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.

  • Article
  • Published:

Non-invasive intravital imaging of cellular differentiation with a bright red-excitable fluorescent protein

Abstract

A method for non-invasive visualization of genetically labeled cells in animal disease models with micrometer-level resolution would greatly facilitate development of cell-based therapies. Imaging of fluorescent proteins (FPs) using red excitation light in the 'optical window' above 600 nm is one potential method for visualizing implanted cells. However, previous efforts to engineer FPs with peak excitation beyond 600 nm have resulted in undesirable reductions in brightness. Here we report three new red-excitable monomeric FPs obtained by structure-guided mutagenesis of mNeptune. Two of these, mNeptune2 and mNeptune2.5, demonstrate improved maturation and brighter fluorescence than mNeptune, whereas the third, mCardinal, has a red-shifted excitation spectrum without reduction in brightness. We show that mCardinal can be used to non-invasively and longitudinally visualize the differentiation of myoblasts into myocytes in living mice with high anatomical detail.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Spectral characteristics of new far-red FPs.
Figure 2: Structural basis of red-shifting in mCardinal.
Figure 3: Comparison of far-red FPs for deep-tissue imaging.
Figure 4: Non-invasive longitudinal visualization of muscle regeneration in living mice.
Figure 5: Comparison of mCardinal with mNeptune1, iRFP and Clover GFP for non-invasive visualization of muscle regeneration in living mice.

Similar content being viewed by others

Accession codes

Primary accessions

Protein Data Bank

Referenced accessions

NCBI Reference Sequence

Protein Data Bank

References

  1. Schenkman, K.A., Marble, D.R., Feigl, E.O. & Burns, D.H. Near-infrared spectroscopic measurement of myoglobin oxygen saturation in the presence of hemoglobin using partial least-squares analysis. Appl. Spectrosc. 53, 325–331 (1999).

    Article  CAS  Google Scholar 

  2. Tromberg, B.J. et al. Non-invasive in vivo characterization of breast tumors using photon migration spectroscopy. Neoplasia 2, 26–40 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Monici, M. Cell and tissue autofluorescence research and diagnostic applications. Biotechnol. Annu. Rev. 11, 227–256 (2005).

    Article  CAS  PubMed  Google Scholar 

  4. Deliolanis, N.C. et al. In vivo tomographic imaging of red-shifted fluorescent proteins. Biomed. Opt. Express 2, 887–900 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Kawai, Y., Sato, M. & Umezawa, Y. Single color fluorescent indicators of protein phosphorylation for multicolor imaging of intracellular signal flow dynamics. Anal. Chem. 76, 6144–6149 (2004).

    Article  CAS  PubMed  Google Scholar 

  6. Sakaue-Sawano, A. et al. Visualizing spatiotemporal dynamics of multicellular cell-cycle progression. Cell 132, 487–498 (2008).

    Article  CAS  PubMed  Google Scholar 

  7. Lin, M.Z. et al. Autofluorescent proteins with excitation in the optical window for intravital imaging in mammals. Chem. Biol. 16, 1169–1179 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Morozova, K.S. et al. Far-red fluorescent protein excitable with red lasers for flow cytometry and superresolution STED nanoscopy. Biophys. J. 99, L13–L15 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Shcherbo, D. et al. Far-red fluorescent tags for protein imaging in living tissues. Biochem. J. 418, 567–574 (2009).

    Article  CAS  PubMed  Google Scholar 

  10. Subach, O.M. et al. A photoswitchable orange-to-far-red fluorescent protein, PSmOrange. Nat. Methods 8, 771–777 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Shu, X. et al. Mammalian expression of infrared fluorescent proteins engineered from a bacterial phytochrome. Science 324, 804–807 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  12. Lin, M.Z. Beyond the rainbow: new fluorescent proteins brighten the infrared scene. Nat. Methods 8, 726–728 (2011).

    Article  CAS  PubMed  Google Scholar 

  13. Filonov, G.S. et al. Bright and stable near-infrared fluorescent protein for in vivo imaging. Nat. Biotechnol. 29, 757–761 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Shcherbo, D. et al. Near-infrared fluorescent proteins. Nat. Methods 7, 827–829 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Jeffrey, G.A. An Introduction to Hydrogen Bonding (Oxford University Press, New York, 1997).

  16. Rice, B.W., Cable, M.D. & Nelson, M.B. In vivo imaging of light-emitting probes. J. Biomed. Opt. 6, 432–440 (2001).

    Article  CAS  PubMed  Google Scholar 

  17. Gilbert, P.M. & Blau, H.M. Engineering a stem cell house into a home. Stem Cell Res. Ther. 2, 3 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Schroeder, T. Imaging stem-cell-driven regeneration in mammals. Nature 453, 345–351 (2008).

    Article  CAS  PubMed  Google Scholar 

  19. Sacco, A., Doyonnas, R., Kraft, P., Vitorovic, S. & Blau, H.M. Self-renewal and expansion of single transplanted muscle stem cells. Nature 456, 502–506 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Contag, C.H. & Bachmann, M.H. Advances in in vivo bioluminescence imaging of gene expression. Annu. Rev. Biomed. Eng. 4, 235–260 (2002).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Condeelis, J. & Weissleder, R. In vivo imaging in cancer. Cold Spring Harb. Perspect. Biol. 2, a003848 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Harms, G.S., Cognet, L., Lommerse, P.H., Blab, G.A. & Schmidt, T. Autofluorescent proteins in single-molecule research: applications to live cell imaging microscopy. Biophys. J. 80, 2396–2408 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Shinde, R., Perkins, J. & Contag, C.H. Luciferin derivatives for enhanced in vitro and in vivo bioluminescence assays. Biochemistry 45, 11103–11112 (2006).

    Article  CAS  PubMed  Google Scholar 

  25. Inoue, Y., Kiryu, S., Watanabe, M., Tojo, A. & Ohtomo, K. Timing of imaging after D-luciferin injection affects the longitudinal assessment of tumor growth using in vivo bioluminescence imaging. Int. J. Biomed. Imaging 2010, 471408 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  26. Brunk, U.T. & Terman, A. Lipofuscin: mechanisms of age-related accumulation and influence on cell function. Free Radic. Biol. Med. 33, 611–619 (2002).

    Article  CAS  PubMed  Google Scholar 

  27. Eldred, G.E. & Katz, M.L. Fluorophores of the human retinal pigment epithelium: separation and spectral characterization. Exp. Eye Res. 47, 71–86 (1988).

    Article  CAS  PubMed  Google Scholar 

  28. Murdaugh, L.S. et al. Compositional studies of human RPE lipofuscin. J. Mass Spectrom. 45, 1139–1147 (2010).

    Article  CAS  PubMed  Google Scholar 

  29. Nighswander-Rempel, S.P., Kupriyanov, V.V. & Shaw, R.A. Relative contributions of hemoglobin and myoglobin to near-infrared spectroscopic images of cardiac tissue. Appl. Spectrosc. 59, 190–193 (2005).

    Article  CAS  PubMed  Google Scholar 

  30. Chalfie, M. & Kain, S.R. Green Fluorescent Protein: Properties, Applications, and Protocols 2nd edn. (Wiley, 2006).

  31. Shaner, N.C., Steinbach, P.A. & Tsien, R.Y. A guide to choosing fluorescent proteins. Nat. Methods 2, 905–909 (2005).

    Article  CAS  PubMed  Google Scholar 

  32. Walter, T.S. et al. Lysine methylation as a routine rescue strategy for protein crystallization. Structure 14, 1617–1622 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Kabsch, W. XDS. Acta Crystallogr. D Biol. Crystallogr. 66, 125–132 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Adams, P.D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Emsley, P., Lohkamp, B., Scott, W.G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Pražnikar, J., Afonine, P.V., Guncar, G., Adams, P.D. & Turk, D. Averaged kick maps: less noise, more signal and probably less bias. Acta Crystallogr. D Biol. Crystallogr. 65, 921–931 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  38. Bass, R.B., Strop, P., Barclay, M. & Rees, D.C. Crystal structure of Escherichia coli MscS, a voltage-modulated and mechanosensitive channel. Science 298, 1582–1587 (2002).

    Article  CAS  PubMed  Google Scholar 

  39. Vagin, A. & Teplyakov, A. Molecular replacement with MOLREP. Acta Crystallogr. D Biol. Crystallogr. 66, 22–25 (2010).

    Article  CAS  PubMed  Google Scholar 

  40. 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 

  41. Mello, C.C., Kramer, J.M., Stinchcomb, D. & Ambros, V. Efficient gene transfer in C. elegans: extrachromosomal maintenance and integration of transforming sequences. EMBO J. 10, 3959–3970 (1991).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Kerr, R. et al. Optical imaging of calcium transients in neurons and pharyngeal muscle of C. elegans. Neuron 26, 583–594 (2000).

    Article  CAS  PubMed  Google Scholar 

  43. National Research Council (US) Committee on Guidelines for the Use of Animals in Neuroscience and Behavioral Research. Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research (National Academies Press, 2003).

Download references

Acknowledgements

We thank F. Kanokwan for technical support, F. Fang and L. Lisowski for cell sorting, C. Ran and X. Chen for two-photon imaging, F. Zhang for hydrodynamic injection and S. Classen for help with data collection at ALS Beamline 12.3.1. We also thank K. Yusa (Wellcome Trust Sanger Institute) for pCMV-piggyBAC, M. Kay (Stanford University) for minicircle plasmid, N. Deliolanis and C. Vinegoni for helpful suggestions on mouse imaging, members of the laboratory of E. Mellins for help with protein purification and members of the Lin laboratory for advice and assistance. This work was supported by a seed grant from the Center for Biological Imaging at Stanford (J.C.), US National Institutes of Health (NIH) grants 1R01NS076860-01 (J.C., M.Z.L.), T32 HD007249 (R.D.H.) and 5R01AG020961-08 (S.Y.C., H.M.B.), the Florida State University Research Foundation (P.J.C., M.A.B., M.W.D.), the Chambers Family Foundation and the Pachyonychia Congenita Project (E.G.-G., C.H.C.), an Irvington Postdoctoral Fellowship from the Cancer Research Institute (J.S.B.), the University of Hawaii (N.J.A., H.-L.N.), the Howard Hughes Medical Institute (K.S., K.C.G.) and the Burroughs Wellcome Fund and the Rita Allen Foundation (M.Z.L.).

Author information

Authors and Affiliations

Authors

Contributions

J.C. performed protein mutagenesis and characterization, cell imaging, mouse imaging and data analysis and cowrote the paper. S.Y.C. performed myoblast and stem cell purification and transfection. R.D.H. and E.G.-G. assisted with animal experiments. P.L. performed worm culture and made transgenic worms. P.J.C., M.A.B. and M.W.D. performed microscopy of FP fusions. A.J.L. prepared mCardinal proteins for crystallization. J.S.B. and N.J.A. obtained structures of mCardinal and mCardinal-V218E, respectively. H.-L.N., K.C.G., M.W.D., C.H.C., K.S. and H.M.B. provided supervision. M.Z.L. performed protein mutagenesis and characterization, analyzed data, cowrote the paper, provided supervision and directed the project.

Corresponding author

Correspondence to Michael Z Lin.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–16 and Supplementary Table 1 (PDF 11836 kb)

Time-lapse laser-scanning confocal fluorescence microscopy imaging of a mCardinal-18aa-actin fusion protein targeting actin filaments in a fox lung fibroblast

Frames were acquired 18 s apart with a 633 nm laser and a 60× objective. Playback was encoded at 15 frames per s. (MOV 4127 kb)

Time-lapse laser-scanning confocal fluorescence microscopy imaging of a PDHA1-10-aa-mCardinal fusion protein targeting mitochondria in an NIH3T3 fibroblast

Frames were acquired 15 s apart with a 633 nm laser and a 60× objective. Playback was encoded at 15 frames per s. (MOV 1805 kb)

Fast time-lapse laser-scanning confocal fluorescence microscopy imaging of freely moving C. elegans with mCardinal-labeled pharynx

Frames were acquired 125 ms apart with a 635 nm laser and a 20× objective (NA = 0.75). Power measured at the specimen was 100 μW, corresponding to an irradiance of 0.030 J/cm2 per frame. Playback was encoded at 7 frames per s. (MOV 13440 kb)

Fast time-lapse laser-scanning confocal fluorescence microscopy imaging of partially immobilized C. elegans with mCardinal-labeled pharynx. Worms were partially immobilized by dotting cyanoacrylate glue on their tails

1 mg/mL serotonin in M9 was added to keep the sample hydrated during recordings and to induce pharyngeal pumping. Frames were acquired 125 ms apart with a 635 nm laser and a 20× objective (NA = 0.75). Power measured at the specimen was 100 μW, corresponding to an irradiance of 0.030 J/cm2 per frame. Playback was encoded at 7 frames per s. (MOV 11800 kb)

Source data

Rights and permissions

Reprints and permissions

About this article

Cite this article

Chu, J., Haynes, R., Corbel, S. et al. Non-invasive intravital imaging of cellular differentiation with a bright red-excitable fluorescent protein. Nat Methods 11, 572–578 (2014). https://doi.org/10.1038/nmeth.2888

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nmeth.2888

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing