Abstract
The advent of fluorescent proteins (FPs) for genetic labeling of molecules and cells has revolutionized fluorescence microscopy. Genetic manipulations have created a vast array of bright and stable FPs spanning blue to red spectral regions. Common to autofluorescent FPs is their tight β-barrel structure, which provides the rigidity and chemical environment needed for effectual fluorescence. Despite the common structure, each FP has unique properties. Thus, there is no single 'best' FP for every circumstance, and each FP has advantages and disadvantages. To guide decisions about which FP is right for a given application, we have quantitatively characterized the brightness, photostability, pH stability and monomeric properties of more than 40 FPs to enable straightforward and direct comparison between them. We focus on popular and/or top-performing FPs in each spectral region.
Similar content being viewed by others
References
Goldman, R.D., Swedlow, J. & Spector, D.L. Live Cell Imaging: A Laboratory Manual 2nd edn. (Cold Spring Harbor Laboratory Press, 2010).
Prasher, D.C., Eckenrode, V.K., Ward, W.W., Prendergast, F.G. & Cormier, M.J. Primary structure of the Aequorea victoria green-fluorescent protein. Gene 111, 229–233 (1992).
Chalfie, M., Tu, Y., Euskirchen, G., Ward, W.W. & Prasher, D.C. Green fluorescent protein as a marker for gene expression. Science 263, 802–805 (1994).
Matz, M.V. et al. Fluorescent proteins from nonbioluminescent Anthozoa species. Nat. Biotechnol. 17, 969–973 (1999).
Tsien, R.Y. The green fluorescent protein. Annu. Rev. Biochem. 67, 509–544 (1998).
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).
Patterson, G.H., Knobel, S.M., Sharif, W.D., Kain, S.R. & Piston, D.W. Use of the green fluorescent protein and its mutants in quantitative fluorescence microscopy. Biophys. J. 73, 2782–2790 (1997).
Kremers, G.J., Gilbert, S.G., Cranfill, P.J., Davidson, M.W. & Piston, D.W. Fluorescent proteins at a glance. J. Cell Sci. 124, 157–160 (2011).
Shu, X., Shaner, N.C., Yarbrough, C.A., Tsien, R.Y. & Remington, S.J. Novel chromophores and buried charges control color in mFruits. Biochemistry 45, 9639–9647 (2006).
Ormö, M. et al. Crystal structure of the Aequorea victoria green fluorescent protein. Science 273, 1392–1395 (1996).
Follenius-Wund, A. et al. Fluorescent derivatives of the GFP chromophore give a new insight into the GFP fluorescence process. Biophys. J. 85, 1839–1850 (2003).
Cormack, B.P., Valdivia, R.H. & Falkow, S. FACS-optimized mutants of the green fluorescent protein (GFP). Gene 173, 33–38 (1996).
Cubitt, A.B. et al. Understanding, improving and using green fluorescent proteins. Trends Biochem. Sci. 20, 448–455 (1995).
Pédelacq, J.D., Cabantous, S., Tran, T., Terwilliger, T.C. & Waldo, G.S. Engineering and characterization of a superfolder green fluorescent protein. Nat. Biotechnol. 24, 79–88 (2006).
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).
Yang, T.T. et al. Improved fluorescence and dual color detection with enhanced blue and green variants of the green fluorescent protein. J. Biol. Chem. 273, 8212–8216 (1998).
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).
Shaner, N.C. et al. Improving the photostability of bright monomeric orange and red fluorescent proteins. Nat. Methods 5, 545–551 (2008).
Kremers, G.-J. & Piston, D. Photoconversion of purified fluorescent proteins and dual-probe optical highlighting in live cells. J. Vis. Exp. 40, 1995 (2010).
Piston, D.W., Patterson, G.H. & Knobel, S.M. Quantitative imaging of the green fluorescent protein (GFP). Methods Cell Biol. 58, 31–48 (1999).
Patterson, G.H. & Piston, D.W. Photobleaching in two-photon excitation microscopy. Biophys. J. 78, 2159–2162 (2000).
Hirschfeld, T. Quantum efficiency independence of the time integrated emission from a fluorescent molecule. Appl. Opt. 15, 3135–3139 (1976).
Song, L., Hennink, E.J., Young, I.T. & Tanke, H.J. Photobleaching kinetics of fluorescein in quantitative fluorescence microscopy. Biophys. J. 68, 2588–2600 (1995).
Sandison, D.R., Piston, D.W., Williams, R.M. & Webb, W.W. Quantitative comparison of background rejection, signal-to-noise ratio, and resolution in confocal and full-field laser scanning microscopes. Appl. Opt. 34, 3576–3588 (1995).
Wu, Y. et al. Spatially isotropic four-dimensional imaging with dual-view plane illumination microscopy. Nat. Biotechnol. 31, 1032–1038 (2013).
Bogdanov, A.M. et al. Cell culture medium affects GFP photostability: a solution. Nat. Methods 6, 859–860 (2009).
Dempsey, W.P. et al. In vivo single-cell labeling by confined primed conversion. Nat. Methods 12, 645–648 (2015).
Axelrod, D. Chapter 7: Total internal reflection fluorescence microscopy. Methods Cell Biol. 89, 169–221 (2008).
Swedlow, J.R., Sedat, J.W. & Agard, D.A. Multiple chromosomal populations of topoisomerase II detected in vivo by time-lapse, three-dimensional wide-field microscopy. Cell 73, 97–108 (1993).
Gustafsson, M.G. et al. Three-dimensional resolution doubling in wide-field fluorescence microscopy by structured illumination. Biophys. J. 94, 4957–4970 (2008).
Hiraoka, Y., Sedat, J.W. & Agard, D.A. Determination of three-dimensional imaging properties of a light microscope system. Partial confocal behavior in epifluorescence microscopy. Biophys. J. 57, 325–333 (1990).
Carlton, P.M. et al. Fast live simultaneous multiwavelength four-dimensional optical microscopy. Proc. Natl. Acad. Sci. USA 107, 16016–16022 (2010).
Tomer, R., Khairy, K. & Keller, P.J. Light sheet microscopy in cell biology. Methods Mol. Biol. 931, 123–137 (2013).
Ward, W.W. Biochemical and physical properties of green fluorescent protein. Methods Biochem. Anal. 47, 39–65 (2006).
Costantini, L.M., Fossati, M., Francolini, M. & Snapp, E.L. Assessing the tendency of fluorescent proteins to oligomerize under physiologic conditions. Traffic 13, 643–649 (2012).
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).
Li, X. et al. Generation of destabilized green fluorescent protein as a transcription reporter. J. Biol. Chem. 273, 34970–34975 (1998).
Terskikh, A. et al. “Fluorescent timer”: protein that changes color with time. Science 290, 1585–1588 (2000).
Valentin, G. et al. Photoconversion of YFP into a CFP-like species during acceptor photobleaching FRET experiments. Nat. Methods 2, 801 (2005).
Kremers, G.J., Hazelwood, K.L., Murphy, C.S., Davidson, M.W. & Piston, D.W. Photoconversion in orange and red fluorescent proteins. Nat. Methods 6, 355–358 (2009).
Chen, S.X. et al. Quantification of factors influencing fluorescent protein expression using RMCE to generate an allelic series in the ROSA26 locus in mice. Dis. Model. Mech. 4, 537–547 (2011).
Katayama, H., Yamamoto, A., Mizushima, N., Yoshimori, T. & Miyawaki, A. GFP-like proteins stably accumulate in lysosomes. Cell Struct. Funct. 33, 1–12 (2008).
Ai, H.W., Shaner, N.C., Cheng, Z., Tsien, R.Y. & Campbell, R.E. Exploration of new chromophore structures leads to the identification of improved blue fluorescent proteins. Biochemistry 46, 5904–5910 (2007).
Subach, O.M., Cranfill, P.J., Davidson, M.W. & Verkhusha, V.V. An enhanced monomeric blue fluorescent protein with the high chemical stability of the chromophore. PLoS One 6, e28674 (2011).
Goedhart, J. et al. Bright cyan fluorescent protein variants identified by fluorescence lifetime screening. Nat. Methods 7, 137–139 (2010).
Goedhart, J. et al. Structure-guided evolution of cyan fluorescent proteins towards a quantum yield of 93%. Nat. Commun. 3, 751 (2012).
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).
Markwardt, M.L. et al. An improved cerulean fluorescent protein with enhanced brightness and reduced reversible photoswitching. PLoS One 6, e17896 (2011).
Ai, H.W., Henderson, J.N., Remington, S.J. & Campbell, R.E. Directed evolution of a monomeric, bright and photostable version of Clavularia cyan fluorescent protein: structural characterization and applications in fluorescence imaging. Biochem. J. 400, 531–540 (2006).
Zapata-Hommer, O. & Griesbeck, O. Efficiently folding and circularly permuted variants of the Sapphire mutant of GFP. BMC Biotechnol. 3, 5 (2003).
Cubitt, A.B., Woollenweber, L.A. & Heim, R. Understanding structure-function relationships in the Aequorea victoria green fluorescent protein. Methods Cell Biol. 58, 19–30 (1999).
Nguyen, A.W. & Daugherty, P.S. Evolutionary optimization of fluorescent proteins for intracellular FRET. Nat. Biotechnol. 23, 355–360 (2005).
Lam, A.J. et al. Improving FRET dynamic range with bright green and red fluorescent proteins. Nat. Methods 9, 1005–1012 (2012).
Shaner, N.C. et al. A bright monomeric green fluorescent protein derived from Branchiostoma lanceolatum. Nat. Methods 10, 407–409 (2013).
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).
Sakaue-Sawano, A. et al. Visualizing spatiotemporal dynamics of multicellular cell-cycle progression. Cell 132, 487–498 (2008).
Merzlyak, E.M. et al. Bright monomeric red fluorescent protein with an extended fluorescence lifetime. Nat. Methods 4, 555–557 (2007).
Haas, J., Park, E.C. & Seed, B. Codon usage limitation in the expression of HIV-1 envelope glycoprotein. Curr. Biol. 6, 315–324 (1996).
Kredel, S. et al. mRuby, a bright monomeric red fluorescent protein for labeling of subcellular structures. PLoS One 4, e4391 (2009).
Campbell, R.E. et al. A monomeric red fluorescent protein. Proc. Natl. Acad. Sci. USA 99, 7877–7882 (2002).
Shemiakina, I.I. et al. A monomeric red fluorescent protein with low cytotoxicity. Nat. Commun. 3, 1204 (2012).
Shcherbo, D. et al. Far-red fluorescent tags for protein imaging in living tissues. Biochem. J. 418, 567–574 (2009).
Lin, M.Z. et al. Autofluorescent proteins with excitation in the optical window for intravital imaging in mammals. Chem. Biol. 16, 1169–1179 (2009).
Chu, J. et al. Non-invasive intravital imaging of cellular differentiation with a bright red-excitable fluorescent protein. Nat. Methods 11, 572–578 (2014).
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).
Acknowledgements
This work was supported in part by NIH grants R01DK085064, R01DK098659, S10OD010681 and P20GM072048 to D.W.P.
Author information
Authors and Affiliations
Contributions
D.W.P., A.U. and M.W.D. designed the research; P.J.C., B.R.S., M.A.B., J.R.A., Z.L., H.M.d.G. and G.-J.K. performed experiments and analyzed data; D.W.P. wrote and edited the paper with comments from all authors.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing financial interests.
Supplementary information
Supplementary Text and Figures
Supplementary Tables 1 and 2 and Supplementary Note (PDF 3313 kb)
Source data
Rights and permissions
About this article
Cite this article
Cranfill, P., Sell, B., Baird, M. et al. Quantitative assessment of fluorescent proteins. Nat Methods 13, 557–562 (2016). https://doi.org/10.1038/nmeth.3891
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/nmeth.3891
- Springer Nature America, Inc.
This article is cited by
-
Bright and stable monomeric green fluorescent protein derived from StayGold
Nature Methods (2024)
-
Structural biases in disordered proteins are prevalent in the cell
Nature Structural & Molecular Biology (2024)
-
StayGold variants for molecular fusion and membrane-targeting applications
Nature Methods (2024)
-
A short guide on blue fluorescent proteins: limits and perspectives
Applied Microbiology and Biotechnology (2024)
-
Multidimensional characterization of inducible promoters and a highly light-sensitive LOV-transcription factor
Nature Communications (2023)