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.
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
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
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).
Nguyen, A.W. & Daugherty, P.S. Evolutionary optimization of fluorescent proteins for intracellular FRET. Nat. Biotechnol. 23, 355–360 (2005).
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).
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).
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).
Zapata-Hommer, O. & Griesbeck, O. Efficiently folding and circularly permuted variants of the Sapphire mutant of GFP. BMC Biotechnol. 3, 5 (2003).
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).
Chudakov, D.M. et al. Photoswitchable cyan fluorescent protein for protein tracking. Nat. Biotechnol. 22, 1435–1439 (2004).
Chudakov, D.M. et al. Kindling fluorescent proteins for precise in vivo photolabeling. Nat. Biotechnol. 21, 191–194 (2003).
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).
Matz, M.V. et al. Fluorescent proteins from nonbioluminescent Anthozoa species. Nat. Biotechnol. 17, 969–973 (1999).
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).
Verkhusha, V.V. & Sorkin, A. Conversion of the monomeric red fluorescent protein into a photoactivatable probe. Chem. Biol. 12, 279–285 (2005).
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).
Patterson, G.H. & Lippincott-Schwartz, J. Selective photolabeling of proteins using photoactivatable GFP. Methods 32, 445–450 (2004).
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).
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).
Tsien, R.Y. The green fluorescent protein. Annu. Rev. Biochem. 67, 509–544 (1998).
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).
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).
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).
Verkhusha, V.V. & Lukyanov, K.A. The molecular properties and applications of Anthozoa fluorescent proteins and chromoproteins. Nat. Biotechnol. 22, 289–296 (2004).
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).
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).
Zhu, H. et al. Ubiquitous expression of mRFP1 in transgenic mice. Genesis 42, 86–90 (2005).
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).
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).
Hiraoka, Y., Shimi, T. & Haraguchi, T. Multispectral imaging fluorescence microscopy for living cells. Cell Struct. Funct. 27, 367–374 (2002).
Habuchi, S. et al. Reversible single-molecule photoswitching in the GFP-like fluorescent protein Dronpa. Proc. Natl. Acad. Sci. USA 102, 9511–9516 (2005).
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).
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).
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
Authors and Affiliations
Corresponding author
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.
Rights 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
Published:
Issue Date:
DOI: https://doi.org/10.1038/nmeth819
This article is cited by
-
Intravital imaging to study cancer progression and metastasis
Nature Reviews Cancer (2023)
-
Rhizosphere frame system enables nondestructive live-imaging of legume-rhizobium interactions in the soil
Journal of Plant Research (2023)
-
Using the organelle glue technique to engineer the plant cell metabolome
Plant Cell Reports (2023)
-
Practical Guidance for Developing Small-Molecule Optical Probes for In Vivo Imaging
Molecular Imaging and Biology (2023)
-
Fluorescence microscopic visualization of functionalized hydrogels
NPG Asia Materials (2022)