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. 2016 Jul;13(7):557-62.
doi: 10.1038/nmeth.3891. Epub 2016 May 30.

Quantitative assessment of fluorescent proteins

Paula J Cranfill  1   2 , Brittney R Sell  1 , Michelle A Baird  1 , John R Allen  1 , Zeno Lavagnino  2   3 , H Martijn de Gruiter  4 , Gert-Jan Kremers  4 , Michael W Davidson  1 , Alessandro Ustione  2   3 , David W Piston  2   3
Affiliations

Quantitative assessment of fluorescent proteins

Paula J Cranfill et al. Nat Methods. 2016 Jul.

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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Figures

Figure 1
Figure 1
Excitation and emission spectra of several commonly used FPs across the visible region. The peak values of the excitation spectra (A) are normalized to EGFP = 1 by the extinction coefficient, and the emission spectra peaks (B) are normalized to EGFP = 1 by their relative brightness (extinction coefficient × quantum yield).
Figure 2
Figure 2
Log-Log plot of photobleaching rates vs. illumination power as measured by laser scanning microscopy – similar power dependence is seen with widefield lamp or LED illumination (see Table 2). Data points are plotted as average and standard deviation as described in the methods. Panel (A) shows the excitation intensity dependence for a few common cyan, green, yellow FPs, while (B) shows the behavior for orange and red FPs. For each FP, the fit value of the exponent α is given on the legend.
Figure 3
Figure 3
Widefield fluorescence images of FP-CytERM fusion proteins expressed in HeLa cells. (A) mEGFP (A206K); (B) mCherry; (C) mCitrine (A206K); (D) mOrange2; (E) mNeonGreen; (F) mKate2; (G) EGFP (A206); (H) mKO2; (I) mTagBFP2; (J) mTagRFP-T; (K) Citrine (A206); (L) DsRed2. Scale bar = 10 µm.
See this image and copyright information in PMC

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