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Optics Express

Optics Express

  • Editor: C. Martijn de Sterke
  • Vol. 20, Iss. 24 — Nov. 19, 2012
  • pp: 26806–26827

Quantitative multi-color FRET measurements by Fourier lifetime excitation-emission matrix spectroscopy

Ming Zhao, Run Huang, and Leilei Peng  »View Author Affiliations

Optics Express, Vol. 20, Issue 24, pp. 26806-26827 (2012)

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Förster resonant energy transfer (FRET) is extensively used to probe macromolecular interactions and conformation changes. The established FRET lifetime analysis method measures the FRET process through its effect on the donor lifetime. In this paper we present a method that directly probes the time-resolved FRET signal with frequency domain Fourier lifetime excitation-emission matrix (FLEEM) measurements. FLEEM separates fluorescent signals by their different phonon energy pathways from excitation to emission. The FRET process generates a unique signal channel that is initiated by donor excitation but ends with acceptor emission. Time-resolved analysis of the FRET EEM channel allows direct measurements on the FRET process, unaffected by free fluorophores that might be present in the sample. Together with time-resolved analysis on non-FRET channels, i.e. donor and acceptor EEM channels, time resolved EEM analysis allows precise quantification of FRET in the presence of free fluorophores. The method is extended to three-color FRET processes, where quantification with traditional methods remains challenging because of the significantly increased complexity in the three-way FRET interactions. We demonstrate the time-resolved EEM analysis method with quantification of three-color FRET in incompletely hybridized triple-labeled DNA oligonucleotides. Quantitative measurements of the three-color FRET process in triple-labeled dsDNA are obtained in the presence of free single-labeled ssDNA and double-labeled dsDNA. The results establish a quantification method for studying multi-color FRET between multiple macromolecules in biochemical equilibrium.

© 2012 OSA

OCIS Codes
(170.2520) Medical optics and biotechnology : Fluorescence microscopy
(170.6280) Medical optics and biotechnology : Spectroscopy, fluorescence and luminescence

ToC Category:
Medical Optics and Biotechnology

Original Manuscript: July 24, 2012
Revised Manuscript: October 14, 2012
Manuscript Accepted: November 6, 2012
Published: November 13, 2012

Virtual Issues
Vol. 7, Iss. 12 Virtual Journal for Biomedical Optics

Ming Zhao, Run Huang, and Leilei Peng, "Quantitative multi-color FRET measurements by Fourier lifetime excitation-emission matrix spectroscopy," Opt. Express 20, 26806-26827 (2012)

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  1. T. Forster, “Zwischenmolekulare energiewanderung und fluoreszenz,” Ann. Phys. (Berlin)437(1-2), 55–75 (1948). [CrossRef]
  2. L. Stryer, “Fluorescence energy transfer as a spectroscopic ruler,” Annu. Rev. Biochem.47(1), 819–846 (1978). [CrossRef] [PubMed]
  3. M. Elangovan, R. N. Day, and A. Periasamy, “Nanosecond fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy to localize the protein interactions in a single living cell,” J. Microsc.205(1), 3–14 (2002). [CrossRef] [PubMed]
  4. S. C. Blanchard, H. D. Kim, R. L. Gonzalez, J. D. Puglisi, and S. Chu, “tRNA dynamics on the ribosome during translation,” Proc. Natl. Acad. Sci. U.S.A.101(35), 12893–12898 (2004). [CrossRef] [PubMed]
  5. S. Kumar, D. Alibhai, A. Margineanu, R. Laine, G. Kennedy, J. McGinty, S. Warren, D. Kelly, Y. Alexandrov, I. Munro, C. Talbot, D. W. Stuckey, C. Kimberly, B. Viellerobe, F. Lacombe, E. W. F. Lam, H. Taylor, M. J. Dallman, G. Stamp, E. J. Murray, F. Stuhmeier, A. Sardini, M. Katan, D. S. Elson, M. A. A. Neil, C. Dunsby, and P. M. W. French, “FLIM FRET technology for drug discovery: automated multiwell-plate high-content analysis, multiplexed readouts and application in situ,” ChemPhysChem12(3), 609–626 (2011). [CrossRef] [PubMed]
  6. E. A. Jares-Erijman and T. M. Jovin, “FRET imaging,” Nat. Biotechnol.21(11), 1387–1395 (2003). [CrossRef] [PubMed]
  7. D. W. Piston and G. J. Kremers, “Fluorescent protein FRET: the good, the bad and the ugly,” Trends Biochem. Sci.32(9), 407–414 (2007). [CrossRef] [PubMed]
  8. J. R. Lakowicz and A. Balter, “Theory of phase-modulation fluorescence spectroscopy for excited-state processes,” Biophys. Chem.16(2), 99–115 (1982). [CrossRef] [PubMed]
  9. J. R. Lakowicz and A. Balter, “Analysis of excited-state processes by phase-modulation fluorescence spectroscopy,” Biophys. Chem.16(2), 117–132 (1982). [CrossRef] [PubMed]
  10. S. P. Laptenok, J. W. Borst, K. M. Mullen, I. H. M. van Stokkum, A. J. Visser, and H. van Amerongen, “Global analysis of Forster resonance energy transfer in live cells measured by fluorescence lifetime imaging microscopy exploiting the rise time of acceptor fluorescence,” Phys. Chem. Chem. Phys.12(27), 7593–7602 (2010). [CrossRef] [PubMed]
  11. J. W. Borst, S. P. Laptenok, A. H. Westphal, R. Kühnemuth, H. Hornen, N. V. Visser, S. Kalinin, J. Aker, A. van Hoek, C. A. M. Seidel, and A. J. W. G. Visser, “Structural changes of yellow cameleon domains observed by quantitative FRET analysis and polarized fluorescence correlation spectroscopy,” Biophys. J.95(11), 5399–5411 (2008). [CrossRef] [PubMed]
  12. J. R. Lakowicz, Principles of fluorescence spectroscopy, third edition (Springer, 2006).
  13. J. R. Lakowicz, G. Laczko, H. Cherek, E. Gratton, and M. Limkeman, “Analysis of fluorescence decay kinetics from variable-frequency phase shift and modulation data,” Biophys. J.46(4), 463–477 (1984). [CrossRef] [PubMed]
  14. H. M. Watrob, C. P. Pan, and M. D. Barkley, “Two-step FRET as a structural tool,” J. Am. Chem. Soc.125(24), 7336–7343 (2003). [CrossRef] [PubMed]
  15. D. Klostermeier, P. Sears, C. H. Wong, D. P. Millar, and J. R. Williamson, “A three-fluorophore FRET assay for high-throughput screening of small-molecule inhibitors of ribosome assembly,” Nucleic Acids Res.32(9), 2707–2715 (2004). [CrossRef] [PubMed]
  16. Y. S. Sun, H. Wallrabe, C. F. Booker, R. N. Day, and A. Periasamy, “Three-color spectral FRET microscopy localizes three interacting proteins in living cells,” Biophys. J.99(4), 1274–1283 (2010). [CrossRef] [PubMed]
  17. E. Galperin, V. V. Verkhusha, and A. Sorkin, “Three-chromophore FRET microscopy to analyze multiprotein interactions in living cells,” Nat. Methods1(3), 209–217 (2004). [CrossRef] [PubMed]
  18. D. M. Grant, W. Zhang, E. J. McGhee, T. D. Bunney, C. B. Talbot, S. Kumar, I. Munro, C. Dunsby, M. A. Neil, M. Katan, and P. M. French, “Multiplexed FRET to image multiple signaling events in live cells,” Biophys. J.95(10), L69–L71 (2008). [CrossRef] [PubMed]
  19. M. Zhao and L. Peng, “Multiplexed fluorescence lifetime measurements by frequency-sweeping Fourier spectroscopy,” Opt. Lett.35(17), 2910–2912 (2010). [CrossRef] [PubMed]
  20. J. Lee, S. Lee, K. Ragunathan, C. Joo, T. Ha, and S. Hohng, “Single-molecule four-color FRET,” Angew. Chem. Int. Ed.49(51), 9922–9925 (2010). [CrossRef]
  21. D. W. Millican and L. B. McGown, “Fluorescence lifetime selectivity in excitation emission matrices for qualitative-analysis of a 2-component system,” Anal. Chem.61(6), 580–583 (1989). [CrossRef]
  22. D. W. Millican and L. B. McGown, “Fluorescence lifetime resolution of spectra in the frequency-domain using multiway analysis,” Anal. Chem.62(20), 2242–2247 (1990). [CrossRef]
  23. J. M. Beechem, “Global analysis of biochemical and biophysical data,” Methods Enzymol.210, 37–54 (1992). [CrossRef] [PubMed]
  24. T. Ha, I. Rasnik, W. Cheng, H. P. Babcock, G. H. Gauss, T. M. Lohman, and S. Chu, “Initiation and re-initiation of DNA unwinding by the Escherichia coli Rep helicase,” Nature419(6907), 638–641 (2002). [CrossRef] [PubMed]
  25. D. G. Norman, R. J. Grainger, D. Uhrín, and D. M. J. Lilley, “Location of cyanine-3 on double-stranded DNA: Importance for fluorescence resonance energy transfer studies,” Biochemistry39(21), 6317–6324 (2000). [CrossRef] [PubMed]

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