A bright cyan-excitable orange fluorescent protein facilitates dual-emission microscopy and enhances bioluminescence imaging in vivo


Orange-red fluorescent proteins (FPs) are widely used in biomedical research for multiplexed epifluorescence microscopy with GFP-based probes, but their different excitation requirements make multiplexing with new advanced microscopy methods difficult. Separately, orange-red FPs are useful for deep-tissue imaging in mammals owing to the relative tissue transmissibility of orange-red light, but their dependence on illumination limits their sensitivity as reporters in deep tissues. Here we describe CyOFP1, a bright, engineered, orange-red FP that is excitable by cyan light. We show that CyOFP1 enables single-excitation multiplexed imaging with GFP-based probes in single-photon and two-photon microscopy, including time-lapse imaging in light-sheet systems. CyOFP1 also serves as an efficient acceptor for resonance energy transfer from the highly catalytic blue-emitting luciferase NanoLuc. An optimized fusion of CyOFP1 and NanoLuc, called Antares, functions as a highly sensitive bioluminescent reporter in vivo, producing substantially brighter signals from deep tissues than firefly luciferase and other bioluminescent proteins.

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Figure 1: Development of CyOFP1, a cyan-excitable red fluorescent protein.
Figure 2: Structural characterization of CyOFP0.5.
Figure 3: Simultaneous dual-emission two-photon imaging of CyOFP1 with GFP-based reporters.
Figure 4: Development of a BRET system with NanoLuc and CyOFP1.
Figure 5: Antares is superior to other reporters for BLI in mice.

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We thank T. Doyle (Stanford University) for assistance with bioluminescence imaging, J. Chen (Shenzhen Institutes of Advanced Technology) for analysis of light-sheet microscopy data, S. Classen (ALS SIBYLS) for help with synchrotron X-ray data collection, P. Meisenheimer and T. Kirkland (Promega) for furimazine, and members of the Lin laboratory for general assistance and advice. The MV3 cells were a gift of P. Friedl, University of Texas MD Anderson Cancer Center. The Advanced Light Source (ALS), a national user facility operated by Lawrence Berkeley National Laboratory on behalf of the Department of Energy Office of Basic Energy Sciences, through the Integrated Diffraction Analysis Technologies (IDAT) program, is supported by the DOE Office of Biological and Environmental Research and National Institute of Health project MINOS (R01GM105404). This work was supported by the University of Hawaii at Manoa Undergraduate Research Opportunities Program (M.H. and C.T.T.), a long-term fellowship from the Human Frontier Science Program (T.L.), NIH grants R01HL064274 (M.K.), R01MH080047 (R.Y.), 1U01NS090600 (M.Z.L.), and P50GM107615 (M.Z.L.), Shenzhen Basic Research Foundation grant JCYJ20150521144320987 (J.C.), the Hundred Talents Program award (Y64401) from the Chinese Academy of Sciences (J.C.), a Burroughs Wellcome Foundation Career Award for Medical Scientists (M.Z.L.), and a Rita Allen Foundation Scholar Award (M.Z.L.).

Author information

J.C. engineered and characterized CyOFP1 and Antares, performed bioluminescence experiments, and co-wrote the manuscript. Y.O. performed bioluminescence experiments. A.S., N.A., C.T.T., M.H., and H.-L.N. obtained the crystal structure of CyOFP1. H.D. performed two-photon imaging in mice. J.J.M. and T.L. performed optical characterization of CyOFP1. F.Z. performed injections in mice. B.B.K. characterized CyOFP1 monomericity. E.S.W., K.M.D., and R.F. performed light-sheet microscopy. M.A.B. characterized CyOFP1 fusions. M.W.D., R.F., M.A.K., R.Y., D.S.K., and H.-L.N. provided supervision. M.Z.L. characterized CyOFP1 maturation, assisted in data analysis, and co-wrote the manuscript.

Correspondence to Michael Z Lin.

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Competing interests

J.C. and M.Z.L. have filed a patent application for CyOFP and Antares.

Integrated supplementary information

Supplementary Figure 1 Optical characteristics of CyOFP1

(a) Emission of CyOFP1 compared to other orange FPs, mOrange, DsRed, and tdTomato. The visible spectrum with separated with definitions for blue, green, yellow, orange, and red from the CRC Handbook of Spectroscopic Correlation Charts is shown above. (b) Fluorescence lifetime decay of CyOFP1 following a single pulse from a Ti-Sapphire laser, fit to a monoexponential decay function with τ = 3.66 ns. Emission in each time bin was normalized by the maximum photons counted at peak (71203). (c) Photobleaching kinetics of purified proteins in oil under arc lamp illumination with a 615/20-nm excitation filter. Time was scaled so that emission was normalized to 1,000 photons per s. Each curve is the mean of three independent experiments. (d) pH dependence of CyOFP1 fluorescence showing a pKa of 5.5. (e) Maturation kinetics of CyOFP1. (f) Gel filtration of CyOFP1 at loading concentrations of 0.1, 1, and 10 μM reveals it to be monomeric at 0.1 µM.

Supplementary Figure 2 Role of Lys160 in the large Stokes shift of CyOFP0.5

(a) Left, excitation and emission spectra of CyOFP0.5 and CyOFP0.5 with the single mutation K163M demonstrate the importance of Lys160 in the large Stokes’ shfit. Inset, streaks of bacteria expressing CyOFP0.5 (left) and CyOFP0.5-K160M (right) in visible light. (b) Absorbance spectra of CyOFP at different pHs. Postulated lysine/phenol charge states are: A, cationic/neutral; B, neutral/neutral; C, neutral/anionic. (c) Chromophore structures in DsRed and LSS orange-red FPs. In DsRed, Lys160 donates a hydrogen bond to the anionic form of the chromophore. In LSS orange-red FPs, ESPT occurs to a Glu residue (LSSmKate1) or to a Ser residue followed by relay to a Asp residue (LSSmOrange, mKeima, mBeRFP, LSSmKate2). Numbering follows that of mNeptune.

Supplementary Figure 3 CyOFP1 in single-excitation dual-emission imaging

(a) Absorbance spectra (dashed lines) and emission spectra (solid lines) spectra of EGFP (green lines) and CyOFP1 (orange lines). Absorbance is presented as extinction coefficient while emission spectra are normalized to peak. (b) Single-wavelength dual-color imaging of purified EGFP and CyOFP1 in vitro (10 μM each). (c) Optical section (upper row) and surface rendering (bottom row) of CyOF1P-tractin and cytosolic EGFP in MV3 melanoma cells acquired by single-photon axially swept light-sheet microscopy. Surface reconstruction shows CyOFP1 localization to non-apoptotic membrane blebs. (d) Photostability of cytosolic CyOFP1 under single-photon excitation in light-sheet microscopy over multiple stacks of 126 z-sections each spaced 160 nm apart. Small-intensity fluctuations are due to intensity variations of the laser illumination. (e) Photostability of cytosolic CyOFP1 under two-photon excitation in Bessel beam light-sheet microscopy over multiple stacks of 126 z-sections each spaced 160 nm apart.

Supplementary Figure 4 Optimization of linkers in Antares.

(a) Emission spectra of N-terminal truncations of NanoLuc in CyOFP1-NanoLuc fusions, including CN2. (b) Comparison of CN2 and CN3. (c) Emission spectra of N-terminal truncations of CyOFP1 in NanoLuc-CyOFP1 fusions, including NC2. (d) Emission spectra of linker variants of NanoLuc-CyOFP1 fusions, including NC3. In a-d, constructs were chosen primarily for maximal brightness beyond 600 nm in non-normalized emission from bacterial lysates prepared in parallel (left). When two constructs in a set were similar in output beyond 600 nm, the one with higher apparent BRET efficiency in normalized spectra (right) was chosen.

Supplementary Figure 5 Additional comparisons of Antares with ONL and BRET6

(a) Cells expressing Antares produce larger detectable bioluminescence signal in mouse phantom than ONL. Cells expressing the indicated reporters incubated with the indicated substrates were injected into a mouse phantom at 0.7-cm depth, and images were acquired in an IVIS Spectrum for 1 s in bioluminescence mode. (b) Quantitation of cellular bioluminescence in phantom mice. Total counts were normalized to co-expressed CFP intensity and then normalized to mean counts from Antares with FRZ. ONL produced 4.5% of the detectable emission of Antares with FRZ. Error bars represent standard error of the mean (SEM) from 6 replicate measurements. (c) Antares with FRZ produces more detectable signal than BRET6 with CTZ upon IV administration of 0.33 μmol of each substrate. (d) Quantification of experiment in (c). Total counts were normalized to mean counts from Antares with FRZ. Error bars represent SEM of measurements from multiple mice (n = 10 each). (e) Antares with FRZ produces more detectable signal than BRET6 with CTZ upon IP administration of 0.33 μmol of each substrate. (f) Quantification of experiment in (e). Total counts were normalized to mean counts from Antares with FRZ. Error bars represent SEM of measurements from multiple mice (n = 6 each). Scale bar, 1 cm.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–5 and Supplementary Tables 1–2 (PDF 1552 kb)

Single-photon dual-emission microscopy with CyOFP and EGFP.

Time-lapse imaging of CyOFP-tractin and cytosolic EGFP in a MV3 cell cultured in a three-dimensional environment with a single-photon axially swept light-sheet microscope. One of 126 optical sections taken through the cell is shown over repeated imaging stacks. Playback is at 5 frames per s. Intensities were corrected for photobleaching by histogram renormalization in ImageJ as in Chen et al. (ref. 11). (MP4 127 kb)

Two-photon dual-emission microscopy with CyOFP and EGFP.

Time-lapse imaging of CyOFP-tractin and cytosolic EGFP in a MV3 cell cultured in a three-dimensional environment on a two-photon Bessel-beam light-sheet microscope. One of 126 optical sections taken through the cell is shown over repeated imaging stacks. Playback is at 5 frames per s. Intensities were corrected for photobleaching by histogram renormalization in ImageJ as in Chen et al. (ref. 11). (MP4 116 kb)

Two-photon dual-emission microscopy with CyOFP and GCaMP6s.

Sections through Layer-2/3 of a mouse cortex were acquired by two-photon excitation at 940 nm through a cranial window, 4 weeks after infection by an AAV coexpressing CyOFP and GCaMP6s. GCaMP6s is at left and CyOFP at right. Playback is at 3 sections per s. (MP4 7494 kb)

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Chu, J., Oh, Y., Sens, A. et al. A bright cyan-excitable orange fluorescent protein facilitates dual-emission microscopy and enhances bioluminescence imaging in vivo. Nat Biotechnol 34, 760–767 (2016) doi:10.1038/nbt.3550

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