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Intensity Range Based Quantitative FRET Data Analysis to Localize Protein Molecules in Live Cell Nuclei

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Förster (fluorescence) resonance energy transfer (FRET) is an ideal technique to estimate the distance between interacting protein molecules in live specimens using intensity-based microscopy. The spectral overlap of donor and acceptor— essential for FRET—also generates a contamination of the FRET signal. There are a number of algorithms available to remove this spectral bleedthrough (SBT) contamination and in this paper we compare two popular algorithms to estimate the SBT element and to calculate a more precise level of energy transfer efficiency, and with that a more accurate distance estimate.

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REFERENCES

  1. R. M. Clegg (1996). In X. F. Wang and B. Herman (Eds.), Fluorescence Imaging Spectroscopy and Microscopy, Wiley, New York, pp. 179–251.

  2. T. Förster (1948). Intermolecular energy migration and fluorescence. Ann. Phys. (Leipzig) 2, 55–75.

    Google Scholar 

  3. L. Stryer (1978). Fluorescence energy transfer as a spectroscopic ruler. Annu. Rev. Biochem. 47, 819–846.

    Article  PubMed  CAS  Google Scholar 

  4. E. A. Jares-Erijman and T.M. Jovin (2003). FRET imaging. Nat. Biotechnol. 21(11), 1387–1395.

    Article  PubMed  CAS  Google Scholar 

  5. B. A. Cunningham (2001). Finding a mate. The Scientist 15, 7–26.

    Google Scholar 

  6. G. W. Gordon, G. Berry, X. H. Liang, B. Levine, and B. Herman (1998). Quantitative fluorescence resonance energy transfer measurements using fluorescence microscopy. Biophys. J. 74, 2702–2713.

    PubMed  CAS  Google Scholar 

  7. A. Periasamy and R. N. Day (2005). Molecular Imaging: FRET Microscopy and Spectroscopy. Oxford University Press: New York.

    Google Scholar 

  8. H. Wallrabe, M. Elangovan, A. Burchard, A. Periasamy, and M. Barroso (2003). Confocal FRET microscopy to measure clustering of receptor-ligand complexes in endocytic membranes. Biophys. J. 85, 559–571.

    PubMed  CAS  Google Scholar 

  9. H. Wallrabe and A. Periasamy (2005). FRET-FLIM microscopy and spectroscopy in the biomedical sciences. Curr. Opin. Biotech. 16, 19–27.

    Article  PubMed  CAS  Google Scholar 

  10. A. Hoppe, K. A. Christensen, and J. A. Swanson (2002). Fluorescence resonance energy transfer-based stoichiometry in living cells. Biophys. J. 83, 3652–3664.

    PubMed  CAS  Google Scholar 

  11. J. R. Lakowicz (1999). Principles of Fluorescence Spectroscopy, 2nd ed., Plenum Press, New York.

    Google Scholar 

  12. A. Periasamy (2001). Methods in Cellular Imaging, Oxford University Press, New York.

    Google Scholar 

  13. A. B. Cubitt, R. Heim, S. R. Adams, A. E. Boyd, L. A. Gross, and R. Y. Tsien (1999). Understanding, improving and using green fluorescent proteins. Trends Biochem. Sci. 20, 448–455.

    Article  Google Scholar 

  14. R. N. Day (1998). Visualization of Pit-1 transcription factor interactions in the living cell nucleus by fluorescence resonance energy transfer microscopy. Mol. Endo. 12, 1410–1419.

    Article  CAS  Google Scholar 

  15. J. Lippincott-Schwartz and G. H. Patterson (2003). Development and use of fluorescent protein markers in living cells. Science 300(5616), 87–91.

    Article  PubMed  CAS  Google Scholar 

  16. A. Miyawaki and R. Y. Tsien (2000). Monitoring protein conformations and interactions by fluorescence resonance energy transfer between mutants of green fluorescent protein. Methods Enzymol. 327, 472–500.

    PubMed  CAS  Google Scholar 

  17. Y. Chen, M. Elangovan, and A. Periasamy (2005). In A. Periasamy and R. N. Day (Eds.), Molecular Imaging: FRET Microscopy and Spectroscopy, Oxford University Press, New York, pp. 126–145.

  18. Z. Xia and Y. Liu (2001). Reliable and global measurement of fluorescence resonance energy transfer using fluorescence microscopes. Biophys. J. 81, 2395–2402.

    Article  PubMed  CAS  Google Scholar 

  19. V. S. Kraynov, C. Chamberlain, G. M. Bokoch, M. A. Schwartz, S. Slabaugh, and K. M. Hahn (2000). Localized Rac activation dynamics visualized in living cells. Science 290, 333–337.

    Article  PubMed  CAS  Google Scholar 

  20. L. Tron, J. Szollosi, S. Damjanovich, S. Helliwell, D. Arndt-Jovin, and T. Jovin (1984). Flow cytometric measurement of fluorescence resonance energy transfer on cell surfaces. Quantitative evaluation of the transfer efficiency on a cell-by-cell basis. Biophys. J. 45, 939–946.

    PubMed  CAS  Google Scholar 

  21. F. S. Wouters, P. I. Bastiaens, K. W. Wirtz, and T. M. Jovin (1998). FRET Microscopy demonstrates molecular association of non-specific lipid transfer protein (nsL-TP) with fatty acid oxidation enzymes in peroxisomes. Embo. J. 17, 7179–7189.

    Article  PubMed  CAS  Google Scholar 

  22. D. C. Youvan, C. M. Silva, E. J. Bylina, W. J. Coleman, M. R. Dilworth, and M. M. Yang (1997). Calibration of fluorescence resonance energy transfer in microscopy using genetically engineered GFP derivatives on nickel chelating beads. Biotechnol. et alia 3, 1–18.

    Google Scholar 

  23. M. Elangovan, H. Wallrabe, Y. Chen, R. N. Day, M. Barroso, and A. Periasamy (2003). Characterization of one- and two-photon excitation resonance energy transfer microscopy. Methods 29, 58–73.

    Article  PubMed  CAS  Google Scholar 

  24. D. Lew, H. Brady, K. Klausing, K. Yaginuma, L. E. Theill, C. Stauber, M. Karin, and P. Mellon (1992). GHF-1-promoter-targeted immortalization of a somatotropic progenitor cell results in dwarfism in transgenic mice. Genes Dev. 7, 683–693.

    Article  Google Scholar 

  25. A. J. Lincoln, C. Williams, and P. F. Johnson (1994). A revised sequence of the rat c/ebp gene. Genes Dev. 8, 1131–1132.

    Article  PubMed  CAS  Google Scholar 

  26. M. Barroso and E. S. Sztul (1994). Basolateral to apical transcytosis in polarized cells is indirect and involves BFA and trimeric G protein sensitive passage through the apical endosome. J. Cell Biol. 124, 83–100.

    Article  PubMed  CAS  Google Scholar 

  27. A. K. Kenworthy, N. Petranova, and M. Edidin (2000). High-resolution FRET microscopy of cholera toxin B-subunit and GPI-anchored proteins in cell plasma membranes. Mol. Biol. Cell 11, 1645–1655.

    PubMed  CAS  Google Scholar 

  28. R. B. Sekar and A. Periasamy (2003). Fluorescence resonance energy transfer (FRET) microscopy imaging of live protein localization. J. Cell Biol. 160, 629–633.

    Article  PubMed  CAS  Google Scholar 

  29. A. Periasamy, and R. N. Day (1999). Visualizing protein interactions in living cells using digitized GFP imaging and FRET microscopy. Meth. Cell Biol. 58, 293–314.

    CAS  Google Scholar 

  30. J. D. Mills, J. R. Stone, J. D. Rubin, D. E. Melon, D. O. Okonkwo, A. Periasamy, and G. A. Helm (2003). Illuminating protein interactions in tissue using confocal and two-photon excitation fluorescent resonance energy transfer (FRET) microscopy. J. Biomed. Opt. 8, 347–356.

    Article  PubMed  CAS  Google Scholar 

  31. C. Barney and G. Danuser (2003). FRET or no FRET: A quantitative comparison. Biophys. J. 84, 3992–4010.

    PubMed  Google Scholar 

  32. B. Herman, G. Gordon, N. Mahajan, and V. Centonze (2001). In A. Periasamy (Ed.), Methods in Cellular Imaging, Oxford University Press, New York, pp. 257–272.

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ACKNOWLEDGMENTS

We wish to acknowledge the funding provided by the National Center for Research Resources (NCRR-NIH, RR021202) and the University of Virginia. We also wish to thank Drs. Margarida Barroso, Richard Day and Horst Wallrabe for their valuable suggestions.

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Authors and Affiliations

  1. W.M. Keck Center for Cellular Imaging, University of Virginia, Gilmer Hall, Charlottesville, Virginia 22904., U.S.

    Ye Chen & Ammasi Periasamy

  2. Departments of Biology and Biomedical Engineering, University of Virginia, Gilmer Hall, Charlottesville, Virginia 22904., U.S.

    Ammasi Periasamy

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  1. Ye Chen
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  2. Ammasi Periasamy
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Correspondence to Ammasi Periasamy.

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Chen, Y., Periasamy, A. Intensity Range Based Quantitative FRET Data Analysis to Localize Protein Molecules in Live Cell Nuclei. J Fluoresc 16, 95–104 (2006). https://doi.org/10.1007/s10895-005-0024-1

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  • DOI: https://doi.org/10.1007/s10895-005-0024-1

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