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Quantitative assessment of fluorescent proteins

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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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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