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Quantitative assessment of fluorescent proteins

Nature Methods volume 13, pages 557–562 (2016)Cite this article

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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.

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Figure 1: Excitation and emission spectra of several commonly used FPs across the visible region.
Figure 2: Log–log plot of photobleaching rates versus illumination power as measured by laser-scanning microscopy.
Figure 3: Wide-field fluorescence images of FP–CytERM fusion proteins expressed in live cells.

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Acknowledgements

This work was supported in part by NIH grants R01DK085064, R01DK098659, S10OD010681 and P20GM072048 to D.W.P.

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Author notes

  1. Paula J Cranfill, Brittney R Sell, Michelle A Baird & John R Allen

    Present address: Present addresses: H. Lee Moffitt Cancer Center, Tampa, Florida, USA (P.J.C.); Department of Biochemistry and Molecular Medicine, The George Washington University, Washington, DC, USA (B.R.S.); US National Heart, Lung, and Blood Institute, NIH, Bethesda, Maryland, USA (M.A.B.); Nikon Instruments, Melville, New York, USA (J.R.A.).,

  2. Michael W Davidson: Deceased.

Authors and Affiliations

  1. National High Field Magnet Lab, Florida State University, Tallahassee, Florida, USA

    Paula J Cranfill, Brittney R Sell, Michelle A Baird, John R Allen & Michael W Davidson

  2. Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee, USA

    Paula J Cranfill, Zeno Lavagnino, Alessandro Ustione & David W Piston

  3. Department of Cell Biology and Physiology, Washington University, St. Louis, Missouri, USA

    Zeno Lavagnino, Alessandro Ustione & David W Piston

  4. Erasmus Optical Imaging Centre, Josephine Nefkens Institute, Erasmus MC, Rotterdam, the Netherlands

    H Martijn de Gruiter & Gert-Jan Kremers

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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.

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Correspondence to David W Piston.

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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

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