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Multiphoton-evoked color change of DsRed as an optical highlighter for cellular and subcellular labeling

Nature Biotechnology volume 19, pages 645–649 (2001)Cite this article

Abstract

DsRed, a recently cloned red fluorescent protein, has attracted great interest as an expression tracer and fusion partner for multicolor imaging. We report that three-photon excitation (λ <760 nm) rapidly changes the fluorescence of DsRed from red to green when viewed subsequently by conventional (one-photon) epifluorescence. Mechanistically, three-photon excitation (λ <760 nm) selectively bleaches the mature, red-emitting form of DsRed, thereby enhancing emission from the immature green form through reduction of fluorescence resonance energy transfer (FRET). The “greening” effect occurs in live mammalian cells at the cellular and subcellular levels, and the resultant color change persists for >30 h without affecting cell viability. This technique allows individual cells, organelles, and fusion proteins to be optically marked and has potential utility for studying cell lineage, organelle dynamics, and protein trafficking, as well as for selective retrieval of cells from a population. We describe optimal parameters to induce the color change of DsRed, and demonstrate applications that show the potential of this optical highlighter.

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Figure 1: Multiphoton excitation and bleaching of red fluorescence of DsRed.
Figure 2: DsRed-expressing cells display vivid green fluorescence after three-photon photobleaching.
Figure 3: Mechanism of “greening”.
Figure 4: Optimal parameters for “greening”.
Figure 5: Applications of the greening technique for labeling subcellular regions.

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References

  1. Tsien, R.Y. The green fluorescent protein. Annu. Rev. Biochem. 67, 509–544 (1998).

    Article  CAS  Google Scholar 

  2. Matz, M.V. et al. Fluorescent proteins from nonbioluminescent Anthozoa species. Nat. Biotechnol. 17, 969–973 (1999).

    Article  CAS  Google Scholar 

  3. Fradkov, A.F. et al. Novel fluorescent protein from Discosoma coral and its mutants possesses a unique far-red fluorescence. FEBS Lett. 479, 127–130 (2000).

    Article  CAS  Google Scholar 

  4. 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).

    Article  CAS  Google Scholar 

  5. Gross, L.A., Baird, G.S., Hoffman, R.C., Baldridge, K.K. & Tsien, R.Y. The structure of the chromophore within DsRed, a red fluorescent protein from coral. Proc. Natl. Acad. Sci. USA 97, 11990–11995 (2000).

    Article  CAS  Google Scholar 

  6. Denk, W., Piston, D.W. & Webb, W.W. Two-photon molecular excitation in laser scanning microscopy. In Handbook of biological confocal microscopy. (ed. Pawley, J.) 445–458 (Plenum Press, New York; 1995).

    Chapter  Google Scholar 

  7. Jakobs, S., Subramaniam, V., Schonle, A., Jovin, T.M. & Hell, S.W. EGFP and DsRed expressing cultures of Escherichia coli imaged by confocal, two-photon and fluorescence lifetime microscopy. FEBS Lett. 479, 131–135 (2000).

    Article  CAS  Google Scholar 

  8. Heikal, A.A., Hess, S.T., Baird, G.S., Tsien, R.Y. & Webb, W.W. Molecular spectroscopy and dynamics of intrinsically fluorescent proteins: coral red (dsRed) and yellow (Citrine). Proc. Natl. Acad. Sci. USA 97, 11996–12001 (2000).

    Article  CAS  Google Scholar 

  9. Terskikh, A. et al. “Fluorescent timer”; Protein that changes color with time. Science 290, 1585–1588 (2000).

    Article  CAS  Google Scholar 

  10. Wall, M.A., Socolich, M. & Ranganathan, R. The structural basis for red fluorescence in the tetrameric GFP homolog DsRed. Nat. Struct. Biol. 7, 1133–1138 (2000).

    Article  CAS  Google Scholar 

  11. Callamaras, N. & Parker, I. Construction of a confocal microscope for real-time x-y and x-z imaging. Cell Calcium 26, 271–280 (1999).

    Article  CAS  Google Scholar 

  12. Elowitz, M.B., Surette, M.G., Wolf, E., Stock, J. & Leibler, S. Photoactivation turns green fluorescent protein red. Curr. Biol. 7, 809–812 (1997).

    Article  CAS  Google Scholar 

  13. Sawin, K.E. & Nurse, P. Photoactivation of green fluorescent protein. Curr. Biol. 7, R606–607 (1997).

    Article  CAS  Google Scholar 

  14. Xu, C. Two-photon cross sections of indicators. In Imaging neurons. (eds Yuste, R., Lanni, F. & Konnerth, A.) 19.1–19.9 (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; 2000).

    Google Scholar 

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Acknowledgements

We thank Dr. Charlie Glabe for use of the spectrofluorimeter. This work was supported by NIH grants GM48071 and AG16573. G.E.S. is supported by NRSA training grant AG00096.

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Authors and Affiliations

  1. Laboratory of Cellular and Molecular Neurobiology, University of California, 92697-4550, Irvine, CA

    Jonathan S. Marchant & Ian Parker

  2. Department of Neurobiology and Behavior, Laboratory of Molecular Neuropathogenesis, University of California, 92697-4550, Irvine, CA

    Grace E. Stutzmann, Malcolm A. Leissring & Frank M. LaFerla

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Correspondence to Ian Parker.

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Marchant, J., Stutzmann, G., Leissring, M. et al. Multiphoton-evoked color change of DsRed as an optical highlighter for cellular and subcellular labeling. Nat Biotechnol 19, 645–649 (2001). https://doi.org/10.1038/90249

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