Abstract

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

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    [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).
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    [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).
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    [CrossRef] [PubMed]
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    [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).
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  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]

2011 (1)

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]

2010 (4)

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]

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]

M. Zhao and L. Peng, “Multiplexed fluorescence lifetime measurements by frequency-sweeping Fourier spectroscopy,” Opt. Lett.35(17), 2910–2912 (2010).
[CrossRef] [PubMed]

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]

2008 (2)

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]

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]

2007 (1)

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]

2004 (3)

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]

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]

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]

2003 (2)

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]

E. A. Jares-Erijman and T. M. Jovin, “FRET imaging,” Nat. Biotechnol.21(11), 1387–1395 (2003).
[CrossRef] [PubMed]

2002 (2)

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]

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]

2000 (1)

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]

1992 (1)

J. M. Beechem, “Global analysis of biochemical and biophysical data,” Methods Enzymol.210, 37–54 (1992).
[CrossRef] [PubMed]

1990 (1)

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]

1989 (1)

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]

1984 (1)

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]

1982 (2)

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]

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]

1978 (1)

L. Stryer, “Fluorescence energy transfer as a spectroscopic ruler,” Annu. Rev. Biochem.47(1), 819–846 (1978).
[CrossRef] [PubMed]

1948 (1)

T. Forster, “Zwischenmolekulare energiewanderung und fluoreszenz,” Ann. Phys. (Berlin)437(1-2), 55–75 (1948).
[CrossRef]

Aker, J.

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]

Alexandrov, Y.

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]

Alibhai, D.

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]

Babcock, H. P.

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]

Balter, A.

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]

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]

Barkley, M. D.

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]

Beechem, J. M.

J. M. Beechem, “Global analysis of biochemical and biophysical data,” Methods Enzymol.210, 37–54 (1992).
[CrossRef] [PubMed]

Blanchard, S. C.

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]

Booker, C. F.

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]

Borst, J. W.

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]

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]

Bunney, T. D.

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]

Cheng, W.

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]

Cherek, H.

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]

Chu, S.

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]

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]

Dallman, M. J.

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]

Day, R. N.

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]

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]

Dunsby, C.

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]

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]

Elangovan, M.

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]

Elson, D. S.

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]

Forster, T.

T. Forster, “Zwischenmolekulare energiewanderung und fluoreszenz,” Ann. Phys. (Berlin)437(1-2), 55–75 (1948).
[CrossRef]

French, P. M.

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]

French, P. M. W.

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]

Galperin, E.

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]

Gauss, G. H.

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]

Gonzalez, R. L.

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]

Grainger, R. J.

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]

Grant, D. M.

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]

Gratton, E.

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]

Ha, T.

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]

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]

Hohng, S.

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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).
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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).
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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).
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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).
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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).
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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).
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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).
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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).
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[CrossRef] [PubMed]

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).
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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).
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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).
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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).
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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]

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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).
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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).
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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).
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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).
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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).
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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).
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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).
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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).
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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).
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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).
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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).
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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).
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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).
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Ragunathan, K.

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]

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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]

Sardini, A.

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).
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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).
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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).
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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).
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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]

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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]

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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).
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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).
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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]

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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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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]

van Hoek, A.

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]

van Stokkum, I. H. M.

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]

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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]

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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]

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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]

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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]

Visser, N. V.

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]

Wallrabe, H.

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]

Warren, S.

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]

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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]

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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]

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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).
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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]

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Figures (7)

Fig. 1
Fig. 1

Excitation emission matrix (EEM) representation of three-color FRET between fluorescein, Cy3 and Cy5. (a) Photon pathways in a three-color FRET process. Six possible exciter-to-emitter photon pathways are present. (b) EEM representation of the three-color FRET as a function of both excitation and emission wavelengths. Different photon pathways occupy different regions of the EEM. For each photon pathway, the excitation spectrum follows the exciter, and the emission spectrum follows the emitter.

Fig. 2
Fig. 2

Time-resolved EEM analysis sequence of three-color FRET. The analysis is performed on a channel-by-channel basis, with each channel involving only at most one unknown lifetime parameter. EEM channels in the illustration are color-coded by their decay models. The analysis first obtains the longest wavelength acceptor (fluorophore No. 3) lifetime τ3 in ê33, then finds quenched lifetime τ 2 123 of fluorophore No. 2 in FRET channel ê23. The percentage of quenched fluorophore, P2 is then calculated from fluorophore 2 EEM channel ê22. The FRET channel ê12 is next, which yields the quenched lifetime τ 1 123 of fluorophore 1. The percentage of quenched fluorophore No. 1, P1 is extracted from the EEM channel ê11, and finally the FRET channel ê13 serves as a verification of the time-resolved EEM analysis.

Fig. 3
Fig. 3

Structure of the triple-labeled dsDNA. The distances between fluorescein and Cy3, or Cy3 and Cy5 were 15 base-pairs or 5.1 nm.

Fig. 4
Fig. 4

Schematic of the FLEEM system and data processing. A Michelson interferometer is used to modulate a multi-wavelength CW laser source. The modulated output of the interferometer then excites a fluorescent sample. The laser references and fluorescence emission signals at different excitation-emission wavelengths combinantions are digitized and cross-correlated to obtain frequency response of the sample as an EEM.

Fig. 5
Fig. 5

Time-resolved EEM measurements on double-labeled dsDNA. (a) Modulation and phase of quenched Alexa488 (donor EEM channel ê11), in comparison with unquenched Alexa488. The fluorescence lifetime of Alexa488 decreased from 4.1 ± 0.1 ns to 3.0 ± 0.1 ns due to FRET. (b) Modulation and phase of Aelxa546 (acceptor EEM channel ê22). The fluorescence lifetime of Alexa546 was 3.4 ± 0.1 ns, same as pure Alexa546. The acceptor EEM channel is unrelated to FRET, thus the lifetime remains constant. (c) Modulation and phase of the Alexa488-Alexa546 FRET EEM channel ê12. Experimental results in (c) were overlaid with the theoretical model (Eq. (8)). The phase delay in the FRET EEM channel exceeded π/2, which was a signature of FRET. (d) Spectral configuration of the EEM measurement. Error bars in (a~c) represent standard deviations of multiple 46-μs frequency sweeps.

Fig. 6
Fig. 6

Frequency responses of bleedthrough corrected EEM channels from a mixture of two-color FRET complexes and free donors. (a) Cy3 EEM channel ê22 fitted with single exponential decay. The acceptor lifetime was found to be τCy3 = 1.5 ± 0.1 ns. (b) Fluorescein-Cy3 FRET EEM channel ê12 fitted with the FRET frequency response model (Eq. (8)). The quenched lifetime of fluorescein was calculated as τfluo-quench = 1.0 ± 0.1 ns (c) Fluorescein EEM channel ê11 fitted with a double exponential decay model, in which two lifetime, quenched and unquenched lifetime were fixed at knowing values. The percentage of quenched fluorescein was found to be Pfluo = 37 ± 2%. (d) Spectral configuration of the EEM measurement. Error bars in (a~c) represent standard deviations of multiple measurements with 1 ms integration time. Fitted curves of quenched/unquenched donor and acceptor response are plotted in (a~c) for reference.

Fig. 7
Fig. 7

Frequency responses of bleedthrough corrected EEM channels for a three-color FRET mixture with incomplete FRET complexes and free fluorophores. (a) Cy5 EEM channel ê33. The lifetime of the final acceptor Cy5 remained unchanged at τCy5 = 1.8 ± 0.1 ns. (b) Cy3-Cy5 FRET channel ê23. The lifetime of quenched Cy3 was measured as τCy3-quench = 0.8 ± 0.1 ns. (c) Cy3 EEM channel ê22. A double exponential fit found the percentage of quenched Cy3 is PCy3 = 80 ± 5%. (d) Fluorescein-Cy3 FRET channel ê12. The quenched fluorescein lifetime was measured as τfluo-quench = 1.2 ± 0.1 ns. (e) Fluorescein EEM channel ê11. The percentage of quenched fluorescein was measured as Pfluo = 85 ± 2%. (f) Fluorescein-Cy5 FRET channel ê13. This channel contained signal from the two-step FRET (fluorescein-Cy3 then Cy3-Cy5). The measured frequency response was overlaid with the theoretical two-step FRET model, which was a product of individual frequency responses of quenched fluorescein, quenched Cy3 and Cy5. Fitted curves of quenched/unquenched donor and acceptor response were plotted for reference. Error bars represent standard deviations of multiple measurements with 1 ms integration time.

Tables (3)

Tables Icon

Table 1 Intensities, Lifetimes and Percentages of Quenched Donor for Different Donor-acceptor ratio FRET Mixtures

Tables Icon

Table 2 Comparison between EEM Analysis and Double Exponential Fitting of the Donor EEM Channel

Tables Icon

Table 3 Comparison of Three-color FRET with Two-color Controls

Equations (17)

Equations on this page are rendered with MathJax. Learn more.

I = B Em I B Ex ,
( I 11 I 12 I 22 I 13 I 23 I 33 )=( 1 B 12 Em 1 B 13 Em B 23 Em 1 )( I 11 I 12 I 22 I 13 I 23 I 33 )( 1 B 12 Ex 1 B 13 Ex B 23 Ex 1 ),
( I 11 I 12 I 22 I 13 I 23 I 33 )= ( 1 B 12 Em 1 B 13 Em B 23 Em 1 ) 1 ( I 11 I 12 I 22 I 13 I 23 I 33 ) ( 1 B 12 Ex 1 B 13 Ex B 23 Ex 1 ) 1
I Fluo (t)= I exc (t)R(t)
I Fluo (t)=δ(t)R(t)=u( t )R(t),
I ˜ Fluo (ω)=FT( sin( ω 0 t)R(t) )=δ(ω)× R ˜ ( ω )= R ˜ (ω),
I 12 (t)= k 12 N 1 exc (t) R 2 12 (t) = k 12 ( k 1 12 ) 1 u(t) R 1 12 (t) R 2 12 (t),
I ˜ 12 (ω)= k 12 ( k 1 12 ) 1 R ˜ 1 12 (ω)× R ˜ 2 12 (ω).
I ˜ 1N (ω)= i=1,N1 k i(i+1) ( k i 1...N ) 1 R ˜ 1 (ω)× R ˜ 2 (ω)×× R ˜ N (ω).
EE M 12 =( R ˜ 1 12 ( ω ) k 12 ( k 1 12 ) 1 R ˜ 1 12 ( ω ) R ˜ 2 12 ( ω ) R ˜ 2 12 ( ω ) ).
EE M Mixture = C 12 EE M 12 + C 1 R ˜ 1 1 ( ω ) e ^ 11 + C 2 R ˜ 2 2 ( ω ) e ^ 22 ,
k 1 12 > k 1 1 , and R ˜ 1 12 ( ω ) R ˜ 1 1 ( ω ).
EE M 123 =( R ˜ 1 123 0 0 k 12 ( k 1 123 ) 1 R ˜ 1 123 R ˜ 2 123 R ˜ 2 123 0 k 12 k 23 ( k 1 123 k 2 123 ) 1 R ˜ 1 123 R ˜ 2 123 R ˜ 3 123 + k 13 ( k 1 123 ) 1 R ˜ 1 123 R ˜ 3 123 k 23 ( k 2 123 ) 1 R ˜ 2 123 R ˜ 3 123 R ˜ 3 123 ).
EE M Mixture = C 123 EE M 123 + C 12 EE M 12 + C 23 EE M 23 + C 13 EE M 13 + C 1 R ˜ 1 1 e ^ 11 + C 2 R ˜ 2 2 e ^ 22 + C 3 R ˜ 3 3 e ^ 33
{ P 1 = C 12 + C 123 C 1 + C 13 + C 12 + C 123 1 P 1 = C 1 + C 13 C 1 + C 13 + C 12 + C 123
{ P 2 = C 23 + C 123 C 2 + C 12 + C 23 + C 123 1 P 2 = C 2 + C 12 C 2 + C 12 + C 23 + C 123
{ C 1 : C 12 = C 13 : C 123 C 2 : C 23 = C 12 : C 123

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