Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

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

Abstract

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.

This is a preview of subscription content

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

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.

Accession codes

Primary accessions

NCBI Reference Sequence

Protein Data Bank

References

  1. 1

    Newman, R.H., Fosbrink, M.D. & Zhang, J. Genetically encodable fluorescent biosensors for tracking signaling dynamics in living cells. Chem. Rev. 111, 3614–3666 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. 2

    Depry, C., Mehta, S. & Zhang, J. Multiplexed visualization of dynamic signaling networks using genetically encoded fluorescent protein-based biosensors. Pflugers Arch. 465, 373–381 (2013).

    CAS  Google Scholar 

  3. 3

    Kawano, H., Kogure, T., Abe, Y., Mizuno, H. & Miyawaki, A. Two-photon dual-color imaging using fluorescent proteins. Nat. Methods 5, 373–374 (2008).

    CAS  PubMed  Google Scholar 

  4. 4

    Yang, J. et al. mBeRFP, an improved large stokes shift red fluorescent protein. PLoS One 8, e64849 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. 5

    Shao, L., Kner, P., Rego, E.H. & Gustafsson, M.G. Super-resolution 3D microscopy of live whole cells using structured illumination. Nat. Methods 8, 1044–1046 (2011).

    CAS  Google Scholar 

  6. 6

    Li, D. et al. ADVANCED IMAGING. Extended-resolution structured illumination imaging of endocytic and cytoskeletal dynamics. Science 349, aab3500 (2015).

    PubMed  PubMed Central  Google Scholar 

  7. 7

    Liu, Y. et al. Optical focusing deep inside dynamic scattering media with near-infrared time-reversed ultrasonically encoded (TRUE) light. Nat. Commun. 6, 5904 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. 8

    Si, K., Fiolka, R. & Cui, M. Fluorescence imaging beyond the ballistic regime by ultrasound pulse guided digital phase conjugation. Nat. Photonics 6, 657–661 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. 9

    Ji, N., Milkie, D.E. & Betzig, E. Adaptive optics via pupil segmentation for high-resolution imaging in biological tissues. Nat. Methods 7, 141–147 (2010).

    CAS  PubMed  Google Scholar 

  10. 10

    Katona, G. et al. Fast two-photon in vivo imaging with three-dimensional random-access scanning in large tissue volumes. Nat. Methods 9, 201–208 (2012).

    CAS  PubMed  Google Scholar 

  11. 11

    Planchon, T.A. et al. Rapid three-dimensional isotropic imaging of living cells using Bessel beam plane illumination. Nat. Methods 8, 417–423 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. 12

    Gao, L. et al. Noninvasive imaging beyond the diffraction limit of 3D dynamics in thickly fluorescent specimens. Cell 151, 1370–1385 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. 13

    Welf, E.S. et al. Quantitative multiscale cell imaging in controlled 3D microenvironments. Dev. Cell 36, 462–475 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. 14

    Chen, B.C. et al. Lattice light-sheet microscopy: imaging molecules to embryos at high spatiotemporal resolution. Science 346, 1257998 (2014).

    PubMed  PubMed Central  Google Scholar 

  15. 15

    Dean, K.M., Roudot, P., Welf, E.S., Danuser, G. & Fiolka, R. Deconvolution-free subcellular imaging with axially swept light sheet microscopy. Biophys. J. 108, 2807–2815 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. 16

    Dean, K.M. & Fiolka, R. Uniform and scalable light-sheets generated by extended focusing. Opt. Express 22, 26141–26152 (2014).

    PubMed  Google Scholar 

  17. 17

    Close, D.M., Xu, T., Sayler, G.S. & Ripp, S. In vivo bioluminescent imaging (BLI): noninvasive visualization and interrogation of biological processes in living animals. Sensors (Basel) 11, 180–206 (2011).

    CAS  Google Scholar 

  18. 18

    Zhao, H. et al. Emission spectra of bioluminescent reporters and interaction with mammalian tissue determine the sensitivity of detection in vivo. J. Biomed. Opt. 10, 41210 (2005).

    PubMed  Google Scholar 

  19. 19

    Contag, C.H. et al. Visualizing gene expression in living mammals using a bioluminescent reporter. Photochem. Photobiol. 66, 523–531 (1997).

    CAS  PubMed  Google Scholar 

  20. 20

    Ando, Y., Niwa, K., Yamada, N., Enomoto, T. & Irie, T. Firefly bioluminescence quantum yield and colour change by pH-sensitive green emission. Nat. Photonics 2, 44–47 (2007).

    Google Scholar 

  21. 21

    Branchini, B.R., Magyar, R.A., Murtiashaw, M.H., Anderson, S.M. & Zimmer, M. Site-directed mutagenesis of histidine 245 in firefly luciferase: a proposed model of the active site. Biochemistry 37, 15311–15319 (1998).

    CAS  PubMed  Google Scholar 

  22. 22

    Liang, Y., Walczak, P. & Bulte, J.W. Comparison of red-shifted firefly luciferase Ppy RE9 and conventional Luc2 as bioluminescence imaging reporter genes for in vivo imaging of stem cells. J. Biomed. Opt. 17, 016004 (2012).

    PubMed  PubMed Central  Google Scholar 

  23. 23

    Mazo-Vargas, A., Park, H., Aydin, M. & Buchler, N.E. Measuring fast gene dynamics in single cells with time-lapse luminescence microscopy. Mol. Biol. Cell 25, 3699–3708 (2014).

    PubMed  PubMed Central  Google Scholar 

  24. 24

    Mezzanotte, L. et al. Evaluating reporter genes of different luciferases for optimized in vivo bioluminescence imaging of transplanted neural stem cells in the brain. Contrast Media Mol. Imaging 8, 505–513 (2013).

    CAS  PubMed  Google Scholar 

  25. 25

    Matthews, J.C., Hori, K. & Cormier, M.J. Purification and properties of Renilla reniformis luciferase. Biochemistry 16, 85–91 (1977).

    CAS  PubMed  Google Scholar 

  26. 26

    Shimomura, O., Johnson, F.H. & Masugi, T. Cypridina bioluminescence: light-emitting oxyluciferin-luciferase complex. Science 164, 1299–1300 (1969).

    CAS  PubMed  Google Scholar 

  27. 27

    Loening, A.M., Dragulescu-Andrasi, A. & Gambhir, S.S. A red-shifted Renilla luciferase for transient reporter-gene expression. Nat. Methods 7, 5–6 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. 28

    Shimomura, O., Masugi, T., Johnson, F.H. & Haneda, Y. Properties and reaction mechanism of the bioluminescence system of the deep-sea shrimp Oplophorus gracilorostris. Biochemistry 17, 994–998 (1978).

    CAS  PubMed  Google Scholar 

  29. 29

    Hall, M.P. et al. Engineered luciferase reporter from a deep sea shrimp utilizing a novel imidazopyrazinone substrate. ACS Chem. Biol. 7, 1848–1857 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. 30

    Ward, W.W. & Cormier, M.J. Energy transfer via protein-protein interaction in renilla bioluminescence. Photochem. Photobiol. 27, 389–396 (1978).

    CAS  Google Scholar 

  31. 31

    Dragulescu-Andrasi, A., Chan, C.T., De, A., Massoud, T.F. & Gambhir, S.S. Bioluminescence resonance energy transfer (BRET) imaging of protein-protein interactions within deep tissues of living subjects. Proc. Natl. Acad. Sci. USA 108, 12060–12065 (2011).

    CAS  PubMed  Google Scholar 

  32. 32

    Saito, K. et al. Luminescent proteins for high-speed single-cell and whole-body imaging. Nat. Commun. 3, 1262 (2012).

    PubMed  PubMed Central  Google Scholar 

  33. 33

    Chu, J. et al. Non-invasive intravital imaging of cellular differentiation with a bright red-excitable fluorescent protein. Nat. Methods 11, 572–578 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. 34

    Bruno, T.J. & Svoronos, P.D.N. CRC Handbook of Fundamental Spectroscopic Correlation Charts (CRC Press, Boca Raton, Florida, USA, 2006).

  35. 35

    Chu, J., Xing, Y. & Lin, M.Z. in The Fluorescent Protein Revolution (eds. Day, R. & Davidson, M.) 153–167 (CRC Press, Boca Raton, Florida, USA, 2014).

  36. 36

    Baird, G.S., Zacharias, D.A. & Tsien, R.Y. Biochemistry, mutagenesis, and oligomerization of DsRed, a red fluorescent protein from coral. Proc. Natl. Acad. Sci. USA 97, 11984–11989 (2000).

    CAS  PubMed  Google Scholar 

  37. 37

    Lounis, B. & Moerner, W.E. Single photons on demand from a single molecule at room temperature. Nature 407, 491–493 (2000).

    CAS  PubMed  Google Scholar 

  38. 38

    Lam, A.J. et al. Improving FRET dynamic range with bright green and red fluorescent proteins. Nat. Methods 9, 1005–1012 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. 39

    Miyawaki, A., Shcherbakova, D.M. & Verkhusha, V.V. Red fluorescent proteins: chromophore formation and cellular applications. Curr. Opin. Struct. Biol. 22, 679–688 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. 40

    Olsson, M.H., Søndergaard, C.R., Rostkowski, M. & Jensen, J.H. PROPKA3: consistent treatment of internal and surface residues in empirical pKa predictions. J. Chem. Theory Comput. 7, 525–537 (2011).

    CAS  PubMed  Google Scholar 

  41. 41

    Piatkevich, K.D., Malashkevich, V.N., Almo, S.C. & Verkhusha, V.V. Engineering ESPT pathways based on structural analysis of LSSmKate red fluorescent proteins with large Stokes shift. J. Am. Chem. Soc. 132, 10762–10770 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. 42

    Akerboom, J. et al. Optimization of a GCaMP calcium indicator for neural activity imaging. J. Neurosci. 32, 13819–13840 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. 43

    Chen, T.W. et al. Ultrasensitive fluorescent proteins for imaging neuronal activity. Nature 499, 295–300 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. 44

    Dana, H. et al. Thy1-GCaMP6 transgenic mice for neuronal population imaging in vivo. PLoS One 9, e108697 (2014).

    PubMed  PubMed Central  Google Scholar 

  45. 45

    Morse, D. & Tannous, B.A. A water-soluble coelenterazine for sensitive in vivo imaging of coelenterate luciferases. Mol. Ther. 20, 692–693 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. 46

    Otto-Duessel, M. et al. In vivo testing of Renilla luciferase substrate analogs in an orthotopic murine model of human glioblastoma. Mol. Imaging 5, 57–64 (2006).

    PubMed  Google Scholar 

  47. 47

    Horton, N.G. et al. In vivo three-photon microscopy of subcortical structures within an intact mouse brain. Nat. Photonics 7, 205–209 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. 48

    Sinefeld, D., Paudel, H.P., Ouzounov, D.G., Bifano, T.G. & Xu, C. Adaptive optics in multiphoton microscopy: comparison of two, three and four photon fluorescence. Opt. Express 23, 31472–31483 (2015).

    PubMed  PubMed Central  Google Scholar 

  49. 49

    Shcherbakova, D.M., Hink, M.A., Joosen, L., Gadella, T.W. & Verkhusha, V.V. An orange fluorescent protein with a large Stokes shift for single-excitation multicolor FCCS and FRET imaging. J. Am. Chem. Soc. 134, 7913–7923 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. 50

    Kogure, T. et al. A fluorescent variant of a protein from the stony coral Montipora facilitates dual-color single-laser fluorescence cross-correlation spectroscopy. Nat. Biotechnol. 24, 577–581 (2006).

    CAS  PubMed  Google Scholar 

  51. 51

    Chalfie, M. & Kain, S.R. Green Fluorescent Protein: Properties, Applications, and Protocols (Wiley-Liss, 1998).

  52. 52

    Yasuda, R. Imaging spatiotemporal dynamics of neuronal signaling using fluorescence resonance energy transfer and fluorescence lifetime imaging microscopy. Curr. Opin. Neurobiol. 16, 551–561 (2006).

    CAS  PubMed  Google Scholar 

  53. 53

    Yasuda, R. et al. Supersensitive Ras activation in dendrites and spines revealed by two-photon fluorescence lifetime imaging. Nat. Neurosci. 9, 283–291 (2006).

    CAS  PubMed  Google Scholar 

  54. 54

    McCoy, A.J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  55. 55

    Winn, M.D. et al. Overview of the CCP4 suite and current developments. Acta Crystallogr. D Biol. Crystallogr. 67, 235–242 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  56. 56

    Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).

    Google Scholar 

  57. 57

    Murshudov, G.N., Vagin, A.A. & Dodson, E.J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D Biol. Crystallogr. 53, 240–255 (1997).

    CAS  PubMed  Google Scholar 

  58. 58

    Chen, V.B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D Biol. Crystallogr. 66, 12–21 (2010).

    CAS  PubMed  Google Scholar 

  59. 59

    Laskowski, R.A., Moss, D.S. & Thornton, J.M. Main-chain bond lengths and bond angles in protein structures. J. Mol. Biol. 231, 1049–1067 (1993).

    CAS  PubMed  Google Scholar 

  60. 60

    Pologruto, T.A., Sabatini, B.L. & Svoboda, K. ScanImage: flexible software for operating laser scanning microscopes. Biomed. Eng. Online 2, 13 (2003).

    PubMed  PubMed Central  Google Scholar 

  61. 61

    Brainard, D.H. The Psychophysics Toolbox. Spat. Vis. 10, 433–436 (1997).

    CAS  PubMed  Google Scholar 

  62. 62

    Faul, F., Erdfelder, E., Lang, A.G. & Buchner, A. G*Power 3: a flexible statistical power analysis program for the social, behavioral, and biomedical sciences. Behav. Res. Methods 39, 175–191 (2007).

    PubMed  Google Scholar 

Download references

Acknowledgements

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

Affiliations

Authors

Contributions

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.

Corresponding author

Correspondence to Michael Z Lin.

Ethics declarations

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)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

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). https://doi.org/10.1038/nbt.3550

Download citation

Further reading

Search

Quick links