We report the engineering of mScarlet, a truly monomeric red fluorescent protein with record brightness, quantum yield (70%) and fluorescence lifetime (3.9 ns). We developed mScarlet starting with a consensus synthetic template and using improved spectroscopic screening techniques; mScarlet's crystal structure reveals a planar and rigidified chromophore. mScarlet outperforms existing red fluorescent proteins as a fusion tag, and it is especially useful as a Förster resonance energy transfer (FRET) acceptor in ratiometric imaging.
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We thank R. Breedijk for technical assistance with advanced microscopy; J. Pfeilschifter for her help with cloning fusion constructs and QY determination; and J. Goedhart and other Molecular Cytology lab members for discussions and useful suggestions. We thank R. van Amerongen for proofreading the manuscript. The ESRF is acknowledged for access to beam lines via its in-house research program. This work was supported by 'Middelgroot' investment grant 834.09.003 (M.A.H.); CW-Echo grants 711.011.018 (M.A.H. and T.W.J.G.) and 711.013.009 (J.G. and T.W.J.G.); ALW-VIDI grant 864.09.015 (M.P.) from the Netherlands Organization for Scientific Research (NWO); grant 12149 (T.W.J.G.) from the Foundation for Technological Sciences (STW) from the Netherlands; and the European Union's Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement 706443 (K.E.W.).
The authors declare no competing financial interests.
Integrated supplementary information
Supplementary Figure 1 The evolution from mRed7 to the mScarlet series depicted as amino acid alignment.
The intermediates in the evolution of mRed7 to the mScarlet variants are given in order of occurrence. The chromophore is shown in grey boxes. Amino acid mutations highlighted in blue originate from quantum yield OmniChange mutagenesis, in green from OmniChange side mutagenesis, in magenta from random mutageneses. Amino acid residues highlighted in red, orange and brown are specific for mScarlet, mScarlet-I and mScarlet-H, respectively.
(a) Systematic representation of the used screening construct for bacterial and mammalian expression. To correct for protein concentration, mTurquoise2 is fused to an RFP of interest. To minimize FRET in bacteria, the FPs are linked through a large spatial linker. The linker is followed by a P2A sequence to exclude FRET in mammalian cells. (b - f) Bacteria transformed with the vector described in (a) with three different RFP mutants, grown in a 3 × 3 square on a Petri dish. Three images are recorded sequentially with red settings (b), green setting (c), cyan settings (d). Ratios of red/cyan (e) and red/green (f) fluorescence intensities are calculated. An elevated ratio red/cyan (e *) shows a variant with an increased brightness. An elevated ratio red/green (f +) represents an RFP mutant that has decreased dead-end green component, thereby improved maturation. Ratio images are pseudo-colored using the rainbow LUT displayed.
(a) Normalized absorbance spectra of purified RFPs (b) Fluorescence emission spectra of RFPs area normalized to the calculated brightness (ε˙QY) (c) Fluorescence emission spectra of RFPs area normalized to the brightness observed in mammalian cells (Table 1). (a, b, c) mScarlet (solid red), mScarlet-I (dotted red), mScarlet-H (dashed red), mRuby2 (green), mKate2 (magenta), TagRFP-T (dark yellow), mApple (light blue), mCherry (dark blue) and dTomato (grey). Source data
Time-resolved fluorescence decay curves of purified mScarlet (a), mScarlet-I (b) and mScarlet-H (c) in phosphate buffer. The measured decay curves (gray dots) were analyzed using a mono- and bi-exponential fit model (black line, Eq. 2) including the instrumental response function (blue line), the parameters are listed in Supplementary Table 2. The residuals of the fits are shown for a mono- (middle, red) and bi-exponential (bottom, black) fit model. Source data
Mammalian cells produce mTurquoise2 and an RFP variant of interest separately in a 1:1 ratio. Dot plots show the mean fluorescence intensity of individual cells in the RFP channel (y-axis) against the CFP channel (x-axis). A higher RFP/CFP ratio corresponds to an increased RFP brightness in cells. The expression vector mTurquoise2-P2A-RFP that was used for transfection with the different RFP variants: mScarlet (a), mScarlet-I (b), mScarlet-H (c), mRuby3 (d), mRuby2 (e), mKate2 (f), TagRFP-T (g), mApple (h), mCherry (i) or dTomato (j) is shown in Supplementary Figure 2. (k) A C- and N-terminus polypeptide can influence the brightness of FPs in cells, as is observed for mRuby2, but not for mScarlet. Red dots represent mScarlet (mTurquoise2-P2A-mScarlet, shown in panel a), dark red dots represent mScarlet with a C-terminus fused polypeptide (mScarlet-T2A-mTurquoise2), light green dots represent mRuby2 (mTurquoise2-P2A-mRuby2, shown in panel e), and dark green dots represent mRuby2 with a C-terminus fused polypeptide (mRuby2-T2A-mTurquoise2). (l) Photochromic behavior of RFPs interfering with brightness analysis in cells. Cells containing TagRFP-T are imaged multiple times in the red and cyan channels. Due to blue light induced photochromic behavior, the red fluorescence intensity increased while the mTurquoise2 fluorescence intensity remained constant. The consecutive measurements are displayed in the order: yellow, orange, brown, and black, respectively. Source data
The normalized spectral-integrated fluorescence intensity for mScarlet (a), mScarlet-I (b) and mScarlet-H (c) were plotted against pH and fitted using Equation 3 (red curve), the parameters are listed in Supplementary Table 2. Data points indicate measurements from four concentration series. Source data
Fluorescence intensity rise of recently divided mammalian cells separately producing mTurquoise2 and an RFP variant of interest in a 1:1 ratio. The delay between the cyan fluorescence intensity (cyan) and red fluorescence intensity (red) corresponds to the initial delay time of the synthesis of RFP compared to mTurquoise2. The expression vector mTq2-P2A-RFP that was used for transfection with the different RFP variants: (a) mScarlet, n=32; (b) mScarlet-I, n=26; (c) mScarlet-H, n=47; (d) mRuby3, n=27; (e) mRuby2, n=42; (f) mKate2, n=32; (g) TagRFP-T, n=45; (h) mApple, n=45; (i) mCherry, n=30; or (j) dTomato, n=32; is shown in Supplementary Figure 2a. Source data
Photon emission rate under continuous widefield (a) or confocal spinning disk (b) illumination: mScarlet (solid red), mScarlet-I (dotted red), mScarlet-H (dashed red), mRuby2 (green), mKate2 (magenta), TagRFP-T (dark yellow), mApple (light blue), mCherry (dark blue) and dTomato (grey). *All RFPs are bleached with the same illumination intensity, and the time axis is computationally normalized to an initial photon emission rate of 1000 photons s-1 molecule-1. Source data
Supplementary Figure 9 Quantification of photostability under widefield and spinning disk imaging conditions in living cells.
The time to reduce the emission rate from 1000 to 500 photons s-1 molecule-1 (t1/2) was calculated per cell under continuous widefield (a) and confocal spinning disk (b) imaging conditions. Each dot represents one cell (total number of cells is indicated above the horizontal axis). Thick lines represent the median t1/2 value in seconds. *All RFPs are bleached with the same illumination intensity, and the time axis is computationally normalized to an initial emission rate of 1000 photons s-1 molecule-1. Only the variants that lack photochromic behavior are shown (Fig. 2h and Supplementary Fig. 17). Source data
Supplementary Figure 10 Assessment of oligomeric state of RFPs in living mammalian cells by OSER assay.
U-2 OS cells were transfected with plasmids encoding fusions of CytERM to different FPs: (a) mScarlet, (b) mScarlet-I, (c) mScarlet-H, (d) dTomato, (e) mCherry, (f) mKate2, (g) mRuby2, thereby anchoring the FP to the membrane of the ER. To enhance visualization of cells with bright OSER structures, the gamma of all images was adjusted to 0.8. Scale bars are 10 μm. (h) Table displaying the results of the OSER assay. 'Normal looking cells' are cells with reticular shaped ER, without nuclear envelope (NE) thickenings, without whorl (OSER) structures, and without incorrect localization. * FPs that are monomeric according to definition given by Costantini et al.22. 22. Costantini, L. M., Fossati, M., Francolini, M. & Snapp, E. L. Traffic 13, 643–649 (2012).
The intensity ratio of the whorls structure (OSER) over the mean intensity of the nuclear envelope (NE) was measured in cells expressing different CytERM-FP constructs. Each dot represents an OSER structure. Thick lines in each FP variant represent the median intensity ratio OSER/NE. The bar at 2.3±0.6 represents the monomeric threshold22. 22. Costantini, L. M., Fossati, M., Francolini, M. & Snapp, E. L. Traffic 13, 643–649 (2012). Source data
U-2 OS cells were co-transfected with plasmids encoding fusions CytERM-mScarlet and mTurquoise2–Giantin (a-c), CytERM-mRuby2 and mTurquoise2–Giantin (d-f), or CytERM-mRuby3 and mTurquoise2–Giantin (g-i). a, d, g: RFP channel showing localization of CytERM fusion, b, e, h: CFP channel showing localization of Golgi marker, c, f, i: Overlay of Red and Cyan channels. (a-i) boxed area is zoomed in the upper right corner. Scale bars are 10 μm.
Supplementary Figure 13 mScarlet-I and mScarlet-H as fusion tag to visualize intracellular structures in living cells.
U-2 OS cells (a-f, i and j) or HeLa cells (g, h) were transfected with plasmids encoding fusion constructs with mScarlet-I (a, c, e, g and i) or mScarlet-H (b, d, f, h, and j). (a, b) LifeAct–7aa–mScarlet-I/-H (F-Actin), (c, d) MTS1–4aa–mScarlet-I/-H(mitochondria), (e, f) mScarlet-I/-H–7aa–Giantin (Golgi apparatus), (g, h) mScarlet-I/-H– 7aa–α-tubulin (microtubules), and (i, j) mScarlet-I/-H–SRL (direct fusion, peroxisomes). Scale bars are 10 μm. The fluorescence intensities were pseudo-colored according to the indicated Scarlet-LUT (bottom).
The percentage of positive (fluorescent) HeLa cells was calculated 2 and 6 days after transfection. The percentage of positive cells at day 6 relative to day 2 were normalized to EGFP. The lines represent the mean values of three independent experiments from transfections on three different days, the individual experiments are shown as dots. Source data
U-2 OS cells were co-transfected with plasmids encoding mScarlet–H2A (Histon 2A, nuclei) and mScarlet-H–NES (nuclear export sequence, cytoplasm). Wide-field fluorescence lifetime imaging showing (a) steady-state fluorescence intensity image, (b) the phase fluorescence lifetime image (τφ), and (c) τφ histogram. τφ is pseudo-colored as indicated by the LUT in the τφ-histogram. The unmixed fluorescence intensity image of (d) mScarlet-H–NES and (e) mScarlet–H2A. The overlay of the unmixed fluorescence intensity images of mScarlet-H–NES (green pseudo-color) and mScarlet–H2A (blue pseudo-color). Scale bar is 10 μm.
Fluorescence emission spectra were measured in U-2 OS cells expressing YFP–RFP fusions. Each spectrum (colored line, Fig 2g) corresponds to the mean spectrum based on multiple cells. The FRET spectra (upper colored lines) were unmixed into the YFP donor component (black line) and the total RFP acceptor component (grey line) and subsequently normalized to donor emission maxima. The component arising from direct acceptor excitation in the FRET spectra (lower colored line) were measured in cells only containing the acceptor, and normalized to the average donor emission maximum. Hence, the grey areas represent the net sensitized emission from the RFP acceptors, the mean values are presented in Figure 2h and the values and statistics from individual cells are shown in Supplementary Figure 18a. The RFP spectra and their sample size are presented in the following order: (a) mScarlet, n=50; (b) mScarlet-I, n=47; (c) mScarlet-H, n=41; (d) mRuby2, n=37; (e) mKate2, n=45; (f) TagRFP-T, n=43; (g) mApple, n=56; and (h) mCherry, n=54. Source data
U-2 OS cells were transfected with the plasmid shown in Supplementary Figure 2a. The cells were widefield illuminated with alternating light of 556/20 nm and 448/20 nm light for multiple illumination cycles. Photochromic RFPs exhibit a fast decrease and subsequent recovery of the red fluorescence intensity upon alternating excitation with yellow and blue light, respectively. The photochromic amplitudes represented by the blue arrows were calculated using Equation 4. In this figure only one representative recording is shown for each RFP, amplitudes and statistics or all measurements are presented in Supplementary Figure 18b. RFPs are displayed in the following order: (a) mScarlet, (b) mScarlet-I, (c) mScarlet-H, (d) mRuby3, (e) mRuby2, (f) mKate2, (g) TagRFP-T, (h) mApple, (i) mCherry, and (j) dTomato. Source data
Supplementary Figure 18 Quantification of net sensitized emission and photochromic amplitude of RFPs.
(a) Boxplot of the net sensitized emission from each RFP calculated for individual cells (see grey area in Supplementary Figure 16) relative to mCherry (100%). The mean values of the net sensitized emission is presented in Figure 2h and listed in Supplementary Table 2. (b). Boxplot of all determined photochromic amplitudes (see blue arrows in Supplementary Figure 17) based on 2-4 recordings each with 3 illumination cycles. The mean values of the photochromic amplitudes is presented in Figure 2h and listed in Table 1. Source data
Fluorescence emission spectra measured in HeLa cells expressing GR-RhoA sensors constructs were recorded, each spectrum corresponds to the mean spectrum based on measurements from multiple cells. The FRET spectra (colored lines) are normalized to the intensity at the wavelength where the GFP donor spectrum (black lines) peaks. (a) FRET spectrum of the wtGR-RhoA sensor in unstimulated cells (blue dashed line, n=26) and constitutive active caGR-RhoA sensor in the GTP-locked state (blue solid line, n=28) with mCherry as acceptor; (b) FRET spectrum of wtGR-RhoA sensor in unstimulated cells (red dashed line, n=28) and caGR-RhoA sensor in the GTP-locked state (red solid line, n=36) with mScarlet-I as acceptor. Source data
(a) Visualization of the hydrophobic AB5 (green) and hydrophilic AC5 (orange) interaction interfaces in the crystal structure of mScarlet. (b) Orthogonal view (rotation around the horizontal axis of a). 5. Verkhusha, V. V. & Lukyanov, K. A. Nat. Biotechnol. 22, 289–296 (2004).
Supplementary Figures 1–20, Supplementary Tables 1–3, Supplementary Notes 1 and 2, Supplementary Results, and Supplementary Discussion. (PDF 45222 kb)
Live cell imaging of HeLa cells transfected with mScarlet–7aa–α-tubulin. The Scarlet-LUT was used. (MOV 1059 kb)
U-2 OS cells were co-transfected with the multimeric FRET biosensor with mScarletI as acceptor and unlabeled histamine1 receptor. The cells were stimulated with histamine in the same manner as for Figure 2j. The FRET ratio is shown (left) and pseudo-colored with the fire-LUT. The FRET ratio multiplied with the sum of the fluorescence intensity of both channels (right). (MOV 4220 kb)
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Bindels, D., Haarbosch, L., van Weeren, L. et al. mScarlet: a bright monomeric red fluorescent protein for cellular imaging. Nat Methods 14, 53–56 (2017). https://doi.org/10.1038/nmeth.4074
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