 September 2005 (10.1042/BJ20051314).

Download references

Acknowledgements

Thanks to S. Adams for helpful advice on choosing filter sets. N.C.S. is a Howard Hughes Medical Institute Predoctoral Fellow. This work was additionally supported by US National Institutes of Health (NS27177 and GM72033) and Howard Hughes Medical Institutes.

Author information

Affiliations

Authors

Corresponding author

Correspondence to Roger Y Tsien.

Ethics declarations

Competing interests

Fluorescent proteins described in the paper that originate from the Tsien lab are covered by pending US and international patents. These patents, when issued, will be property of the University of California.

Supplementary information

Supplementary Fig. 1

mCherry and Emerald photobleaching curves.

Supplementary Table 1

FPs properties in detail.

Supplementary Table 2

Mutations of AFPs relative to wild-type GFP.

Supplementary Table 3

Starting points for advanced FP applications.

Supplementary Discussion

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Shaner, N., Steinbach, P. & Tsien, R. A guide to choosing fluorescent proteins. Nat Methods 2, 905–909 (2005). https://doi.org/10.1038/nmeth819

Download citation

Further reading

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