Abstract

We present a novel excitation-emission multiplexed fluorescence lifetime microscopy (FLIM) method that surpasses current FLIM techniques in multiplexing capability. The method employs Fourier multiplexing to simultaneously acquire confocal fluorescence lifetime images of multiple excitation wavelength and emission color combinations at 44,000 pixels/sec. The system is built with low-cost CW laser sources and standard PMTs with versatile spectral configuration, which can be implemented as an add-on to commercial confocal microscopes. The Fourier lifetime confocal method allows fast multiplexed FLIM imaging, which makes it possible to monitor multiple biological processes in live cells. The low cost and compatibility with commercial systems could also make multiplexed FLIM more accessible to biological research community.

© 2014 Optical Society of America

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    [CrossRef] [PubMed]

2013

H. T. Chen, E. Gratton, “A practical implementation of multifrequency widefield frequency-domain fluorescence lifetime imaging microscopy,” Microsc. Res. Tech. 76(3), 282–289 (2013).
[CrossRef] [PubMed]

S.-E. Kim, H. Huang, M. Zhao, X. Zhang, A. Zhang, M. V. Semonov, B. T. MacDonald, X. Zhang, J. G. Abreu, L. Peng, X. He, “Wnt stabilization of β-catenin reveals principles for morphogen receptor-scaffold assemblies,” Science 340(6134), 867–870 (2013).
[CrossRef] [PubMed]

F. Fereidouni, K. Reitsma, H. C. Gerritsen, “High speed multispectral fluorescence lifetime imaging,” Opt. Express 21(10), 11769–11782 (2013).
[CrossRef] [PubMed]

2012

M. Zhao, R. Huang, L. L. Peng, “Quantitative multi-color FRET measurements by Fourier lifetime excitation-emission matrix spectroscopy,” Opt. Express 20(24), 26806–26827 (2012).
[CrossRef] [PubMed]

R. A. Colyer, O. H. W. Siegmund, A. S. Tremsin, J. V. Vallerga, S. Weiss, X. Michalet, “Phasor imaging with a widefield photon-counting detector,” J. Biomed. Opt. 17(1), 016008 (2012).
[CrossRef] [PubMed]

2011

D. D. U. Li, J. Arlt, D. Tyndall, R. Walker, J. Richardson, D. Stoppa, E. Charbon, R. K. Henderson, “Video-rate fluorescence lifetime imaging camera with CMOS single-photon avalanche diode arrays and high-speed imaging algorithm,” J. Biomed. Opt. 16(9), 096012 (2011).
[CrossRef] [PubMed]

K. Greger, M. J. Neetz, E. G. Reynaud, E. H. K. Stelzer, “Three-dimensional fluorescence lifetime imaging with a single plane illumination microscope provides an improved signal to noise ratio,” Opt. Express 19(21), 20743–20750 (2011).
[CrossRef] [PubMed]

2010

2008

O. M. Subach, I. S. Gundorov, M. Yoshimura, F. V. Subach, J. Zhang, D. Grüenwald, E. A. Souslova, D. M. Chudakov, V. V. Verkhusha, “Conversion of red fluorescent protein into a bright blue probe,” Chem. Biol. 15(10), 1116–1124 (2008).
[CrossRef] [PubMed]

A. Elder, S. Schlachter, C. F. Kaminski, “Theoretical investigation of the photon efficiency in frequency-domain fluorescence lifetime imaging microscopy,” J. Opt. Soc. Am. A 25(2), 452–462 (2008).
[CrossRef] [PubMed]

R. A. Colyer, C. Lee, E. Gratton, “A novel fluorescence lifetime imaging system that optimizes photon efficiency,” Microsc. Res. Tech. 71(3), 201–213 (2008).
[CrossRef] [PubMed]

M. A. Digman, V. R. Caiolfa, M. Zamai, E. Gratton, “The phasor approach to fluorescence lifetime imaging analysis,” Biophys. J. 94(2), L14–L16 (2008).
[CrossRef] [PubMed]

2007

2006

A. Esposito, H. C. Gerritsen, T. Oggier, F. Lustenberger, F. S. Wouters, “Innovating lifetime microscopy: a compact and simple tool for life sciences, screening, and diagnostics,” J. Biomed. Opt. 11(3), 034016 (2006).
[CrossRef] [PubMed]

2005

T. A. Laurence, X. X. Kong, M. Jäger, S. Weiss, “Probing structural heterogeneities and fluctuations of nucleic acids and denatured proteins,” Proc. Natl. Acad. Sci. U.S.A. 102(48), 17348–17353 (2005).
[CrossRef] [PubMed]

B. Treanor, P. M. P. Lanigan, K. Suhling, T. Schreiber, I. Munro, M. A. A. Neil, D. Phillips, D. M. Davis, P. M. W. French, “Imaging fluorescence lifetime heterogeneity applied to GFP-tagged MHC protein at an immunological synapse,” J. Microsc. 217(1), 36–43 (2005).
[CrossRef] [PubMed]

2004

M. J. Booth, T. Wilson, “Low-cost, frequency-domain, fluorescence lifetime confocal microscopy,” J. Microsc. 214(1), 36–42 (2004).
[CrossRef] [PubMed]

W. Becker, A. Bergmann, M. A. Hink, K. König, K. Benndorf, C. Biskup, “Fluorescence lifetime imaging by time-correlated single-photon counting,” Microsc. Res. Tech. 63(1), 58–66 (2004).
[CrossRef] [PubMed]

D. S. Elson, I. Munro, J. Requejo-Isidro, J. McGinty, C. Dunsby, N. Galletly, G. W. Stamp, M. A. A. Neil, M. J. Lever, P. A. Kellett, A. Dymoke-Bradshaw, J. Hares, P. M. W. French, “Real-time time-domain fluorescence lifetime imaging including single-shot acquisition with a segmented optical image intensifier,” New J. Phys. 6, 180 (2004).
[CrossRef]

2003

2002

A. Furtado, R. Henry, “Measurement of green fluorescent protein concentration in single cells by image analysis,” Anal. Biochem. 310(1), 84–92 (2002).
[CrossRef] [PubMed]

2001

Q. S. Hanley, V. Subramaniam, D. J. Arndt-Jovin, T. M. Jovin, “Fluorescence lifetime imaging: multi-point calibration, minimum resolvable differences, and artifact suppression,” Cytometry 43(4), 248–260 (2001).
[CrossRef] [PubMed]

P. Herman, B. P. Maliwal, H. J. Lin, J. R. Lakowicz, “Frequency-domain fluorescence microscopy with the LED as a light source,” J. Microsc. 203(2), 176–181 (2001).
[CrossRef] [PubMed]

2000

1998

K. Carlsson, A. Liljeborg, “Simultaneous confocal lifetime imaging of multiple fluorophores using the intensity-modulated multiple-wavelength scanning (IMS) technique,” J. Microsc. 191(2), 119–127 (1998).
[CrossRef] [PubMed]

1997

K. Carlsson, A. Liljeborg, “Confocal fluorescence microscopy using spectral and lifetime information to simultaneously record four fluorophores with high channel separation,” J. Microsc. 185(1), 37–46 (1997).
[CrossRef]

1995

K. D. Niswender, S. M. Blackman, L. Rohde, M. A. Magnuson, D. W. Piston, “Quantitative imaging of Green Fluorescent Protein in cultured cells: comparison of microscopic techniques, use in fusion proteins and detection limits,” J. Microsc. 180(2), 109–116 (1995).

P. T. C. So, T. French, W. M. Yu, K. M. Berland, C. Y. Dong, E. Gratton, “Time-resolved fluorescence microscopy using two-photon excitation,” Bioimaging 3(2), 49–63 (1995).
[CrossRef]

1993

T. W. J. Gadella, T. M. Jovin, R. M. Clegg, “Fluorescengce lifetime imaging microscopy (FLIM) - spatial resolution of microstructures on the nanosecond time-scale,” Biophys. Chem. 48(2), 221–239 (1993).
[CrossRef]

1983

H. Holthöfer, “Lectin binding sites in kidney. a comparative study of 14 animal species,” J. Histochem. Cytochem. 31(4), 531–537 (1983).
[CrossRef] [PubMed]

Abreu, J. G.

S.-E. Kim, H. Huang, M. Zhao, X. Zhang, A. Zhang, M. V. Semonov, B. T. MacDonald, X. Zhang, J. G. Abreu, L. Peng, X. He, “Wnt stabilization of β-catenin reveals principles for morphogen receptor-scaffold assemblies,” Science 340(6134), 867–870 (2013).
[CrossRef] [PubMed]

Arlt, J.

D. D. U. Li, J. Arlt, D. Tyndall, R. Walker, J. Richardson, D. Stoppa, E. Charbon, R. K. Henderson, “Video-rate fluorescence lifetime imaging camera with CMOS single-photon avalanche diode arrays and high-speed imaging algorithm,” J. Biomed. Opt. 16(9), 096012 (2011).
[CrossRef] [PubMed]

D. U. Li, J. Arlt, J. Richardson, R. Walker, A. Buts, D. Stoppa, E. Charbon, R. Henderson, “Real-time fluorescence lifetime imaging system with a 32 × 32 013μm CMOS low dark-count single-photon avalanche diode array,” Opt. Express 18(10), 10257–10269 (2010).
[CrossRef] [PubMed]

Arndt-Jovin, D. J.

Q. S. Hanley, V. Subramaniam, D. J. Arndt-Jovin, T. M. Jovin, “Fluorescence lifetime imaging: multi-point calibration, minimum resolvable differences, and artifact suppression,” Cytometry 43(4), 248–260 (2001).
[CrossRef] [PubMed]

Auksorius, E.

Becker, W.

W. Becker, A. Bergmann, M. A. Hink, K. König, K. Benndorf, C. Biskup, “Fluorescence lifetime imaging by time-correlated single-photon counting,” Microsc. Res. Tech. 63(1), 58–66 (2004).
[CrossRef] [PubMed]

Benndorf, K.

W. Becker, A. Bergmann, M. A. Hink, K. König, K. Benndorf, C. Biskup, “Fluorescence lifetime imaging by time-correlated single-photon counting,” Microsc. Res. Tech. 63(1), 58–66 (2004).
[CrossRef] [PubMed]

Bergmann, A.

W. Becker, A. Bergmann, M. A. Hink, K. König, K. Benndorf, C. Biskup, “Fluorescence lifetime imaging by time-correlated single-photon counting,” Microsc. Res. Tech. 63(1), 58–66 (2004).
[CrossRef] [PubMed]

Berland, K. M.

P. T. C. So, T. French, W. M. Yu, K. M. Berland, C. Y. Dong, E. Gratton, “Time-resolved fluorescence microscopy using two-photon excitation,” Bioimaging 3(2), 49–63 (1995).
[CrossRef]

Birch, D. J. S.

A. J. W. G. Visser, S. P. Laptenok, N. V. Visser, A. van Hoek, D. J. S. Birch, J.-C. Brochon, J. W. Borst, “Time-resolved FRET fluorescence spectroscopy of visible fluorescent protein pairs,” Eur. Biophys. J. 39(2), 241–253 (2010).
[CrossRef] [PubMed]

Biskup, C.

W. Becker, A. Bergmann, M. A. Hink, K. König, K. Benndorf, C. Biskup, “Fluorescence lifetime imaging by time-correlated single-photon counting,” Microsc. Res. Tech. 63(1), 58–66 (2004).
[CrossRef] [PubMed]

Blackman, S. M.

K. D. Niswender, S. M. Blackman, L. Rohde, M. A. Magnuson, D. W. Piston, “Quantitative imaging of Green Fluorescent Protein in cultured cells: comparison of microscopic techniques, use in fusion proteins and detection limits,” J. Microsc. 180(2), 109–116 (1995).

Booth, M. J.

M. J. Booth, T. Wilson, “Low-cost, frequency-domain, fluorescence lifetime confocal microscopy,” J. Microsc. 214(1), 36–42 (2004).
[CrossRef] [PubMed]

Boppart, S. A.

Borst, J. W.

A. J. W. G. Visser, S. P. Laptenok, N. V. Visser, A. van Hoek, D. J. S. Birch, J.-C. Brochon, J. W. Borst, “Time-resolved FRET fluorescence spectroscopy of visible fluorescent protein pairs,” Eur. Biophys. J. 39(2), 241–253 (2010).
[CrossRef] [PubMed]

Brochon, J.-C.

A. J. W. G. Visser, S. P. Laptenok, N. V. Visser, A. van Hoek, D. J. S. Birch, J.-C. Brochon, J. W. Borst, “Time-resolved FRET fluorescence spectroscopy of visible fluorescent protein pairs,” Eur. Biophys. J. 39(2), 241–253 (2010).
[CrossRef] [PubMed]

Bunney, T. D.

Buts, A.

Caiolfa, V. R.

M. A. Digman, V. R. Caiolfa, M. Zamai, E. Gratton, “The phasor approach to fluorescence lifetime imaging analysis,” Biophys. J. 94(2), L14–L16 (2008).
[CrossRef] [PubMed]

Carlsson, K.

K. Carlsson, A. Liljeborg, “Simultaneous confocal lifetime imaging of multiple fluorophores using the intensity-modulated multiple-wavelength scanning (IMS) technique,” J. Microsc. 191(2), 119–127 (1998).
[CrossRef] [PubMed]

K. Carlsson, A. Liljeborg, “Confocal fluorescence microscopy using spectral and lifetime information to simultaneously record four fluorophores with high channel separation,” J. Microsc. 185(1), 37–46 (1997).
[CrossRef]

Charbon, E.

D. D. U. Li, J. Arlt, D. Tyndall, R. Walker, J. Richardson, D. Stoppa, E. Charbon, R. K. Henderson, “Video-rate fluorescence lifetime imaging camera with CMOS single-photon avalanche diode arrays and high-speed imaging algorithm,” J. Biomed. Opt. 16(9), 096012 (2011).
[CrossRef] [PubMed]

D. U. Li, J. Arlt, J. Richardson, R. Walker, A. Buts, D. Stoppa, E. Charbon, R. Henderson, “Real-time fluorescence lifetime imaging system with a 32 × 32 013μm CMOS low dark-count single-photon avalanche diode array,” Opt. Express 18(10), 10257–10269 (2010).
[CrossRef] [PubMed]

Chen, H. T.

H. T. Chen, E. Gratton, “A practical implementation of multifrequency widefield frequency-domain fluorescence lifetime imaging microscopy,” Microsc. Res. Tech. 76(3), 282–289 (2013).
[CrossRef] [PubMed]

Chudakov, D. M.

O. M. Subach, I. S. Gundorov, M. Yoshimura, F. V. Subach, J. Zhang, D. Grüenwald, E. A. Souslova, D. M. Chudakov, V. V. Verkhusha, “Conversion of red fluorescent protein into a bright blue probe,” Chem. Biol. 15(10), 1116–1124 (2008).
[CrossRef] [PubMed]

Clegg, R. M.

T. W. J. Gadella, T. M. Jovin, R. M. Clegg, “Fluorescengce lifetime imaging microscopy (FLIM) - spatial resolution of microstructures on the nanosecond time-scale,” Biophys. Chem. 48(2), 221–239 (1993).
[CrossRef]

Cole, M. J.

Colyer, R. A.

R. A. Colyer, O. H. W. Siegmund, A. S. Tremsin, J. V. Vallerga, S. Weiss, X. Michalet, “Phasor imaging with a widefield photon-counting detector,” J. Biomed. Opt. 17(1), 016008 (2012).
[CrossRef] [PubMed]

R. A. Colyer, C. Lee, E. Gratton, “A novel fluorescence lifetime imaging system that optimizes photon efficiency,” Microsc. Res. Tech. 71(3), 201–213 (2008).
[CrossRef] [PubMed]

Courtney, P.

Davis, D. M.

B. Treanor, P. M. P. Lanigan, K. Suhling, T. Schreiber, I. Munro, M. A. A. Neil, D. Phillips, D. M. Davis, P. M. W. French, “Imaging fluorescence lifetime heterogeneity applied to GFP-tagged MHC protein at an immunological synapse,” J. Microsc. 217(1), 36–43 (2005).
[CrossRef] [PubMed]

de Beule, P. A. A.

Digman, M. A.

M. A. Digman, V. R. Caiolfa, M. Zamai, E. Gratton, “The phasor approach to fluorescence lifetime imaging analysis,” Biophys. J. 94(2), L14–L16 (2008).
[CrossRef] [PubMed]

Dong, C. Y.

P. T. C. So, T. French, W. M. Yu, K. M. Berland, C. Y. Dong, E. Gratton, “Time-resolved fluorescence microscopy using two-photon excitation,” Bioimaging 3(2), 49–63 (1995).
[CrossRef]

Dowling, K.

Dunsby, C.

Dymoke-Bradshaw, A.

D. S. Elson, I. Munro, J. Requejo-Isidro, J. McGinty, C. Dunsby, N. Galletly, G. W. Stamp, M. A. A. Neil, M. J. Lever, P. A. Kellett, A. Dymoke-Bradshaw, J. Hares, P. M. W. French, “Real-time time-domain fluorescence lifetime imaging including single-shot acquisition with a segmented optical image intensifier,” New J. Phys. 6, 180 (2004).
[CrossRef]

Elder, A.

Elson, D. S.

D. S. Elson, I. Munro, J. Requejo-Isidro, J. McGinty, C. Dunsby, N. Galletly, G. W. Stamp, M. A. A. Neil, M. J. Lever, P. A. Kellett, A. Dymoke-Bradshaw, J. Hares, P. M. W. French, “Real-time time-domain fluorescence lifetime imaging including single-shot acquisition with a segmented optical image intensifier,” New J. Phys. 6, 180 (2004).
[CrossRef]

Esposito, A.

A. Esposito, H. C. Gerritsen, T. Oggier, F. Lustenberger, F. S. Wouters, “Innovating lifetime microscopy: a compact and simple tool for life sciences, screening, and diagnostics,” J. Biomed. Opt. 11(3), 034016 (2006).
[CrossRef] [PubMed]

Fereidouni, F.

French, P. M. W.

D. M. Grant, J. McGinty, E. J. McGhee, T. D. Bunney, D. M. Owen, C. B. Talbot, W. Zhang, S. Kumar, I. Munro, P. M. P. Lanigan, G. T. Kennedy, C. Dunsby, A. I. Magee, P. Courtney, M. Katan, M. A. A. Neil, P. M. W. French, “High speed optically sectioned fluorescence lifetime imaging permits study of live cell signaling events,” Opt. Express 15(24), 15656–15673 (2007).
[CrossRef] [PubMed]

D. M. Owen, E. Auksorius, H. B. Manning, C. B. Talbot, P. A. A. de Beule, C. Dunsby, M. A. A. Neil, P. M. W. French, “Excitation-resolved hyperspectral fluorescence lifetime imaging using a UV-extended supercontinuum source,” Opt. Lett. 32(23), 3408–3410 (2007).
[CrossRef] [PubMed]

B. Treanor, P. M. P. Lanigan, K. Suhling, T. Schreiber, I. Munro, M. A. A. Neil, D. Phillips, D. M. Davis, P. M. W. French, “Imaging fluorescence lifetime heterogeneity applied to GFP-tagged MHC protein at an immunological synapse,” J. Microsc. 217(1), 36–43 (2005).
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D. S. Elson, I. Munro, J. Requejo-Isidro, J. McGinty, C. Dunsby, N. Galletly, G. W. Stamp, M. A. A. Neil, M. J. Lever, P. A. Kellett, A. Dymoke-Bradshaw, J. Hares, P. M. W. French, “Real-time time-domain fluorescence lifetime imaging including single-shot acquisition with a segmented optical image intensifier,” New J. Phys. 6, 180 (2004).
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P. T. C. So, T. French, W. M. Yu, K. M. Berland, C. Y. Dong, E. Gratton, “Time-resolved fluorescence microscopy using two-photon excitation,” Bioimaging 3(2), 49–63 (1995).
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A. Furtado, R. Henry, “Measurement of green fluorescent protein concentration in single cells by image analysis,” Anal. Biochem. 310(1), 84–92 (2002).
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T. W. J. Gadella, T. M. Jovin, R. M. Clegg, “Fluorescengce lifetime imaging microscopy (FLIM) - spatial resolution of microstructures on the nanosecond time-scale,” Biophys. Chem. 48(2), 221–239 (1993).
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D. S. Elson, I. Munro, J. Requejo-Isidro, J. McGinty, C. Dunsby, N. Galletly, G. W. Stamp, M. A. A. Neil, M. J. Lever, P. A. Kellett, A. Dymoke-Bradshaw, J. Hares, P. M. W. French, “Real-time time-domain fluorescence lifetime imaging including single-shot acquisition with a segmented optical image intensifier,” New J. Phys. 6, 180 (2004).
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Grüenwald, D.

O. M. Subach, I. S. Gundorov, M. Yoshimura, F. V. Subach, J. Zhang, D. Grüenwald, E. A. Souslova, D. M. Chudakov, V. V. Verkhusha, “Conversion of red fluorescent protein into a bright blue probe,” Chem. Biol. 15(10), 1116–1124 (2008).
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O. M. Subach, I. S. Gundorov, M. Yoshimura, F. V. Subach, J. Zhang, D. Grüenwald, E. A. Souslova, D. M. Chudakov, V. V. Verkhusha, “Conversion of red fluorescent protein into a bright blue probe,” Chem. Biol. 15(10), 1116–1124 (2008).
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Q. S. Hanley, V. Subramaniam, D. J. Arndt-Jovin, T. M. Jovin, “Fluorescence lifetime imaging: multi-point calibration, minimum resolvable differences, and artifact suppression,” Cytometry 43(4), 248–260 (2001).
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D. S. Elson, I. Munro, J. Requejo-Isidro, J. McGinty, C. Dunsby, N. Galletly, G. W. Stamp, M. A. A. Neil, M. J. Lever, P. A. Kellett, A. Dymoke-Bradshaw, J. Hares, P. M. W. French, “Real-time time-domain fluorescence lifetime imaging including single-shot acquisition with a segmented optical image intensifier,” New J. Phys. 6, 180 (2004).
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S.-E. Kim, H. Huang, M. Zhao, X. Zhang, A. Zhang, M. V. Semonov, B. T. MacDonald, X. Zhang, J. G. Abreu, L. Peng, X. He, “Wnt stabilization of β-catenin reveals principles for morphogen receptor-scaffold assemblies,” Science 340(6134), 867–870 (2013).
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Henderson, R. K.

D. D. U. Li, J. Arlt, D. Tyndall, R. Walker, J. Richardson, D. Stoppa, E. Charbon, R. K. Henderson, “Video-rate fluorescence lifetime imaging camera with CMOS single-photon avalanche diode arrays and high-speed imaging algorithm,” J. Biomed. Opt. 16(9), 096012 (2011).
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A. Furtado, R. Henry, “Measurement of green fluorescent protein concentration in single cells by image analysis,” Anal. Biochem. 310(1), 84–92 (2002).
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P. Herman, B. P. Maliwal, H. J. Lin, J. R. Lakowicz, “Frequency-domain fluorescence microscopy with the LED as a light source,” J. Microsc. 203(2), 176–181 (2001).
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W. Becker, A. Bergmann, M. A. Hink, K. König, K. Benndorf, C. Biskup, “Fluorescence lifetime imaging by time-correlated single-photon counting,” Microsc. Res. Tech. 63(1), 58–66 (2004).
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Jäger, M.

T. A. Laurence, X. X. Kong, M. Jäger, S. Weiss, “Probing structural heterogeneities and fluctuations of nucleic acids and denatured proteins,” Proc. Natl. Acad. Sci. U.S.A. 102(48), 17348–17353 (2005).
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Jones, R.

Jovin, T. M.

Q. S. Hanley, V. Subramaniam, D. J. Arndt-Jovin, T. M. Jovin, “Fluorescence lifetime imaging: multi-point calibration, minimum resolvable differences, and artifact suppression,” Cytometry 43(4), 248–260 (2001).
[CrossRef] [PubMed]

T. W. J. Gadella, T. M. Jovin, R. M. Clegg, “Fluorescengce lifetime imaging microscopy (FLIM) - spatial resolution of microstructures on the nanosecond time-scale,” Biophys. Chem. 48(2), 221–239 (1993).
[CrossRef]

Juskaitis, R.

Kaminski, C. F.

Katan, M.

Kellett, P. A.

D. S. Elson, I. Munro, J. Requejo-Isidro, J. McGinty, C. Dunsby, N. Galletly, G. W. Stamp, M. A. A. Neil, M. J. Lever, P. A. Kellett, A. Dymoke-Bradshaw, J. Hares, P. M. W. French, “Real-time time-domain fluorescence lifetime imaging including single-shot acquisition with a segmented optical image intensifier,” New J. Phys. 6, 180 (2004).
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Kim, S.-E.

S.-E. Kim, H. Huang, M. Zhao, X. Zhang, A. Zhang, M. V. Semonov, B. T. MacDonald, X. Zhang, J. G. Abreu, L. Peng, X. He, “Wnt stabilization of β-catenin reveals principles for morphogen receptor-scaffold assemblies,” Science 340(6134), 867–870 (2013).
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T. A. Laurence, X. X. Kong, M. Jäger, S. Weiss, “Probing structural heterogeneities and fluctuations of nucleic acids and denatured proteins,” Proc. Natl. Acad. Sci. U.S.A. 102(48), 17348–17353 (2005).
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W. Becker, A. Bergmann, M. A. Hink, K. König, K. Benndorf, C. Biskup, “Fluorescence lifetime imaging by time-correlated single-photon counting,” Microsc. Res. Tech. 63(1), 58–66 (2004).
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Kumar, S.

Lakowicz, J. R.

P. Herman, B. P. Maliwal, H. J. Lin, J. R. Lakowicz, “Frequency-domain fluorescence microscopy with the LED as a light source,” J. Microsc. 203(2), 176–181 (2001).
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Laptenok, S. P.

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T. A. Laurence, X. X. Kong, M. Jäger, S. Weiss, “Probing structural heterogeneities and fluctuations of nucleic acids and denatured proteins,” Proc. Natl. Acad. Sci. U.S.A. 102(48), 17348–17353 (2005).
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R. A. Colyer, C. Lee, E. Gratton, “A novel fluorescence lifetime imaging system that optimizes photon efficiency,” Microsc. Res. Tech. 71(3), 201–213 (2008).
[CrossRef] [PubMed]

Lever, M. J.

D. S. Elson, I. Munro, J. Requejo-Isidro, J. McGinty, C. Dunsby, N. Galletly, G. W. Stamp, M. A. A. Neil, M. J. Lever, P. A. Kellett, A. Dymoke-Bradshaw, J. Hares, P. M. W. French, “Real-time time-domain fluorescence lifetime imaging including single-shot acquisition with a segmented optical image intensifier,” New J. Phys. 6, 180 (2004).
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M. J. Cole, J. Siegel, S. E. D. Webb, R. Jones, K. Dowling, P. M. W. French, M. J. Lever, L. O. D. Sucharov, M. A. A. Neil, R. Juskaitis, T. Wilson, “Whole-field optically sectioned fluorescence lifetime imaging,” Opt. Lett. 25(18), 1361–1363 (2000).
[CrossRef] [PubMed]

Li, D. D. U.

D. D. U. Li, J. Arlt, D. Tyndall, R. Walker, J. Richardson, D. Stoppa, E. Charbon, R. K. Henderson, “Video-rate fluorescence lifetime imaging camera with CMOS single-photon avalanche diode arrays and high-speed imaging algorithm,” J. Biomed. Opt. 16(9), 096012 (2011).
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Li, D. U.

Liljeborg, A.

K. Carlsson, A. Liljeborg, “Simultaneous confocal lifetime imaging of multiple fluorophores using the intensity-modulated multiple-wavelength scanning (IMS) technique,” J. Microsc. 191(2), 119–127 (1998).
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P. Herman, B. P. Maliwal, H. J. Lin, J. R. Lakowicz, “Frequency-domain fluorescence microscopy with the LED as a light source,” J. Microsc. 203(2), 176–181 (2001).
[CrossRef] [PubMed]

Lustenberger, F.

A. Esposito, H. C. Gerritsen, T. Oggier, F. Lustenberger, F. S. Wouters, “Innovating lifetime microscopy: a compact and simple tool for life sciences, screening, and diagnostics,” J. Biomed. Opt. 11(3), 034016 (2006).
[CrossRef] [PubMed]

MacDonald, B. T.

S.-E. Kim, H. Huang, M. Zhao, X. Zhang, A. Zhang, M. V. Semonov, B. T. MacDonald, X. Zhang, J. G. Abreu, L. Peng, X. He, “Wnt stabilization of β-catenin reveals principles for morphogen receptor-scaffold assemblies,” Science 340(6134), 867–870 (2013).
[CrossRef] [PubMed]

Magee, A. I.

Magnuson, M. A.

K. D. Niswender, S. M. Blackman, L. Rohde, M. A. Magnuson, D. W. Piston, “Quantitative imaging of Green Fluorescent Protein in cultured cells: comparison of microscopic techniques, use in fusion proteins and detection limits,” J. Microsc. 180(2), 109–116 (1995).

Maliwal, B. P.

P. Herman, B. P. Maliwal, H. J. Lin, J. R. Lakowicz, “Frequency-domain fluorescence microscopy with the LED as a light source,” J. Microsc. 203(2), 176–181 (2001).
[CrossRef] [PubMed]

Manning, H. B.

Marks, D. L.

McGhee, E. J.

McGinty, J.

D. M. Grant, J. McGinty, E. J. McGhee, T. D. Bunney, D. M. Owen, C. B. Talbot, W. Zhang, S. Kumar, I. Munro, P. M. P. Lanigan, G. T. Kennedy, C. Dunsby, A. I. Magee, P. Courtney, M. Katan, M. A. A. Neil, P. M. W. French, “High speed optically sectioned fluorescence lifetime imaging permits study of live cell signaling events,” Opt. Express 15(24), 15656–15673 (2007).
[CrossRef] [PubMed]

D. S. Elson, I. Munro, J. Requejo-Isidro, J. McGinty, C. Dunsby, N. Galletly, G. W. Stamp, M. A. A. Neil, M. J. Lever, P. A. Kellett, A. Dymoke-Bradshaw, J. Hares, P. M. W. French, “Real-time time-domain fluorescence lifetime imaging including single-shot acquisition with a segmented optical image intensifier,” New J. Phys. 6, 180 (2004).
[CrossRef]

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R. A. Colyer, O. H. W. Siegmund, A. S. Tremsin, J. V. Vallerga, S. Weiss, X. Michalet, “Phasor imaging with a widefield photon-counting detector,” J. Biomed. Opt. 17(1), 016008 (2012).
[CrossRef] [PubMed]

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D. M. Grant, J. McGinty, E. J. McGhee, T. D. Bunney, D. M. Owen, C. B. Talbot, W. Zhang, S. Kumar, I. Munro, P. M. P. Lanigan, G. T. Kennedy, C. Dunsby, A. I. Magee, P. Courtney, M. Katan, M. A. A. Neil, P. M. W. French, “High speed optically sectioned fluorescence lifetime imaging permits study of live cell signaling events,” Opt. Express 15(24), 15656–15673 (2007).
[CrossRef] [PubMed]

B. Treanor, P. M. P. Lanigan, K. Suhling, T. Schreiber, I. Munro, M. A. A. Neil, D. Phillips, D. M. Davis, P. M. W. French, “Imaging fluorescence lifetime heterogeneity applied to GFP-tagged MHC protein at an immunological synapse,” J. Microsc. 217(1), 36–43 (2005).
[CrossRef] [PubMed]

D. S. Elson, I. Munro, J. Requejo-Isidro, J. McGinty, C. Dunsby, N. Galletly, G. W. Stamp, M. A. A. Neil, M. J. Lever, P. A. Kellett, A. Dymoke-Bradshaw, J. Hares, P. M. W. French, “Real-time time-domain fluorescence lifetime imaging including single-shot acquisition with a segmented optical image intensifier,” New J. Phys. 6, 180 (2004).
[CrossRef]

Neetz, M. J.

Neil, M. A. A.

D. M. Grant, J. McGinty, E. J. McGhee, T. D. Bunney, D. M. Owen, C. B. Talbot, W. Zhang, S. Kumar, I. Munro, P. M. P. Lanigan, G. T. Kennedy, C. Dunsby, A. I. Magee, P. Courtney, M. Katan, M. A. A. Neil, P. M. W. French, “High speed optically sectioned fluorescence lifetime imaging permits study of live cell signaling events,” Opt. Express 15(24), 15656–15673 (2007).
[CrossRef] [PubMed]

D. M. Owen, E. Auksorius, H. B. Manning, C. B. Talbot, P. A. A. de Beule, C. Dunsby, M. A. A. Neil, P. M. W. French, “Excitation-resolved hyperspectral fluorescence lifetime imaging using a UV-extended supercontinuum source,” Opt. Lett. 32(23), 3408–3410 (2007).
[CrossRef] [PubMed]

B. Treanor, P. M. P. Lanigan, K. Suhling, T. Schreiber, I. Munro, M. A. A. Neil, D. Phillips, D. M. Davis, P. M. W. French, “Imaging fluorescence lifetime heterogeneity applied to GFP-tagged MHC protein at an immunological synapse,” J. Microsc. 217(1), 36–43 (2005).
[CrossRef] [PubMed]

D. S. Elson, I. Munro, J. Requejo-Isidro, J. McGinty, C. Dunsby, N. Galletly, G. W. Stamp, M. A. A. Neil, M. J. Lever, P. A. Kellett, A. Dymoke-Bradshaw, J. Hares, P. M. W. French, “Real-time time-domain fluorescence lifetime imaging including single-shot acquisition with a segmented optical image intensifier,” New J. Phys. 6, 180 (2004).
[CrossRef]

M. J. Cole, J. Siegel, S. E. D. Webb, R. Jones, K. Dowling, P. M. W. French, M. J. Lever, L. O. D. Sucharov, M. A. A. Neil, R. Juskaitis, T. Wilson, “Whole-field optically sectioned fluorescence lifetime imaging,” Opt. Lett. 25(18), 1361–1363 (2000).
[CrossRef] [PubMed]

Niswender, K. D.

K. D. Niswender, S. M. Blackman, L. Rohde, M. A. Magnuson, D. W. Piston, “Quantitative imaging of Green Fluorescent Protein in cultured cells: comparison of microscopic techniques, use in fusion proteins and detection limits,” J. Microsc. 180(2), 109–116 (1995).

Oggier, T.

A. Esposito, H. C. Gerritsen, T. Oggier, F. Lustenberger, F. S. Wouters, “Innovating lifetime microscopy: a compact and simple tool for life sciences, screening, and diagnostics,” J. Biomed. Opt. 11(3), 034016 (2006).
[CrossRef] [PubMed]

Oldenburg, A. L.

Owen, D. M.

Peng, L.

S.-E. Kim, H. Huang, M. Zhao, X. Zhang, A. Zhang, M. V. Semonov, B. T. MacDonald, X. Zhang, J. G. Abreu, L. Peng, X. He, “Wnt stabilization of β-catenin reveals principles for morphogen receptor-scaffold assemblies,” Science 340(6134), 867–870 (2013).
[CrossRef] [PubMed]

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

Peng, L. L.

Phillips, D.

B. Treanor, P. M. P. Lanigan, K. Suhling, T. Schreiber, I. Munro, M. A. A. Neil, D. Phillips, D. M. Davis, P. M. W. French, “Imaging fluorescence lifetime heterogeneity applied to GFP-tagged MHC protein at an immunological synapse,” J. Microsc. 217(1), 36–43 (2005).
[CrossRef] [PubMed]

Piston, D. W.

K. D. Niswender, S. M. Blackman, L. Rohde, M. A. Magnuson, D. W. Piston, “Quantitative imaging of Green Fluorescent Protein in cultured cells: comparison of microscopic techniques, use in fusion proteins and detection limits,” J. Microsc. 180(2), 109–116 (1995).

Reitsma, K.

Requejo-Isidro, J.

D. S. Elson, I. Munro, J. Requejo-Isidro, J. McGinty, C. Dunsby, N. Galletly, G. W. Stamp, M. A. A. Neil, M. J. Lever, P. A. Kellett, A. Dymoke-Bradshaw, J. Hares, P. M. W. French, “Real-time time-domain fluorescence lifetime imaging including single-shot acquisition with a segmented optical image intensifier,” New J. Phys. 6, 180 (2004).
[CrossRef]

Reynaud, E. G.

Reynolds, J. J.

Richardson, J.

D. D. U. Li, J. Arlt, D. Tyndall, R. Walker, J. Richardson, D. Stoppa, E. Charbon, R. K. Henderson, “Video-rate fluorescence lifetime imaging camera with CMOS single-photon avalanche diode arrays and high-speed imaging algorithm,” J. Biomed. Opt. 16(9), 096012 (2011).
[CrossRef] [PubMed]

D. U. Li, J. Arlt, J. Richardson, R. Walker, A. Buts, D. Stoppa, E. Charbon, R. Henderson, “Real-time fluorescence lifetime imaging system with a 32 × 32 013μm CMOS low dark-count single-photon avalanche diode array,” Opt. Express 18(10), 10257–10269 (2010).
[CrossRef] [PubMed]

Rohde, L.

K. D. Niswender, S. M. Blackman, L. Rohde, M. A. Magnuson, D. W. Piston, “Quantitative imaging of Green Fluorescent Protein in cultured cells: comparison of microscopic techniques, use in fusion proteins and detection limits,” J. Microsc. 180(2), 109–116 (1995).

Schlachter, S.

Schreiber, T.

B. Treanor, P. M. P. Lanigan, K. Suhling, T. Schreiber, I. Munro, M. A. A. Neil, D. Phillips, D. M. Davis, P. M. W. French, “Imaging fluorescence lifetime heterogeneity applied to GFP-tagged MHC protein at an immunological synapse,” J. Microsc. 217(1), 36–43 (2005).
[CrossRef] [PubMed]

Semonov, M. V.

S.-E. Kim, H. Huang, M. Zhao, X. Zhang, A. Zhang, M. V. Semonov, B. T. MacDonald, X. Zhang, J. G. Abreu, L. Peng, X. He, “Wnt stabilization of β-catenin reveals principles for morphogen receptor-scaffold assemblies,” Science 340(6134), 867–870 (2013).
[CrossRef] [PubMed]

Siegel, J.

Siegmund, O. H. W.

R. A. Colyer, O. H. W. Siegmund, A. S. Tremsin, J. V. Vallerga, S. Weiss, X. Michalet, “Phasor imaging with a widefield photon-counting detector,” J. Biomed. Opt. 17(1), 016008 (2012).
[CrossRef] [PubMed]

So, P. T. C.

P. T. C. So, T. French, W. M. Yu, K. M. Berland, C. Y. Dong, E. Gratton, “Time-resolved fluorescence microscopy using two-photon excitation,” Bioimaging 3(2), 49–63 (1995).
[CrossRef]

Souslova, E. A.

O. M. Subach, I. S. Gundorov, M. Yoshimura, F. V. Subach, J. Zhang, D. Grüenwald, E. A. Souslova, D. M. Chudakov, V. V. Verkhusha, “Conversion of red fluorescent protein into a bright blue probe,” Chem. Biol. 15(10), 1116–1124 (2008).
[CrossRef] [PubMed]

Stamp, G. W.

D. S. Elson, I. Munro, J. Requejo-Isidro, J. McGinty, C. Dunsby, N. Galletly, G. W. Stamp, M. A. A. Neil, M. J. Lever, P. A. Kellett, A. Dymoke-Bradshaw, J. Hares, P. M. W. French, “Real-time time-domain fluorescence lifetime imaging including single-shot acquisition with a segmented optical image intensifier,” New J. Phys. 6, 180 (2004).
[CrossRef]

Stelzer, E. H. K.

Stoppa, D.

D. D. U. Li, J. Arlt, D. Tyndall, R. Walker, J. Richardson, D. Stoppa, E. Charbon, R. K. Henderson, “Video-rate fluorescence lifetime imaging camera with CMOS single-photon avalanche diode arrays and high-speed imaging algorithm,” J. Biomed. Opt. 16(9), 096012 (2011).
[CrossRef] [PubMed]

D. U. Li, J. Arlt, J. Richardson, R. Walker, A. Buts, D. Stoppa, E. Charbon, R. Henderson, “Real-time fluorescence lifetime imaging system with a 32 × 32 013μm CMOS low dark-count single-photon avalanche diode array,” Opt. Express 18(10), 10257–10269 (2010).
[CrossRef] [PubMed]

Subach, F. V.

O. M. Subach, I. S. Gundorov, M. Yoshimura, F. V. Subach, J. Zhang, D. Grüenwald, E. A. Souslova, D. M. Chudakov, V. V. Verkhusha, “Conversion of red fluorescent protein into a bright blue probe,” Chem. Biol. 15(10), 1116–1124 (2008).
[CrossRef] [PubMed]

Subach, O. M.

O. M. Subach, I. S. Gundorov, M. Yoshimura, F. V. Subach, J. Zhang, D. Grüenwald, E. A. Souslova, D. M. Chudakov, V. V. Verkhusha, “Conversion of red fluorescent protein into a bright blue probe,” Chem. Biol. 15(10), 1116–1124 (2008).
[CrossRef] [PubMed]

Subramaniam, V.

Q. S. Hanley, V. Subramaniam, D. J. Arndt-Jovin, T. M. Jovin, “Fluorescence lifetime imaging: multi-point calibration, minimum resolvable differences, and artifact suppression,” Cytometry 43(4), 248–260 (2001).
[CrossRef] [PubMed]

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Suhling, K.

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Chem. Biol.

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[CrossRef] [PubMed]

Cytometry

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[CrossRef] [PubMed]

J. Biomed. Opt.

D. D. U. Li, J. Arlt, D. Tyndall, R. Walker, J. Richardson, D. Stoppa, E. Charbon, R. K. Henderson, “Video-rate fluorescence lifetime imaging camera with CMOS single-photon avalanche diode arrays and high-speed imaging algorithm,” J. Biomed. Opt. 16(9), 096012 (2011).
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Supplementary Material (1)

» Media 1: MP4 (2189 KB)     

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Figures (6)

Fig. 1
Fig. 1

Schematics of Fourier confocal FLIM. (a) Schematic of the optical and signal acquisition system. (b) Fourier transform interferometer with double passed delay line. The interferometer has a 23 μs scan repetition time and no beam walk-off. (c) Wavelength-dependent interference modulation sweep twice between 0 to 150 MHz within 23 μs.

Fig. 2
Fig. 2

Analog-digital hybrid data processing of Fourier confocal FLIM. (a) Frequency-sweeping laser excitation and fluorescence emission signals from the sample. (b) Down-mixed emission signals. (c) Measured modulation and phase signal of GFP in comparison to those of fluorescein.

Fig. 3
Fig. 3

3D confocal lifetime image of triple-labeled mouse kidney slice. (a) False color intensity image of an x-y plane slice. (b) Lifetime x-y slice image of DAPI. (c) Lifetime x-y slice image of Alexa488 WGA. (d) Lifetime x-y slice image of Alexa568 phalloidin. (e) Sample x-z section. Top row: false color intensity image, showing co-localization of Alexa488 WGA and Alexa568 phalloidin at the basal area of proximal tubules. 2nd row: lifetime image of DAPI. 3rd row: lifetime image of Alexa488 WGA, showing decreased lifetime in the basal area of proximal tubules due to FRET between Alexa488 and Alexa568. Bottom row: lifetime image of Alexa568 phalloidin. The image size is 800-by-800 pixels with 0.15 μm pixel size, acquired at the full 44,000 pixel/s speed and processed without average. The full 3D data set is shown in Media 1. Scale bar 20 μm.

Fig. 4
Fig. 4

Monte Carlo simulation of F factor for heterodyne Fourier confocal FLIM compared to single frequency homodyne frequency domain lifetime method, at 100% and 80% excitation modulation depth. For 1~4 ns lifetimes Fourier confocal FLIM requires 5,000~1,0000 photons to achieve 10% lifetime accuracy. Homodyne lifetime F-factor curves are recalculated with same parameters as in Ref [10].

Fig. 5
Fig. 5

GFP lifetime accuracy in live cell imaging under different laser excitation power. (a) Histograms of GFP lifetime distribution narrow with increasing excitation power. (b) The standard deviation of GFP lifetime is inversely proportional to the square root of excitation power. (c) GFP lifetime image of HeLa cells at 200 μW excitation power. (d) GFP lifetime image of HeLa cells at 30 μW excitation power. GFP fluorescence lifetime was measured at 2.25 ± 0.16 ns at 200 μW excitation power and 2.3 ± 0.3 ns at 30 μW excitation power. Image size is 400-by-400 pixels with 0.3 μm pixel size. The image set was acquired at full 44,000 pixel/s speed. Lifetimes were fitted from data smoothed by a 2-by-2 pixel wide Gaussian window. The intensity image was processed at the original resolution without smoothing.

Fig. 6
Fig. 6

Lifetime images of HeLa cells expressing fluorescence proteins of blue and red colors. (a) TagBFP, 50 μW 405 nm excitation, 457 ± 20 nm emission. The excitation power and lifetime accuracy are lower because TagBFP is less photostable than GFP and mCherry; (b) mCherry, 200 μW 561 nm excitation, 593 ± 20 nm emission; (c) Lifetime histograms of three FPs; (d) Bar chart showing lifetime accuracy of three FPs. TagBFP: 2.79 ± 0.25 ns; GFP: 2.25 ± 0.16 ns; mCherry 1.80 ± 0.09 ns. All lifetimes match with literature values [3537]. All images were acquired at the full 44,000 pixel/s speed. Lifetimes were fitted from data smoothed by a 2-by-2 pixel wide Gaussian window. The intensity images were processed at the original resolution. Scale bar 20 μm.

Equations (7)

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

I ˜ i j e x e m = I i e x sin [ ( ω i + Δ ω i ) t ] × I ˜ j e m .
I ˜ j e m = k β k j I k e x m k j ( ω k ) sin [ ω k t + φ k j ( ω k ) ] .
I ˜ i j e x e m = ( I i e x ) 2 β i j m i j ( ω ) sin [ ( ω i + Δ ω i ) t ] sin [ ω i t + φ i j ( ω i ) ] + I i e x sin [ ( ω i + Δ ω i ) t ] × k i β k j I k e x m k j ( ω k ) sin [ ω k t + φ k j ( ω k ) ] .
I ˜ i j e x e m = ( I i e x ) 2 β i j m i j ( ω ) sin [ Δ ω i t + φ i j ( ω i ) ] .
C ˜ ij ( ω i )= H{ I ij exem ( ω i ) } m st ( ω i )exp[ i ϕ st ( ω i ) ] .
I ˜ ij Cor ( ω i ) β ij m ij ( ω i )exp[ i φ ij ( ω i ) ]= H[ I ij exem ( ω i ) ] C ˜ ij ( ω i ) .
F = Δ τ τ / Δ N N .

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