Abstract

We report the investigation of Foerster’s Resonance Energy Transfer dynamics in GFP based tandem constructs in living T-cells using a combination of Fluorescence Lifetime Imaging Microscopy (FLIM) and Fluorescence Lifetime Micro-Spectroscopy (FLMS) at picosecond time resolution and nanometer spectral resolution. The involvement of multiple lifetimes of CFP in energy transfer was analyzed by plotting pre-exponential factors of individual lifetimes along the wavelength resulting in the Decay Associated Spectra (DAS). A change in the amplitude of pre-exponential factors from positive to negative at the acceptor emission maxima was used as a confirmation of FRET in the multiexponential lifetime analysis.

© 2006 Optical Society of America

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    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
  4. M. Tramier, I. Gautier, T. Piolot, S. Ravalet, K. Kemnitz, J. Coppey, C. Durieux, V. Mignotte, and M. Coppey-Moisan, "Picosecond-hetero-FRET microscopy to probe protein-protein interactions in live cells," Biophys. J. 83, 3570-3577 (2002).
    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
  17. R. R. Duncan, A. Bergmann, M. A. Cousin, D. K. Apps, and M. J. Shipston, "Multi-dimensional time-correlated single photon counting (TCSPC) fluorescence lifetime imaging microscopy (FLIM) to detect FRET in cells," J. Microsc. 215, 1-12 (2004).
    [CrossRef] [PubMed]
  18. W. Holzer, A. Penzkofer, M. Fuhrmann, and P. Hegemann, "Spectroscopic characterization of flavin mononucleotide bound to the LOV1 domain of Phot1 from Chlamydomonas reinhardtii," Photochem. Photobiol. 75, 479-487 (2002).
    [CrossRef] [PubMed]
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2005 (1)

J. W. Borst, M. A. Hink, A. V. Hoek, and A. J. W. G. Visser, "Effects of Refractive index and viscocity on Flourescence and Anisotropy Decays of Enhanced Cyan and Yellow Fluorescent Proteins," J. Fluoresc. 15, 153-160 (2005).
[CrossRef] [PubMed]

2004 (2)

M. Tramier, K. Kemnitz, C. Durieux, and M. Coppey-Moisan, "Picosecond time-resolved microspectrofluorometry in live cells exemplified by complex fluorescence dynamics of popular probes ethidium and cyan fluorescent protein," J. Microsc. 213, 110-118 (2004).
[CrossRef] [PubMed]

R. R. Duncan, A. Bergmann, M. A. Cousin, D. K. Apps, and M. J. Shipston, "Multi-dimensional time-correlated single photon counting (TCSPC) fluorescence lifetime imaging microscopy (FLIM) to detect FRET in cells," J. Microsc. 215, 1-12 (2004).
[CrossRef] [PubMed]

2003 (3)

J. Lippincott-Schwartz and G. H. Patterson, "Development and use of fluorescent protein markers in living cells," Science 300, 87-90 (2003).
[CrossRef] [PubMed]

P. Kapusta, R. Erdmann, U. Ortmann, and M. Wahl, "Time-resolved fluorescence anisotropy measurements made simple," J. Fluoresc. 13, 179-183 (2003).
[CrossRef]

Y. Chen, J. D. Mills, and A. Periasamy, "Protein localization in living cells and tissues using FRET and FLIM," Differentiation 71, 528-541 (2003).
[CrossRef] [PubMed]

2002 (5)

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, 3-14 (2002).
[CrossRef] [PubMed]

M. Tramier, I. Gautier, T. Piolot, S. Ravalet, K. Kemnitz, J. Coppey, C. Durieux, V. Mignotte, and M. Coppey-Moisan, "Picosecond-hetero-FRET microscopy to probe protein-protein interactions in live cells," Biophys. J. 83, 3570-3577 (2002).
[CrossRef] [PubMed]

W. Holzer, A. Penzkofer, M. Fuhrmann, and P. Hegemann, "Spectroscopic characterization of flavin mononucleotide bound to the LOV1 domain of Phot1 from Chlamydomonas reinhardtii," Photochem. Photobiol. 75, 479-487 (2002).
[CrossRef] [PubMed]

M. H. Seifert, D. Ksiazek, M. K. Azim, P. Smialowski, N. Budisa, and T. A. Holak, "Slow exchange in the chromophore of a green fluorescent protein variant," J. Am. Chem. Soc. 124, 7932-7942 (2002).
[CrossRef] [PubMed]

S. Habuchi, M. Cotlet, J. Hofkens, G. Dirix, J. Michiels, J. Vanderleyden, V. Subramaniam, and F. C. D. Schryver, "Resonance Energy Transfer in a Calcium Concentration-Dependent Cameleon Protein," Biophys. J. 83, 3499-3506 (2002).
[CrossRef] [PubMed]

2001 (1)

I. Gautier, M. Tramier, C. Durieux, J. Coppey, R. B. Pansu, J. C. Nicolas, K. Kemnitz, and M. Coppey-Moisan, "Homo-FRET microscopy in living cells to measure monomer-dimer transition of GFP-tagged proteins," Biophys. J. 80, 3000-3008 (2001).
[CrossRef] [PubMed]

2000 (2)

S. I. Murata, J. Kusba, G. Piszczek, I. Gryczynski, and J. R. Lakowicz, "Donor fluorescence decay analysis for energy transfer in double-helical DNA with various acceptor concentrations," Biopolymers 57, 306-315 (2000).
[CrossRef] [PubMed]

T. Kuner and G. J. Augustine, "A genetically encoded ratiometric indicator for chloride: capturing chloride transients in cultured hippocampal neurons," Neuron 27, 447-459 (2000).
[CrossRef] [PubMed]

1997 (1)

K. Kemnitz, L. Pfeifer, R. Paul, and M. Coppey-Moisan, "Novel detectors for fluorescence lifetime imaging on the picosecond time scale," J. Fluoresc. 7, 93-98. (1997).
[CrossRef]

1996 (1)

M. Ormo, A. B. Cubitt, K. Kallio, L. A. Gross, R. Y. Tsien, and S. J. Remington, "Crystal structure of the Aequorea victoria green fluorescent protein," Science 273, 1392-1395 (1996).
[CrossRef] [PubMed]

1986 (1)

L. Davenport, J. R. Knutson, and L. Brand, "Excited-state proton transfer of equilenin and dihydroequilenin: interaction with bilayer vesicles," Biochemistry 25, 1186-1195 (1986).
[CrossRef] [PubMed]

1982 (1)

J. R. Knutson, D. G. Walbridge, and L. Brand, "Decay-associated fluorescence spectra and the heterogeneous emission of alcohol dehydrogenase," Biochemistry 21, 4671-4679 (1982).
[CrossRef] [PubMed]

Apps, D. K.

R. R. Duncan, A. Bergmann, M. A. Cousin, D. K. Apps, and M. J. Shipston, "Multi-dimensional time-correlated single photon counting (TCSPC) fluorescence lifetime imaging microscopy (FLIM) to detect FRET in cells," J. Microsc. 215, 1-12 (2004).
[CrossRef] [PubMed]

Augustine, G. J.

T. Kuner and G. J. Augustine, "A genetically encoded ratiometric indicator for chloride: capturing chloride transients in cultured hippocampal neurons," Neuron 27, 447-459 (2000).
[CrossRef] [PubMed]

Azim, M. K.

M. H. Seifert, D. Ksiazek, M. K. Azim, P. Smialowski, N. Budisa, and T. A. Holak, "Slow exchange in the chromophore of a green fluorescent protein variant," J. Am. Chem. Soc. 124, 7932-7942 (2002).
[CrossRef] [PubMed]

Bergmann, A.

R. R. Duncan, A. Bergmann, M. A. Cousin, D. K. Apps, and M. J. Shipston, "Multi-dimensional time-correlated single photon counting (TCSPC) fluorescence lifetime imaging microscopy (FLIM) to detect FRET in cells," J. Microsc. 215, 1-12 (2004).
[CrossRef] [PubMed]

Borst, J. W.

J. W. Borst, M. A. Hink, A. V. Hoek, and A. J. W. G. Visser, "Effects of Refractive index and viscocity on Flourescence and Anisotropy Decays of Enhanced Cyan and Yellow Fluorescent Proteins," J. Fluoresc. 15, 153-160 (2005).
[CrossRef] [PubMed]

Brand, L.

L. Davenport, J. R. Knutson, and L. Brand, "Excited-state proton transfer of equilenin and dihydroequilenin: interaction with bilayer vesicles," Biochemistry 25, 1186-1195 (1986).
[CrossRef] [PubMed]

J. R. Knutson, D. G. Walbridge, and L. Brand, "Decay-associated fluorescence spectra and the heterogeneous emission of alcohol dehydrogenase," Biochemistry 21, 4671-4679 (1982).
[CrossRef] [PubMed]

Budisa, N.

M. H. Seifert, D. Ksiazek, M. K. Azim, P. Smialowski, N. Budisa, and T. A. Holak, "Slow exchange in the chromophore of a green fluorescent protein variant," J. Am. Chem. Soc. 124, 7932-7942 (2002).
[CrossRef] [PubMed]

Chen, Y.

Y. Chen, J. D. Mills, and A. Periasamy, "Protein localization in living cells and tissues using FRET and FLIM," Differentiation 71, 528-541 (2003).
[CrossRef] [PubMed]

Coppey, J.

M. Tramier, I. Gautier, T. Piolot, S. Ravalet, K. Kemnitz, J. Coppey, C. Durieux, V. Mignotte, and M. Coppey-Moisan, "Picosecond-hetero-FRET microscopy to probe protein-protein interactions in live cells," Biophys. J. 83, 3570-3577 (2002).
[CrossRef] [PubMed]

I. Gautier, M. Tramier, C. Durieux, J. Coppey, R. B. Pansu, J. C. Nicolas, K. Kemnitz, and M. Coppey-Moisan, "Homo-FRET microscopy in living cells to measure monomer-dimer transition of GFP-tagged proteins," Biophys. J. 80, 3000-3008 (2001).
[CrossRef] [PubMed]

Coppey-Moisan, M.

M. Tramier, K. Kemnitz, C. Durieux, and M. Coppey-Moisan, "Picosecond time-resolved microspectrofluorometry in live cells exemplified by complex fluorescence dynamics of popular probes ethidium and cyan fluorescent protein," J. Microsc. 213, 110-118 (2004).
[CrossRef] [PubMed]

M. Tramier, I. Gautier, T. Piolot, S. Ravalet, K. Kemnitz, J. Coppey, C. Durieux, V. Mignotte, and M. Coppey-Moisan, "Picosecond-hetero-FRET microscopy to probe protein-protein interactions in live cells," Biophys. J. 83, 3570-3577 (2002).
[CrossRef] [PubMed]

I. Gautier, M. Tramier, C. Durieux, J. Coppey, R. B. Pansu, J. C. Nicolas, K. Kemnitz, and M. Coppey-Moisan, "Homo-FRET microscopy in living cells to measure monomer-dimer transition of GFP-tagged proteins," Biophys. J. 80, 3000-3008 (2001).
[CrossRef] [PubMed]

K. Kemnitz, L. Pfeifer, R. Paul, and M. Coppey-Moisan, "Novel detectors for fluorescence lifetime imaging on the picosecond time scale," J. Fluoresc. 7, 93-98. (1997).
[CrossRef]

Cotlet, M.

S. Habuchi, M. Cotlet, J. Hofkens, G. Dirix, J. Michiels, J. Vanderleyden, V. Subramaniam, and F. C. D. Schryver, "Resonance Energy Transfer in a Calcium Concentration-Dependent Cameleon Protein," Biophys. J. 83, 3499-3506 (2002).
[CrossRef] [PubMed]

Cousin, M. A.

R. R. Duncan, A. Bergmann, M. A. Cousin, D. K. Apps, and M. J. Shipston, "Multi-dimensional time-correlated single photon counting (TCSPC) fluorescence lifetime imaging microscopy (FLIM) to detect FRET in cells," J. Microsc. 215, 1-12 (2004).
[CrossRef] [PubMed]

Cubitt, A. B.

M. Ormo, A. B. Cubitt, K. Kallio, L. A. Gross, R. Y. Tsien, and S. J. Remington, "Crystal structure of the Aequorea victoria green fluorescent protein," Science 273, 1392-1395 (1996).
[CrossRef] [PubMed]

Davenport, L.

L. Davenport, J. R. Knutson, and L. Brand, "Excited-state proton transfer of equilenin and dihydroequilenin: interaction with bilayer vesicles," Biochemistry 25, 1186-1195 (1986).
[CrossRef] [PubMed]

Day, R. N.

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, 3-14 (2002).
[CrossRef] [PubMed]

Dirix, G.

S. Habuchi, M. Cotlet, J. Hofkens, G. Dirix, J. Michiels, J. Vanderleyden, V. Subramaniam, and F. C. D. Schryver, "Resonance Energy Transfer in a Calcium Concentration-Dependent Cameleon Protein," Biophys. J. 83, 3499-3506 (2002).
[CrossRef] [PubMed]

Duncan, R. R.

R. R. Duncan, A. Bergmann, M. A. Cousin, D. K. Apps, and M. J. Shipston, "Multi-dimensional time-correlated single photon counting (TCSPC) fluorescence lifetime imaging microscopy (FLIM) to detect FRET in cells," J. Microsc. 215, 1-12 (2004).
[CrossRef] [PubMed]

Durieux, C.

M. Tramier, K. Kemnitz, C. Durieux, and M. Coppey-Moisan, "Picosecond time-resolved microspectrofluorometry in live cells exemplified by complex fluorescence dynamics of popular probes ethidium and cyan fluorescent protein," J. Microsc. 213, 110-118 (2004).
[CrossRef] [PubMed]

M. Tramier, I. Gautier, T. Piolot, S. Ravalet, K. Kemnitz, J. Coppey, C. Durieux, V. Mignotte, and M. Coppey-Moisan, "Picosecond-hetero-FRET microscopy to probe protein-protein interactions in live cells," Biophys. J. 83, 3570-3577 (2002).
[CrossRef] [PubMed]

I. Gautier, M. Tramier, C. Durieux, J. Coppey, R. B. Pansu, J. C. Nicolas, K. Kemnitz, and M. Coppey-Moisan, "Homo-FRET microscopy in living cells to measure monomer-dimer transition of GFP-tagged proteins," Biophys. J. 80, 3000-3008 (2001).
[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, 3-14 (2002).
[CrossRef] [PubMed]

Erdmann, R.

P. Kapusta, R. Erdmann, U. Ortmann, and M. Wahl, "Time-resolved fluorescence anisotropy measurements made simple," J. Fluoresc. 13, 179-183 (2003).
[CrossRef]

Fuhrmann, M.

W. Holzer, A. Penzkofer, M. Fuhrmann, and P. Hegemann, "Spectroscopic characterization of flavin mononucleotide bound to the LOV1 domain of Phot1 from Chlamydomonas reinhardtii," Photochem. Photobiol. 75, 479-487 (2002).
[CrossRef] [PubMed]

Gautier, I.

M. Tramier, I. Gautier, T. Piolot, S. Ravalet, K. Kemnitz, J. Coppey, C. Durieux, V. Mignotte, and M. Coppey-Moisan, "Picosecond-hetero-FRET microscopy to probe protein-protein interactions in live cells," Biophys. J. 83, 3570-3577 (2002).
[CrossRef] [PubMed]

I. Gautier, M. Tramier, C. Durieux, J. Coppey, R. B. Pansu, J. C. Nicolas, K. Kemnitz, and M. Coppey-Moisan, "Homo-FRET microscopy in living cells to measure monomer-dimer transition of GFP-tagged proteins," Biophys. J. 80, 3000-3008 (2001).
[CrossRef] [PubMed]

Gross, L. A.

M. Ormo, A. B. Cubitt, K. Kallio, L. A. Gross, R. Y. Tsien, and S. J. Remington, "Crystal structure of the Aequorea victoria green fluorescent protein," Science 273, 1392-1395 (1996).
[CrossRef] [PubMed]

Gryczynski, I.

S. I. Murata, J. Kusba, G. Piszczek, I. Gryczynski, and J. R. Lakowicz, "Donor fluorescence decay analysis for energy transfer in double-helical DNA with various acceptor concentrations," Biopolymers 57, 306-315 (2000).
[CrossRef] [PubMed]

Habuchi, S.

S. Habuchi, M. Cotlet, J. Hofkens, G. Dirix, J. Michiels, J. Vanderleyden, V. Subramaniam, and F. C. D. Schryver, "Resonance Energy Transfer in a Calcium Concentration-Dependent Cameleon Protein," Biophys. J. 83, 3499-3506 (2002).
[CrossRef] [PubMed]

Hegemann, P.

W. Holzer, A. Penzkofer, M. Fuhrmann, and P. Hegemann, "Spectroscopic characterization of flavin mononucleotide bound to the LOV1 domain of Phot1 from Chlamydomonas reinhardtii," Photochem. Photobiol. 75, 479-487 (2002).
[CrossRef] [PubMed]

Hink, M. A.

J. W. Borst, M. A. Hink, A. V. Hoek, and A. J. W. G. Visser, "Effects of Refractive index and viscocity on Flourescence and Anisotropy Decays of Enhanced Cyan and Yellow Fluorescent Proteins," J. Fluoresc. 15, 153-160 (2005).
[CrossRef] [PubMed]

Hoek, A. V.

J. W. Borst, M. A. Hink, A. V. Hoek, and A. J. W. G. Visser, "Effects of Refractive index and viscocity on Flourescence and Anisotropy Decays of Enhanced Cyan and Yellow Fluorescent Proteins," J. Fluoresc. 15, 153-160 (2005).
[CrossRef] [PubMed]

Hofkens, J.

S. Habuchi, M. Cotlet, J. Hofkens, G. Dirix, J. Michiels, J. Vanderleyden, V. Subramaniam, and F. C. D. Schryver, "Resonance Energy Transfer in a Calcium Concentration-Dependent Cameleon Protein," Biophys. J. 83, 3499-3506 (2002).
[CrossRef] [PubMed]

Holak, T. A.

M. H. Seifert, D. Ksiazek, M. K. Azim, P. Smialowski, N. Budisa, and T. A. Holak, "Slow exchange in the chromophore of a green fluorescent protein variant," J. Am. Chem. Soc. 124, 7932-7942 (2002).
[CrossRef] [PubMed]

Holzer, W.

W. Holzer, A. Penzkofer, M. Fuhrmann, and P. Hegemann, "Spectroscopic characterization of flavin mononucleotide bound to the LOV1 domain of Phot1 from Chlamydomonas reinhardtii," Photochem. Photobiol. 75, 479-487 (2002).
[CrossRef] [PubMed]

Kallio, K.

M. Ormo, A. B. Cubitt, K. Kallio, L. A. Gross, R. Y. Tsien, and S. J. Remington, "Crystal structure of the Aequorea victoria green fluorescent protein," Science 273, 1392-1395 (1996).
[CrossRef] [PubMed]

Kapusta, P.

P. Kapusta, R. Erdmann, U. Ortmann, and M. Wahl, "Time-resolved fluorescence anisotropy measurements made simple," J. Fluoresc. 13, 179-183 (2003).
[CrossRef]

Kemnitz, K.

M. Tramier, K. Kemnitz, C. Durieux, and M. Coppey-Moisan, "Picosecond time-resolved microspectrofluorometry in live cells exemplified by complex fluorescence dynamics of popular probes ethidium and cyan fluorescent protein," J. Microsc. 213, 110-118 (2004).
[CrossRef] [PubMed]

M. Tramier, I. Gautier, T. Piolot, S. Ravalet, K. Kemnitz, J. Coppey, C. Durieux, V. Mignotte, and M. Coppey-Moisan, "Picosecond-hetero-FRET microscopy to probe protein-protein interactions in live cells," Biophys. J. 83, 3570-3577 (2002).
[CrossRef] [PubMed]

I. Gautier, M. Tramier, C. Durieux, J. Coppey, R. B. Pansu, J. C. Nicolas, K. Kemnitz, and M. Coppey-Moisan, "Homo-FRET microscopy in living cells to measure monomer-dimer transition of GFP-tagged proteins," Biophys. J. 80, 3000-3008 (2001).
[CrossRef] [PubMed]

K. Kemnitz, L. Pfeifer, R. Paul, and M. Coppey-Moisan, "Novel detectors for fluorescence lifetime imaging on the picosecond time scale," J. Fluoresc. 7, 93-98. (1997).
[CrossRef]

Knutson, J. R.

L. Davenport, J. R. Knutson, and L. Brand, "Excited-state proton transfer of equilenin and dihydroequilenin: interaction with bilayer vesicles," Biochemistry 25, 1186-1195 (1986).
[CrossRef] [PubMed]

J. R. Knutson, D. G. Walbridge, and L. Brand, "Decay-associated fluorescence spectra and the heterogeneous emission of alcohol dehydrogenase," Biochemistry 21, 4671-4679 (1982).
[CrossRef] [PubMed]

Ksiazek, D.

M. H. Seifert, D. Ksiazek, M. K. Azim, P. Smialowski, N. Budisa, and T. A. Holak, "Slow exchange in the chromophore of a green fluorescent protein variant," J. Am. Chem. Soc. 124, 7932-7942 (2002).
[CrossRef] [PubMed]

Kuner, T.

T. Kuner and G. J. Augustine, "A genetically encoded ratiometric indicator for chloride: capturing chloride transients in cultured hippocampal neurons," Neuron 27, 447-459 (2000).
[CrossRef] [PubMed]

Kusba, J.

S. I. Murata, J. Kusba, G. Piszczek, I. Gryczynski, and J. R. Lakowicz, "Donor fluorescence decay analysis for energy transfer in double-helical DNA with various acceptor concentrations," Biopolymers 57, 306-315 (2000).
[CrossRef] [PubMed]

Lakowicz, J. R.

S. I. Murata, J. Kusba, G. Piszczek, I. Gryczynski, and J. R. Lakowicz, "Donor fluorescence decay analysis for energy transfer in double-helical DNA with various acceptor concentrations," Biopolymers 57, 306-315 (2000).
[CrossRef] [PubMed]

Lippincott-Schwartz, J.

J. Lippincott-Schwartz and G. H. Patterson, "Development and use of fluorescent protein markers in living cells," Science 300, 87-90 (2003).
[CrossRef] [PubMed]

Michiels, J.

S. Habuchi, M. Cotlet, J. Hofkens, G. Dirix, J. Michiels, J. Vanderleyden, V. Subramaniam, and F. C. D. Schryver, "Resonance Energy Transfer in a Calcium Concentration-Dependent Cameleon Protein," Biophys. J. 83, 3499-3506 (2002).
[CrossRef] [PubMed]

Mignotte, V.

M. Tramier, I. Gautier, T. Piolot, S. Ravalet, K. Kemnitz, J. Coppey, C. Durieux, V. Mignotte, and M. Coppey-Moisan, "Picosecond-hetero-FRET microscopy to probe protein-protein interactions in live cells," Biophys. J. 83, 3570-3577 (2002).
[CrossRef] [PubMed]

Mills, J. D.

Y. Chen, J. D. Mills, and A. Periasamy, "Protein localization in living cells and tissues using FRET and FLIM," Differentiation 71, 528-541 (2003).
[CrossRef] [PubMed]

Murata, S. I.

S. I. Murata, J. Kusba, G. Piszczek, I. Gryczynski, and J. R. Lakowicz, "Donor fluorescence decay analysis for energy transfer in double-helical DNA with various acceptor concentrations," Biopolymers 57, 306-315 (2000).
[CrossRef] [PubMed]

Nicolas, J. C.

I. Gautier, M. Tramier, C. Durieux, J. Coppey, R. B. Pansu, J. C. Nicolas, K. Kemnitz, and M. Coppey-Moisan, "Homo-FRET microscopy in living cells to measure monomer-dimer transition of GFP-tagged proteins," Biophys. J. 80, 3000-3008 (2001).
[CrossRef] [PubMed]

Ormo, M.

M. Ormo, A. B. Cubitt, K. Kallio, L. A. Gross, R. Y. Tsien, and S. J. Remington, "Crystal structure of the Aequorea victoria green fluorescent protein," Science 273, 1392-1395 (1996).
[CrossRef] [PubMed]

Ortmann, U.

P. Kapusta, R. Erdmann, U. Ortmann, and M. Wahl, "Time-resolved fluorescence anisotropy measurements made simple," J. Fluoresc. 13, 179-183 (2003).
[CrossRef]

Pansu, R. B.

I. Gautier, M. Tramier, C. Durieux, J. Coppey, R. B. Pansu, J. C. Nicolas, K. Kemnitz, and M. Coppey-Moisan, "Homo-FRET microscopy in living cells to measure monomer-dimer transition of GFP-tagged proteins," Biophys. J. 80, 3000-3008 (2001).
[CrossRef] [PubMed]

Patterson, G. H.

J. Lippincott-Schwartz and G. H. Patterson, "Development and use of fluorescent protein markers in living cells," Science 300, 87-90 (2003).
[CrossRef] [PubMed]

Paul, R.

K. Kemnitz, L. Pfeifer, R. Paul, and M. Coppey-Moisan, "Novel detectors for fluorescence lifetime imaging on the picosecond time scale," J. Fluoresc. 7, 93-98. (1997).
[CrossRef]

Penzkofer, A.

W. Holzer, A. Penzkofer, M. Fuhrmann, and P. Hegemann, "Spectroscopic characterization of flavin mononucleotide bound to the LOV1 domain of Phot1 from Chlamydomonas reinhardtii," Photochem. Photobiol. 75, 479-487 (2002).
[CrossRef] [PubMed]

Periasamy, A.

Y. Chen, J. D. Mills, and A. Periasamy, "Protein localization in living cells and tissues using FRET and FLIM," Differentiation 71, 528-541 (2003).
[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, 3-14 (2002).
[CrossRef] [PubMed]

Pfeifer, L.

K. Kemnitz, L. Pfeifer, R. Paul, and M. Coppey-Moisan, "Novel detectors for fluorescence lifetime imaging on the picosecond time scale," J. Fluoresc. 7, 93-98. (1997).
[CrossRef]

Piolot, T.

M. Tramier, I. Gautier, T. Piolot, S. Ravalet, K. Kemnitz, J. Coppey, C. Durieux, V. Mignotte, and M. Coppey-Moisan, "Picosecond-hetero-FRET microscopy to probe protein-protein interactions in live cells," Biophys. J. 83, 3570-3577 (2002).
[CrossRef] [PubMed]

Piszczek, G.

S. I. Murata, J. Kusba, G. Piszczek, I. Gryczynski, and J. R. Lakowicz, "Donor fluorescence decay analysis for energy transfer in double-helical DNA with various acceptor concentrations," Biopolymers 57, 306-315 (2000).
[CrossRef] [PubMed]

Ravalet, S.

M. Tramier, I. Gautier, T. Piolot, S. Ravalet, K. Kemnitz, J. Coppey, C. Durieux, V. Mignotte, and M. Coppey-Moisan, "Picosecond-hetero-FRET microscopy to probe protein-protein interactions in live cells," Biophys. J. 83, 3570-3577 (2002).
[CrossRef] [PubMed]

Remington, S. J.

M. Ormo, A. B. Cubitt, K. Kallio, L. A. Gross, R. Y. Tsien, and S. J. Remington, "Crystal structure of the Aequorea victoria green fluorescent protein," Science 273, 1392-1395 (1996).
[CrossRef] [PubMed]

Schryver, F. C. D.

S. Habuchi, M. Cotlet, J. Hofkens, G. Dirix, J. Michiels, J. Vanderleyden, V. Subramaniam, and F. C. D. Schryver, "Resonance Energy Transfer in a Calcium Concentration-Dependent Cameleon Protein," Biophys. J. 83, 3499-3506 (2002).
[CrossRef] [PubMed]

Seifert, M. H.

M. H. Seifert, D. Ksiazek, M. K. Azim, P. Smialowski, N. Budisa, and T. A. Holak, "Slow exchange in the chromophore of a green fluorescent protein variant," J. Am. Chem. Soc. 124, 7932-7942 (2002).
[CrossRef] [PubMed]

Shipston, M. J.

R. R. Duncan, A. Bergmann, M. A. Cousin, D. K. Apps, and M. J. Shipston, "Multi-dimensional time-correlated single photon counting (TCSPC) fluorescence lifetime imaging microscopy (FLIM) to detect FRET in cells," J. Microsc. 215, 1-12 (2004).
[CrossRef] [PubMed]

Smialowski, P.

M. H. Seifert, D. Ksiazek, M. K. Azim, P. Smialowski, N. Budisa, and T. A. Holak, "Slow exchange in the chromophore of a green fluorescent protein variant," J. Am. Chem. Soc. 124, 7932-7942 (2002).
[CrossRef] [PubMed]

Subramaniam, V.

S. Habuchi, M. Cotlet, J. Hofkens, G. Dirix, J. Michiels, J. Vanderleyden, V. Subramaniam, and F. C. D. Schryver, "Resonance Energy Transfer in a Calcium Concentration-Dependent Cameleon Protein," Biophys. J. 83, 3499-3506 (2002).
[CrossRef] [PubMed]

Tramier, M.

M. Tramier, K. Kemnitz, C. Durieux, and M. Coppey-Moisan, "Picosecond time-resolved microspectrofluorometry in live cells exemplified by complex fluorescence dynamics of popular probes ethidium and cyan fluorescent protein," J. Microsc. 213, 110-118 (2004).
[CrossRef] [PubMed]

M. Tramier, I. Gautier, T. Piolot, S. Ravalet, K. Kemnitz, J. Coppey, C. Durieux, V. Mignotte, and M. Coppey-Moisan, "Picosecond-hetero-FRET microscopy to probe protein-protein interactions in live cells," Biophys. J. 83, 3570-3577 (2002).
[CrossRef] [PubMed]

I. Gautier, M. Tramier, C. Durieux, J. Coppey, R. B. Pansu, J. C. Nicolas, K. Kemnitz, and M. Coppey-Moisan, "Homo-FRET microscopy in living cells to measure monomer-dimer transition of GFP-tagged proteins," Biophys. J. 80, 3000-3008 (2001).
[CrossRef] [PubMed]

Tsien, R. Y.

M. Ormo, A. B. Cubitt, K. Kallio, L. A. Gross, R. Y. Tsien, and S. J. Remington, "Crystal structure of the Aequorea victoria green fluorescent protein," Science 273, 1392-1395 (1996).
[CrossRef] [PubMed]

Vanderleyden, J.

S. Habuchi, M. Cotlet, J. Hofkens, G. Dirix, J. Michiels, J. Vanderleyden, V. Subramaniam, and F. C. D. Schryver, "Resonance Energy Transfer in a Calcium Concentration-Dependent Cameleon Protein," Biophys. J. 83, 3499-3506 (2002).
[CrossRef] [PubMed]

Visser, A. J. W. G.

J. W. Borst, M. A. Hink, A. V. Hoek, and A. J. W. G. Visser, "Effects of Refractive index and viscocity on Flourescence and Anisotropy Decays of Enhanced Cyan and Yellow Fluorescent Proteins," J. Fluoresc. 15, 153-160 (2005).
[CrossRef] [PubMed]

Wahl, M.

P. Kapusta, R. Erdmann, U. Ortmann, and M. Wahl, "Time-resolved fluorescence anisotropy measurements made simple," J. Fluoresc. 13, 179-183 (2003).
[CrossRef]

Walbridge, D. G.

J. R. Knutson, D. G. Walbridge, and L. Brand, "Decay-associated fluorescence spectra and the heterogeneous emission of alcohol dehydrogenase," Biochemistry 21, 4671-4679 (1982).
[CrossRef] [PubMed]

Biochemistry (2)

J. R. Knutson, D. G. Walbridge, and L. Brand, "Decay-associated fluorescence spectra and the heterogeneous emission of alcohol dehydrogenase," Biochemistry 21, 4671-4679 (1982).
[CrossRef] [PubMed]

L. Davenport, J. R. Knutson, and L. Brand, "Excited-state proton transfer of equilenin and dihydroequilenin: interaction with bilayer vesicles," Biochemistry 25, 1186-1195 (1986).
[CrossRef] [PubMed]

Biophys. J. (3)

I. Gautier, M. Tramier, C. Durieux, J. Coppey, R. B. Pansu, J. C. Nicolas, K. Kemnitz, and M. Coppey-Moisan, "Homo-FRET microscopy in living cells to measure monomer-dimer transition of GFP-tagged proteins," Biophys. J. 80, 3000-3008 (2001).
[CrossRef] [PubMed]

M. Tramier, I. Gautier, T. Piolot, S. Ravalet, K. Kemnitz, J. Coppey, C. Durieux, V. Mignotte, and M. Coppey-Moisan, "Picosecond-hetero-FRET microscopy to probe protein-protein interactions in live cells," Biophys. J. 83, 3570-3577 (2002).
[CrossRef] [PubMed]

S. Habuchi, M. Cotlet, J. Hofkens, G. Dirix, J. Michiels, J. Vanderleyden, V. Subramaniam, and F. C. D. Schryver, "Resonance Energy Transfer in a Calcium Concentration-Dependent Cameleon Protein," Biophys. J. 83, 3499-3506 (2002).
[CrossRef] [PubMed]

Biopolymers (1)

S. I. Murata, J. Kusba, G. Piszczek, I. Gryczynski, and J. R. Lakowicz, "Donor fluorescence decay analysis for energy transfer in double-helical DNA with various acceptor concentrations," Biopolymers 57, 306-315 (2000).
[CrossRef] [PubMed]

Differentiation (1)

Y. Chen, J. D. Mills, and A. Periasamy, "Protein localization in living cells and tissues using FRET and FLIM," Differentiation 71, 528-541 (2003).
[CrossRef] [PubMed]

J. Am. Chem. Soc. (1)

M. H. Seifert, D. Ksiazek, M. K. Azim, P. Smialowski, N. Budisa, and T. A. Holak, "Slow exchange in the chromophore of a green fluorescent protein variant," J. Am. Chem. Soc. 124, 7932-7942 (2002).
[CrossRef] [PubMed]

J. Fluoresc. (3)

P. Kapusta, R. Erdmann, U. Ortmann, and M. Wahl, "Time-resolved fluorescence anisotropy measurements made simple," J. Fluoresc. 13, 179-183 (2003).
[CrossRef]

J. W. Borst, M. A. Hink, A. V. Hoek, and A. J. W. G. Visser, "Effects of Refractive index and viscocity on Flourescence and Anisotropy Decays of Enhanced Cyan and Yellow Fluorescent Proteins," J. Fluoresc. 15, 153-160 (2005).
[CrossRef] [PubMed]

K. Kemnitz, L. Pfeifer, R. Paul, and M. Coppey-Moisan, "Novel detectors for fluorescence lifetime imaging on the picosecond time scale," J. Fluoresc. 7, 93-98. (1997).
[CrossRef]

J. Microsc. (3)

M. Tramier, K. Kemnitz, C. Durieux, and M. Coppey-Moisan, "Picosecond time-resolved microspectrofluorometry in live cells exemplified by complex fluorescence dynamics of popular probes ethidium and cyan fluorescent protein," J. Microsc. 213, 110-118 (2004).
[CrossRef] [PubMed]

R. R. Duncan, A. Bergmann, M. A. Cousin, D. K. Apps, and M. J. Shipston, "Multi-dimensional time-correlated single photon counting (TCSPC) fluorescence lifetime imaging microscopy (FLIM) to detect FRET in cells," J. Microsc. 215, 1-12 (2004).
[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, 3-14 (2002).
[CrossRef] [PubMed]

Neuron (1)

T. Kuner and G. J. Augustine, "A genetically encoded ratiometric indicator for chloride: capturing chloride transients in cultured hippocampal neurons," Neuron 27, 447-459 (2000).
[CrossRef] [PubMed]

Photochem. Photobiol. (1)

W. Holzer, A. Penzkofer, M. Fuhrmann, and P. Hegemann, "Spectroscopic characterization of flavin mononucleotide bound to the LOV1 domain of Phot1 from Chlamydomonas reinhardtii," Photochem. Photobiol. 75, 479-487 (2002).
[CrossRef] [PubMed]

Science (2)

J. Lippincott-Schwartz and G. H. Patterson, "Development and use of fluorescent protein markers in living cells," Science 300, 87-90 (2003).
[CrossRef] [PubMed]

M. Ormo, A. B. Cubitt, K. Kallio, L. A. Gross, R. Y. Tsien, and S. J. Remington, "Crystal structure of the Aequorea victoria green fluorescent protein," Science 273, 1392-1395 (1996).
[CrossRef] [PubMed]

Other (2)

J. R. Lakowicz, Principles of Fluorescence Spectroscopy, Second ed. (Kluwer Academic/Plenum Publishers, 1999).

K. Kemnitz, L. Pfeifer, R. Paul, A. Fink, and A. Bergmann, "Time- and space correlated single photon counting Spectroscopy," in Optical and Imaging Techniques for Biomonitoring, H.-J. Foth, R. Marchesini, H. Podbielska M.D., M. Robert-Nicoud, H. Schneckenburger, eds., SPIE Proc. 2628, 2-11 (1995).

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Figures (7)

Fig. 1.
Fig. 1.

Picosecond FLIM setup for simultaneous detection of donor and acceptor lifetimes using both point and imaging detectors. OCFD: Optical Constant Fraction Discriminator triggered by laser pulse, M: mirrors, ND: neutral density filters, UV: UV lamp for steady state imaging. L: planar convex lens, I: iris to control the area of excitation of the sample and CCD: Charge Coupled Device.

Fig. 2.
Fig. 2.

(a). Comparison of representative fluorescence emission spectra of ECFP (black) and the size variants of tandem constructs C8T (red), C16T (green) and C24T (blue). (b) Comparison of fluorescence intensity decays of ECFP and the size variants of tandem constructs at the emission maximum of CFPin a band of 483.8 ± 3.06 nm. Control ECFP (black), C8T (red), C16T (green) and C24T (blue). The intensity decays of FRET variants were faster compared to ECFP. The fastest decay was detected in the case of C8T indicating best FRET efficiency. The intensity decays had 104 counts at the maximum with χ2 <1.3.

Fig. 3.
Fig. 3.

(a). Decay Associated Spectrum of ECFP. Intensity decays of all measurements were analyzed in 20 emission bands from 470 nm to 590 nm and the pre-exponential factors of lifetimes 3.37±0.03 ns (black) and 1.06±0.03 ns (red) were plotted along the wavelength. (b) The contributions of both the lifetimes 3.37±0.03 ns (black) and 1.06±0.03 ns (red) were calculated and plotted as normalized fractional contribution from the pre-exponential factors along the wavelength. (c) Mean lifetimes of intensity decays of the emission bands were plotted along the wavelength (d) CCD image of Jurkat T-cell expressing ECFP (bar: 10 µm).

Fig. 4.
Fig. 4.

(a). Comparison of CFP and Topaz decays in C8T. The intensity decay of donor CFP (red) in a band of 483.8 ± 3.06 nm and the intensity decay at Topaz (green) emission maximum in a band of 527.3 ± 3.06 nm. (b) DAS of C8T. The decays were fit with a three exponential model with lifetimes of 3.39 ± 0.03 ns (black), 1.31 ± 0.07 ns (red) and 0.16 ± 0.02 ns (green). Intensity decays of all measurements were analyzed in 20 emission bands from 470 nm to 590 nm. At the emission maximum of Topaz (between 520 and 540 nm) the pre-exponential factors of two lifetimes were negative indicating the presence of excited state reactions. (c) Mean lifetimes of intensity decays were calculated along the emission bands and were plotted along the wavelength. A sharp increase was observed for the mean lifetimes at the emission maximum of Topaz. The intensity decays had 104 counts at the maximum with global χ2 <1.3.

Fig. 5.
Fig. 5.

Comparison of DAS for different tandem constructs showed a significant increase in the contributions of τ1 and τ2 and a corresponding reduction in the contribution of τ3 with increase in spacer length. Intensity decays of all measurements were analyzed in 20 emission bands from 470 nm to 590 nm. (a) Plot of DAS for C16T with pre-exponential factors of the lifetimes 3.41 ± 0.04 ns (black), 1.32 ± 0.04 ns (red) and 0.16 ± 0.02 ns (green). (b) Plot of DAS for C24T with pre-exponential factors of the lifetimes 3.42 ± 0.04 ns (black), 1.35 ± 0.04 ns (red) and 0.19 ± 0.02 ns (green). The intensity decays had 104 counts at the maximum.

Fig. 6.
Fig. 6.

Simultaneous analysis of donor and acceptor intensity decays in Jurkat T-cells expressing C8T collected by the imaging detector. The wide field fluorescence emission signal was split into two wavelength bands of CFP and Topaz with Dual Image to detect simultaneously the time resolved images of donor and acceptor. (a) CCD image of the Jurkat cell (bar: 10 µm). (b) Simultaneous analysis of donor and acceptor probes namely, CFP (red) and Topaz (green): Analysis resulted in three lifetimes of 3.23 ns, 1.24 ns and 0.21 ns with the pre-exponential factors of the 0.21 ns showing negative amplitudes for the C8T. The local χ2 values for the linked and unlinked analysis of the donor decay were 1.17 and 1.16. The lifetimes obtained for unlinked donor were 3.17 ns, 1.17 ns and 0.17 ns.

Fig. 7.
Fig. 7.

Rigorous error analysis was performed for different lifetime components obtained for the intensity decay of the donor. The changes in χ2 over the changes in lifetimes were plotted to check the quality of the lifetimes obtained from global analysis. The minimum of the curve was detected to be comparable to the values obtained from the multiexponential analysis. The donor decay of C8T from Fig 5 is presented as an example. (a) χ2 variation of the 3.17 ns component for the unlinked donor decay. (b) χ2 variation of the 1.17 ns lifetime in the unlinked donor decay. (c) χ2 variation of the short lifetime component (0.17 ns) for the unlinked donor decay.

Tables (2)

Tables Icon

Table 1: Multiexponential lifetimes and the percentage of contributions of each lifetime for ECFP and CFP of different size variants of tandem constructs

Tables Icon

Table 2 Efficiency of energy transfer occurring from CFP of different size variants of the tandem constructsa

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