Abstract

Fluorescence lifetime imaging microscopy (FLIM) can be used to quantify molecular reactions in cells by detecting fluorescence resonance energy transfer (FRET). Confocal FLIM systems based on time correlated single photon counting (TCSPC) methods provide high spatial resolution and high sensitivity, but suffer from poor signal to noise ratios (SNR) that complicate quantitative analysis. We extend a global analysis method, originally developed for single frequency domain FLIM data, with a new filtering method optimized for FRET-FLIM data and apply it to TCSPC data. With this approach, the fluorescent lifetimes and relative concentrations of free and interacting molecules can be reliably estimated, even if the SNR is low. The required calibration values of the impulse response function are directly estimated from the data, eliminating the need for reference samples. The proposed method is efficient and robust, and can be routinely applied to analyze FRET-FLIM data acquired in intact cells.

© 2009 Optical Society of America

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    [CrossRef] [PubMed]
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2008 (3)

P. Barber, S. Ameer-Beg, J. Gilbey, L. Carlin, M. Keppler, T. Ng, and B. Vojnovic, "Multiphoton time-domain fluorescence lifetime imaging microscopy: practical application to protein-protein interactions using global analysis," J. R. Soc. Interface 6, S93-S105 (2008).
[CrossRef]

R. A. Colyer, C. Lee, and E. Gratton, "A novel fluorescence lifetime imaging system that optimizes photon efficiency," Microsc. Res. Tech. 71, 201-213 (2008).
[CrossRef]

S. Padilla-Parra, N. Auduge, M. Coppey-Moisan, and M. Tramier, "Quantitative FRET analysis by fast acquisition time domain FLIM at high spatial resolution in living cells," Biophys. J. 95, 2976-2988 (2008).
[CrossRef] [PubMed]

2007 (1)

G. Xouri, A. Squire, M. Dimaki, B. Geverts, P. J. Verveer, S. Taraviras, H. Nishitani, A. B. Houtsmuller, P. I. H. Bastiaens, and Z. Lygerou, "Cdt1 associates dynamically with chromatin throughout G1 and recruits Geminin onto chromatin," EMBO J. 26, 1303-14 (2007).
[CrossRef] [PubMed]

2005 (4)

O. Rocks, A. Peyker, M. Kahms, P. J. Verveer, C. Koerner, M. Lumbierres, J. Kuhlmann, H. Waldmann, A. Wittinghofer, and P. I. H. Bastiaens, "An acylation cycle regulates localization and activity of palmitoylated Ras isoforms," Science 307, 1746-1752 (2005).
[CrossRef] [PubMed]

A. Esposito, H. C. Gerritsen, and F. S. Wouters, "Fluorescence lifetime heterogeneity resolution in the frequency domain by lifetime moments analysis," Biophys. J. 89, 4286-4299 (2005).
[CrossRef] [PubMed]

G. I. Redford and R. M. Clegg, "Polar plot representation for frequency-domain analysis of fluorescence lifetimes," J. Fluoresc. 15, 805-815 (2005).
[CrossRef] [PubMed]

P. Barber, S. Ameer-Beg, J. Gilbey, R. J. Edens, I. Ezike, and B. Vojnovic, "Global and pixel kinetic data analysis for FRET detection by multi-photon time-domain FLIM," Proc. SPIE 5700, 171-181 (2005).
[CrossRef]

2004 (4)

S. Pelet, M. J. R. Previte, L. H. Laiho, and P. T. C. So, "A fast global fitting algorithm for fluorescence lifetime imaging microscopy based on image segmentation," Biophys. J. 87, 2807-2817 (2004).
[CrossRef] [PubMed]

W. Becker, A. Bergmann, M. A. Hink, K. Konig, K. Benndorf, and C. Biskup, "Fluorescence lifetime imaging by time-correlated single-photon counting," Microsc. Res. Tech. 63, 58-66 (2004).
[CrossRef]

M. Peter and S. M. Ameer-Beg, "Imaging molecular interactions by multiphoton FLIM," Biol. Cell 96, 231-236 (2004).
[CrossRef] [PubMed]

A. H. A. Clayton, Q. S. Hanley, and P. J. Verveer, "Graphical representation and multicomponent analysis of single-frequency fluorescence lifetime imaging microscopy data," J. Microsc. 213, 1-5 (2004).
[CrossRef]

2003 (3)

E. Gratton, S. Breusegem, J. Sutin, Q. Ruan, and N. Barry, "Fluorescence lifetime imaging for the two-photon microscope: time-domain and frequency-domain methods," J. Biomed. Opt. 8, 381-390 (2003).
[CrossRef] [PubMed]

P. J. Verveer and P. I. H. Bastiaens, "Evaluation of global analysis algorithms for single frequency fluorescence lifetime imaging microscopy data," J. Microsc. 209, 1-7 (2003).
[CrossRef] [PubMed]

A. R. Reynolds, C. Tischer, P. J. Verveer, O. Rocks, and P. I. H. Bastiaens, "EGFR activation coupled to inhibition of tyrosine phosphatases causes lateral signal propagation," Nat. Cell Biol. 5, 447-453 (2003).
[CrossRef] [PubMed]

2001 (2)

T. Ng, M. Parsons, W. E. Hughes, J. Monypenny, D. Zicha, A. Gautreau, M. Arpin, S. Gschmeissner, P. J. Verveer, P. I. H. Bastiaens, and P. J. Parker, "Ezrin is a downstream effector of trafficking PKC-integrin complexes involved in the control of cell motility," EMBO J. 20, 2723-2741 (2001).
[CrossRef] [PubMed]

F. S. Wouters, P. J. Verveer, and P. I. H. Bastiaens, "Imaging biochemistry inside cells," Trends Cell Biol. 11, 203-211 (2001).
[CrossRef] [PubMed]

2000 (4)

A. Schonle, M. Glatz, and S. W. Hell, "Four-dimensional multiphoton microscopy with time-correlated singlephoton counting," Appl. Opt. 39, 6306-6311 (2000).
[CrossRef]

K. Carlsson, A. Liljeborg, R. M. Andersson, and H. Brismar, "Confocal pH imaging of microscopic specimens using fluorescence lifetimes and phase fluorometry: influence of parameter choice on system performance," J. Microsc. 199, 106-114 (2000).
[CrossRef] [PubMed]

P. J. Verveer, A. Squire, and P. I. H. Bastiaens, "Global analysis of fluorescence lifetime imaging microscopy data," Biophys. J. 78, 2127-2137 (2000).
[CrossRef] [PubMed]

P. J. Verveer, F. S. Wouters, A. R. Reynolds, and P. I. H. Bastiaens, "Quantitative imaging of lateral ErbB1 receptor signal propagation in the plasma membrane," Science 290, 1567-1570 (2000).
[CrossRef] [PubMed]

1999 (2)

F. S. Wouters and P. I. H. Bastiaens, "Fluorescence lifetime imaging of receptor tyrosine kinase activity in cells," Curr. Biol. 9, 1127-1130 (1999).
[CrossRef] [PubMed]

P. I. H. Bastiaens and A. Squire, "Fluorescence lifetime imaging microscopy: spatial resolution of biochemical processes in the cell," Trends Cell Biol. 9, 48-52 (1999).
[CrossRef] [PubMed]

1998 (1)

G. W. Gordon, G. Berry, X. H. Liang, B. Levine, and B. Herman, "Quantitative fluorescence resonance energy transfer measurements using fluorescence microscopy," Biophys. J. 74, 2702-2713 (1998).
[CrossRef] [PubMed]

1996 (1)

R. M. Clegg, "Fluorescence resonance energy tranfer," Fluorescence Imaging Spectroscopy and Microscopy 137, 179-251 (1996).

1984 (1)

D. M. Jameson, E. Gratton, and R. Hall, "The measurement and analysis of heterogeneous emissions by multifrequency phase and modulation fluorometry." Appl. Spec. Rev. 20, 55-106 (1984).
[CrossRef]

Ameer-Beg, S.

P. Barber, S. Ameer-Beg, J. Gilbey, L. Carlin, M. Keppler, T. Ng, and B. Vojnovic, "Multiphoton time-domain fluorescence lifetime imaging microscopy: practical application to protein-protein interactions using global analysis," J. R. Soc. Interface 6, S93-S105 (2008).
[CrossRef]

P. Barber, S. Ameer-Beg, J. Gilbey, R. J. Edens, I. Ezike, and B. Vojnovic, "Global and pixel kinetic data analysis for FRET detection by multi-photon time-domain FLIM," Proc. SPIE 5700, 171-181 (2005).
[CrossRef]

Ameer-Beg, S. M.

M. Peter and S. M. Ameer-Beg, "Imaging molecular interactions by multiphoton FLIM," Biol. Cell 96, 231-236 (2004).
[CrossRef] [PubMed]

Andersson, R. M.

K. Carlsson, A. Liljeborg, R. M. Andersson, and H. Brismar, "Confocal pH imaging of microscopic specimens using fluorescence lifetimes and phase fluorometry: influence of parameter choice on system performance," J. Microsc. 199, 106-114 (2000).
[CrossRef] [PubMed]

Arpin, M.

T. Ng, M. Parsons, W. E. Hughes, J. Monypenny, D. Zicha, A. Gautreau, M. Arpin, S. Gschmeissner, P. J. Verveer, P. I. H. Bastiaens, and P. J. Parker, "Ezrin is a downstream effector of trafficking PKC-integrin complexes involved in the control of cell motility," EMBO J. 20, 2723-2741 (2001).
[CrossRef] [PubMed]

Barber, P.

P. Barber, S. Ameer-Beg, J. Gilbey, L. Carlin, M. Keppler, T. Ng, and B. Vojnovic, "Multiphoton time-domain fluorescence lifetime imaging microscopy: practical application to protein-protein interactions using global analysis," J. R. Soc. Interface 6, S93-S105 (2008).
[CrossRef]

P. Barber, S. Ameer-Beg, J. Gilbey, R. J. Edens, I. Ezike, and B. Vojnovic, "Global and pixel kinetic data analysis for FRET detection by multi-photon time-domain FLIM," Proc. SPIE 5700, 171-181 (2005).
[CrossRef]

Barry, N.

E. Gratton, S. Breusegem, J. Sutin, Q. Ruan, and N. Barry, "Fluorescence lifetime imaging for the two-photon microscope: time-domain and frequency-domain methods," J. Biomed. Opt. 8, 381-390 (2003).
[CrossRef] [PubMed]

Bastiaens, P. I. H.

G. Xouri, A. Squire, M. Dimaki, B. Geverts, P. J. Verveer, S. Taraviras, H. Nishitani, A. B. Houtsmuller, P. I. H. Bastiaens, and Z. Lygerou, "Cdt1 associates dynamically with chromatin throughout G1 and recruits Geminin onto chromatin," EMBO J. 26, 1303-14 (2007).
[CrossRef] [PubMed]

O. Rocks, A. Peyker, M. Kahms, P. J. Verveer, C. Koerner, M. Lumbierres, J. Kuhlmann, H. Waldmann, A. Wittinghofer, and P. I. H. Bastiaens, "An acylation cycle regulates localization and activity of palmitoylated Ras isoforms," Science 307, 1746-1752 (2005).
[CrossRef] [PubMed]

P. J. Verveer and P. I. H. Bastiaens, "Evaluation of global analysis algorithms for single frequency fluorescence lifetime imaging microscopy data," J. Microsc. 209, 1-7 (2003).
[CrossRef] [PubMed]

A. R. Reynolds, C. Tischer, P. J. Verveer, O. Rocks, and P. I. H. Bastiaens, "EGFR activation coupled to inhibition of tyrosine phosphatases causes lateral signal propagation," Nat. Cell Biol. 5, 447-453 (2003).
[CrossRef] [PubMed]

F. S. Wouters, P. J. Verveer, and P. I. H. Bastiaens, "Imaging biochemistry inside cells," Trends Cell Biol. 11, 203-211 (2001).
[CrossRef] [PubMed]

T. Ng, M. Parsons, W. E. Hughes, J. Monypenny, D. Zicha, A. Gautreau, M. Arpin, S. Gschmeissner, P. J. Verveer, P. I. H. Bastiaens, and P. J. Parker, "Ezrin is a downstream effector of trafficking PKC-integrin complexes involved in the control of cell motility," EMBO J. 20, 2723-2741 (2001).
[CrossRef] [PubMed]

P. J. Verveer, A. Squire, and P. I. H. Bastiaens, "Global analysis of fluorescence lifetime imaging microscopy data," Biophys. J. 78, 2127-2137 (2000).
[CrossRef] [PubMed]

P. J. Verveer, F. S. Wouters, A. R. Reynolds, and P. I. H. Bastiaens, "Quantitative imaging of lateral ErbB1 receptor signal propagation in the plasma membrane," Science 290, 1567-1570 (2000).
[CrossRef] [PubMed]

F. S. Wouters and P. I. H. Bastiaens, "Fluorescence lifetime imaging of receptor tyrosine kinase activity in cells," Curr. Biol. 9, 1127-1130 (1999).
[CrossRef] [PubMed]

P. I. H. Bastiaens and A. Squire, "Fluorescence lifetime imaging microscopy: spatial resolution of biochemical processes in the cell," Trends Cell Biol. 9, 48-52 (1999).
[CrossRef] [PubMed]

Becker, W.

W. Becker, A. Bergmann, M. A. Hink, K. Konig, K. Benndorf, and C. Biskup, "Fluorescence lifetime imaging by time-correlated single-photon counting," Microsc. Res. Tech. 63, 58-66 (2004).
[CrossRef]

Benndorf, K.

W. Becker, A. Bergmann, M. A. Hink, K. Konig, K. Benndorf, and C. Biskup, "Fluorescence lifetime imaging by time-correlated single-photon counting," Microsc. Res. Tech. 63, 58-66 (2004).
[CrossRef]

Bergmann, A.

W. Becker, A. Bergmann, M. A. Hink, K. Konig, K. Benndorf, and C. Biskup, "Fluorescence lifetime imaging by time-correlated single-photon counting," Microsc. Res. Tech. 63, 58-66 (2004).
[CrossRef]

Berry, G.

G. W. Gordon, G. Berry, X. H. Liang, B. Levine, and B. Herman, "Quantitative fluorescence resonance energy transfer measurements using fluorescence microscopy," Biophys. J. 74, 2702-2713 (1998).
[CrossRef] [PubMed]

Biskup, C.

W. Becker, A. Bergmann, M. A. Hink, K. Konig, K. Benndorf, and C. Biskup, "Fluorescence lifetime imaging by time-correlated single-photon counting," Microsc. Res. Tech. 63, 58-66 (2004).
[CrossRef]

Breusegem, S.

E. Gratton, S. Breusegem, J. Sutin, Q. Ruan, and N. Barry, "Fluorescence lifetime imaging for the two-photon microscope: time-domain and frequency-domain methods," J. Biomed. Opt. 8, 381-390 (2003).
[CrossRef] [PubMed]

Brismar, H.

K. Carlsson, A. Liljeborg, R. M. Andersson, and H. Brismar, "Confocal pH imaging of microscopic specimens using fluorescence lifetimes and phase fluorometry: influence of parameter choice on system performance," J. Microsc. 199, 106-114 (2000).
[CrossRef] [PubMed]

Carlin, L.

P. Barber, S. Ameer-Beg, J. Gilbey, L. Carlin, M. Keppler, T. Ng, and B. Vojnovic, "Multiphoton time-domain fluorescence lifetime imaging microscopy: practical application to protein-protein interactions using global analysis," J. R. Soc. Interface 6, S93-S105 (2008).
[CrossRef]

Carlsson, K.

K. Carlsson, A. Liljeborg, R. M. Andersson, and H. Brismar, "Confocal pH imaging of microscopic specimens using fluorescence lifetimes and phase fluorometry: influence of parameter choice on system performance," J. Microsc. 199, 106-114 (2000).
[CrossRef] [PubMed]

Clayton, A. H. A.

A. H. A. Clayton, Q. S. Hanley, and P. J. Verveer, "Graphical representation and multicomponent analysis of single-frequency fluorescence lifetime imaging microscopy data," J. Microsc. 213, 1-5 (2004).
[CrossRef]

Clegg, R. M.

G. I. Redford and R. M. Clegg, "Polar plot representation for frequency-domain analysis of fluorescence lifetimes," J. Fluoresc. 15, 805-815 (2005).
[CrossRef] [PubMed]

Clegg, R.M.

R. M. Clegg, "Fluorescence resonance energy tranfer," Fluorescence Imaging Spectroscopy and Microscopy 137, 179-251 (1996).

Colyer, R. A.

R. A. Colyer, C. Lee, and E. Gratton, "A novel fluorescence lifetime imaging system that optimizes photon efficiency," Microsc. Res. Tech. 71, 201-213 (2008).
[CrossRef]

Dimaki, M.

G. Xouri, A. Squire, M. Dimaki, B. Geverts, P. J. Verveer, S. Taraviras, H. Nishitani, A. B. Houtsmuller, P. I. H. Bastiaens, and Z. Lygerou, "Cdt1 associates dynamically with chromatin throughout G1 and recruits Geminin onto chromatin," EMBO J. 26, 1303-14 (2007).
[CrossRef] [PubMed]

Edens, R. J.

P. Barber, S. Ameer-Beg, J. Gilbey, R. J. Edens, I. Ezike, and B. Vojnovic, "Global and pixel kinetic data analysis for FRET detection by multi-photon time-domain FLIM," Proc. SPIE 5700, 171-181 (2005).
[CrossRef]

Esposito, A.

A. Esposito, H. C. Gerritsen, and F. S. Wouters, "Fluorescence lifetime heterogeneity resolution in the frequency domain by lifetime moments analysis," Biophys. J. 89, 4286-4299 (2005).
[CrossRef] [PubMed]

Ezike, I.

P. Barber, S. Ameer-Beg, J. Gilbey, R. J. Edens, I. Ezike, and B. Vojnovic, "Global and pixel kinetic data analysis for FRET detection by multi-photon time-domain FLIM," Proc. SPIE 5700, 171-181 (2005).
[CrossRef]

Gautreau, A.

T. Ng, M. Parsons, W. E. Hughes, J. Monypenny, D. Zicha, A. Gautreau, M. Arpin, S. Gschmeissner, P. J. Verveer, P. I. H. Bastiaens, and P. J. Parker, "Ezrin is a downstream effector of trafficking PKC-integrin complexes involved in the control of cell motility," EMBO J. 20, 2723-2741 (2001).
[CrossRef] [PubMed]

Gerritsen, H. C.

A. Esposito, H. C. Gerritsen, and F. S. Wouters, "Fluorescence lifetime heterogeneity resolution in the frequency domain by lifetime moments analysis," Biophys. J. 89, 4286-4299 (2005).
[CrossRef] [PubMed]

Geverts, B.

G. Xouri, A. Squire, M. Dimaki, B. Geverts, P. J. Verveer, S. Taraviras, H. Nishitani, A. B. Houtsmuller, P. I. H. Bastiaens, and Z. Lygerou, "Cdt1 associates dynamically with chromatin throughout G1 and recruits Geminin onto chromatin," EMBO J. 26, 1303-14 (2007).
[CrossRef] [PubMed]

Gilbey, J.

P. Barber, S. Ameer-Beg, J. Gilbey, L. Carlin, M. Keppler, T. Ng, and B. Vojnovic, "Multiphoton time-domain fluorescence lifetime imaging microscopy: practical application to protein-protein interactions using global analysis," J. R. Soc. Interface 6, S93-S105 (2008).
[CrossRef]

P. Barber, S. Ameer-Beg, J. Gilbey, R. J. Edens, I. Ezike, and B. Vojnovic, "Global and pixel kinetic data analysis for FRET detection by multi-photon time-domain FLIM," Proc. SPIE 5700, 171-181 (2005).
[CrossRef]

Glatz, M.

Gordon, G. W.

G. W. Gordon, G. Berry, X. H. Liang, B. Levine, and B. Herman, "Quantitative fluorescence resonance energy transfer measurements using fluorescence microscopy," Biophys. J. 74, 2702-2713 (1998).
[CrossRef] [PubMed]

Gratton, E.

R. A. Colyer, C. Lee, and E. Gratton, "A novel fluorescence lifetime imaging system that optimizes photon efficiency," Microsc. Res. Tech. 71, 201-213 (2008).
[CrossRef]

E. Gratton, S. Breusegem, J. Sutin, Q. Ruan, and N. Barry, "Fluorescence lifetime imaging for the two-photon microscope: time-domain and frequency-domain methods," J. Biomed. Opt. 8, 381-390 (2003).
[CrossRef] [PubMed]

D. M. Jameson, E. Gratton, and R. Hall, "The measurement and analysis of heterogeneous emissions by multifrequency phase and modulation fluorometry." Appl. Spec. Rev. 20, 55-106 (1984).
[CrossRef]

Gschmeissner, S.

T. Ng, M. Parsons, W. E. Hughes, J. Monypenny, D. Zicha, A. Gautreau, M. Arpin, S. Gschmeissner, P. J. Verveer, P. I. H. Bastiaens, and P. J. Parker, "Ezrin is a downstream effector of trafficking PKC-integrin complexes involved in the control of cell motility," EMBO J. 20, 2723-2741 (2001).
[CrossRef] [PubMed]

Hall, R.

D. M. Jameson, E. Gratton, and R. Hall, "The measurement and analysis of heterogeneous emissions by multifrequency phase and modulation fluorometry." Appl. Spec. Rev. 20, 55-106 (1984).
[CrossRef]

Hanley, Q. S.

A. H. A. Clayton, Q. S. Hanley, and P. J. Verveer, "Graphical representation and multicomponent analysis of single-frequency fluorescence lifetime imaging microscopy data," J. Microsc. 213, 1-5 (2004).
[CrossRef]

Hell, S. W.

Herman, B.

G. W. Gordon, G. Berry, X. H. Liang, B. Levine, and B. Herman, "Quantitative fluorescence resonance energy transfer measurements using fluorescence microscopy," Biophys. J. 74, 2702-2713 (1998).
[CrossRef] [PubMed]

Hink, M. A.

W. Becker, A. Bergmann, M. A. Hink, K. Konig, K. Benndorf, and C. Biskup, "Fluorescence lifetime imaging by time-correlated single-photon counting," Microsc. Res. Tech. 63, 58-66 (2004).
[CrossRef]

Houtsmuller, A. B.

G. Xouri, A. Squire, M. Dimaki, B. Geverts, P. J. Verveer, S. Taraviras, H. Nishitani, A. B. Houtsmuller, P. I. H. Bastiaens, and Z. Lygerou, "Cdt1 associates dynamically with chromatin throughout G1 and recruits Geminin onto chromatin," EMBO J. 26, 1303-14 (2007).
[CrossRef] [PubMed]

Hughes, W. E.

T. Ng, M. Parsons, W. E. Hughes, J. Monypenny, D. Zicha, A. Gautreau, M. Arpin, S. Gschmeissner, P. J. Verveer, P. I. H. Bastiaens, and P. J. Parker, "Ezrin is a downstream effector of trafficking PKC-integrin complexes involved in the control of cell motility," EMBO J. 20, 2723-2741 (2001).
[CrossRef] [PubMed]

Jameson, D. M.

D. M. Jameson, E. Gratton, and R. Hall, "The measurement and analysis of heterogeneous emissions by multifrequency phase and modulation fluorometry." Appl. Spec. Rev. 20, 55-106 (1984).
[CrossRef]

Kahms, M.

O. Rocks, A. Peyker, M. Kahms, P. J. Verveer, C. Koerner, M. Lumbierres, J. Kuhlmann, H. Waldmann, A. Wittinghofer, and P. I. H. Bastiaens, "An acylation cycle regulates localization and activity of palmitoylated Ras isoforms," Science 307, 1746-1752 (2005).
[CrossRef] [PubMed]

Keppler, M.

P. Barber, S. Ameer-Beg, J. Gilbey, L. Carlin, M. Keppler, T. Ng, and B. Vojnovic, "Multiphoton time-domain fluorescence lifetime imaging microscopy: practical application to protein-protein interactions using global analysis," J. R. Soc. Interface 6, S93-S105 (2008).
[CrossRef]

Koerner, C.

O. Rocks, A. Peyker, M. Kahms, P. J. Verveer, C. Koerner, M. Lumbierres, J. Kuhlmann, H. Waldmann, A. Wittinghofer, and P. I. H. Bastiaens, "An acylation cycle regulates localization and activity of palmitoylated Ras isoforms," Science 307, 1746-1752 (2005).
[CrossRef] [PubMed]

Konig, K.

W. Becker, A. Bergmann, M. A. Hink, K. Konig, K. Benndorf, and C. Biskup, "Fluorescence lifetime imaging by time-correlated single-photon counting," Microsc. Res. Tech. 63, 58-66 (2004).
[CrossRef]

Kuhlmann, J.

O. Rocks, A. Peyker, M. Kahms, P. J. Verveer, C. Koerner, M. Lumbierres, J. Kuhlmann, H. Waldmann, A. Wittinghofer, and P. I. H. Bastiaens, "An acylation cycle regulates localization and activity of palmitoylated Ras isoforms," Science 307, 1746-1752 (2005).
[CrossRef] [PubMed]

Laiho, L. H.

S. Pelet, M. J. R. Previte, L. H. Laiho, and P. T. C. So, "A fast global fitting algorithm for fluorescence lifetime imaging microscopy based on image segmentation," Biophys. J. 87, 2807-2817 (2004).
[CrossRef] [PubMed]

Lee, C.

R. A. Colyer, C. Lee, and E. Gratton, "A novel fluorescence lifetime imaging system that optimizes photon efficiency," Microsc. Res. Tech. 71, 201-213 (2008).
[CrossRef]

Levine, B.

G. W. Gordon, G. Berry, X. H. Liang, B. Levine, and B. Herman, "Quantitative fluorescence resonance energy transfer measurements using fluorescence microscopy," Biophys. J. 74, 2702-2713 (1998).
[CrossRef] [PubMed]

Liang, X. H.

G. W. Gordon, G. Berry, X. H. Liang, B. Levine, and B. Herman, "Quantitative fluorescence resonance energy transfer measurements using fluorescence microscopy," Biophys. J. 74, 2702-2713 (1998).
[CrossRef] [PubMed]

Liljeborg, A.

K. Carlsson, A. Liljeborg, R. M. Andersson, and H. Brismar, "Confocal pH imaging of microscopic specimens using fluorescence lifetimes and phase fluorometry: influence of parameter choice on system performance," J. Microsc. 199, 106-114 (2000).
[CrossRef] [PubMed]

Lumbierres, M.

O. Rocks, A. Peyker, M. Kahms, P. J. Verveer, C. Koerner, M. Lumbierres, J. Kuhlmann, H. Waldmann, A. Wittinghofer, and P. I. H. Bastiaens, "An acylation cycle regulates localization and activity of palmitoylated Ras isoforms," Science 307, 1746-1752 (2005).
[CrossRef] [PubMed]

Lygerou, Z.

G. Xouri, A. Squire, M. Dimaki, B. Geverts, P. J. Verveer, S. Taraviras, H. Nishitani, A. B. Houtsmuller, P. I. H. Bastiaens, and Z. Lygerou, "Cdt1 associates dynamically with chromatin throughout G1 and recruits Geminin onto chromatin," EMBO J. 26, 1303-14 (2007).
[CrossRef] [PubMed]

Monypenny, J.

T. Ng, M. Parsons, W. E. Hughes, J. Monypenny, D. Zicha, A. Gautreau, M. Arpin, S. Gschmeissner, P. J. Verveer, P. I. H. Bastiaens, and P. J. Parker, "Ezrin is a downstream effector of trafficking PKC-integrin complexes involved in the control of cell motility," EMBO J. 20, 2723-2741 (2001).
[CrossRef] [PubMed]

Ng, T.

P. Barber, S. Ameer-Beg, J. Gilbey, L. Carlin, M. Keppler, T. Ng, and B. Vojnovic, "Multiphoton time-domain fluorescence lifetime imaging microscopy: practical application to protein-protein interactions using global analysis," J. R. Soc. Interface 6, S93-S105 (2008).
[CrossRef]

T. Ng, M. Parsons, W. E. Hughes, J. Monypenny, D. Zicha, A. Gautreau, M. Arpin, S. Gschmeissner, P. J. Verveer, P. I. H. Bastiaens, and P. J. Parker, "Ezrin is a downstream effector of trafficking PKC-integrin complexes involved in the control of cell motility," EMBO J. 20, 2723-2741 (2001).
[CrossRef] [PubMed]

Nishitani, H.

G. Xouri, A. Squire, M. Dimaki, B. Geverts, P. J. Verveer, S. Taraviras, H. Nishitani, A. B. Houtsmuller, P. I. H. Bastiaens, and Z. Lygerou, "Cdt1 associates dynamically with chromatin throughout G1 and recruits Geminin onto chromatin," EMBO J. 26, 1303-14 (2007).
[CrossRef] [PubMed]

Padilla-Parra, S.

S. Padilla-Parra, N. Auduge, M. Coppey-Moisan, and M. Tramier, "Quantitative FRET analysis by fast acquisition time domain FLIM at high spatial resolution in living cells," Biophys. J. 95, 2976-2988 (2008).
[CrossRef] [PubMed]

Parker, P. J.

T. Ng, M. Parsons, W. E. Hughes, J. Monypenny, D. Zicha, A. Gautreau, M. Arpin, S. Gschmeissner, P. J. Verveer, P. I. H. Bastiaens, and P. J. Parker, "Ezrin is a downstream effector of trafficking PKC-integrin complexes involved in the control of cell motility," EMBO J. 20, 2723-2741 (2001).
[CrossRef] [PubMed]

Parsons, M.

T. Ng, M. Parsons, W. E. Hughes, J. Monypenny, D. Zicha, A. Gautreau, M. Arpin, S. Gschmeissner, P. J. Verveer, P. I. H. Bastiaens, and P. J. Parker, "Ezrin is a downstream effector of trafficking PKC-integrin complexes involved in the control of cell motility," EMBO J. 20, 2723-2741 (2001).
[CrossRef] [PubMed]

Pelet, S.

S. Pelet, M. J. R. Previte, L. H. Laiho, and P. T. C. So, "A fast global fitting algorithm for fluorescence lifetime imaging microscopy based on image segmentation," Biophys. J. 87, 2807-2817 (2004).
[CrossRef] [PubMed]

Peter, M.

M. Peter and S. M. Ameer-Beg, "Imaging molecular interactions by multiphoton FLIM," Biol. Cell 96, 231-236 (2004).
[CrossRef] [PubMed]

Peyker, A.

O. Rocks, A. Peyker, M. Kahms, P. J. Verveer, C. Koerner, M. Lumbierres, J. Kuhlmann, H. Waldmann, A. Wittinghofer, and P. I. H. Bastiaens, "An acylation cycle regulates localization and activity of palmitoylated Ras isoforms," Science 307, 1746-1752 (2005).
[CrossRef] [PubMed]

Previte, M. J. R.

S. Pelet, M. J. R. Previte, L. H. Laiho, and P. T. C. So, "A fast global fitting algorithm for fluorescence lifetime imaging microscopy based on image segmentation," Biophys. J. 87, 2807-2817 (2004).
[CrossRef] [PubMed]

Redford, G. I.

G. I. Redford and R. M. Clegg, "Polar plot representation for frequency-domain analysis of fluorescence lifetimes," J. Fluoresc. 15, 805-815 (2005).
[CrossRef] [PubMed]

Reynolds, A. R.

A. R. Reynolds, C. Tischer, P. J. Verveer, O. Rocks, and P. I. H. Bastiaens, "EGFR activation coupled to inhibition of tyrosine phosphatases causes lateral signal propagation," Nat. Cell Biol. 5, 447-453 (2003).
[CrossRef] [PubMed]

P. J. Verveer, F. S. Wouters, A. R. Reynolds, and P. I. H. Bastiaens, "Quantitative imaging of lateral ErbB1 receptor signal propagation in the plasma membrane," Science 290, 1567-1570 (2000).
[CrossRef] [PubMed]

Rocks, O.

O. Rocks, A. Peyker, M. Kahms, P. J. Verveer, C. Koerner, M. Lumbierres, J. Kuhlmann, H. Waldmann, A. Wittinghofer, and P. I. H. Bastiaens, "An acylation cycle regulates localization and activity of palmitoylated Ras isoforms," Science 307, 1746-1752 (2005).
[CrossRef] [PubMed]

A. R. Reynolds, C. Tischer, P. J. Verveer, O. Rocks, and P. I. H. Bastiaens, "EGFR activation coupled to inhibition of tyrosine phosphatases causes lateral signal propagation," Nat. Cell Biol. 5, 447-453 (2003).
[CrossRef] [PubMed]

Ruan, Q.

E. Gratton, S. Breusegem, J. Sutin, Q. Ruan, and N. Barry, "Fluorescence lifetime imaging for the two-photon microscope: time-domain and frequency-domain methods," J. Biomed. Opt. 8, 381-390 (2003).
[CrossRef] [PubMed]

Schonle, A.

So, P. T. C.

S. Pelet, M. J. R. Previte, L. H. Laiho, and P. T. C. So, "A fast global fitting algorithm for fluorescence lifetime imaging microscopy based on image segmentation," Biophys. J. 87, 2807-2817 (2004).
[CrossRef] [PubMed]

Squire, A.

G. Xouri, A. Squire, M. Dimaki, B. Geverts, P. J. Verveer, S. Taraviras, H. Nishitani, A. B. Houtsmuller, P. I. H. Bastiaens, and Z. Lygerou, "Cdt1 associates dynamically with chromatin throughout G1 and recruits Geminin onto chromatin," EMBO J. 26, 1303-14 (2007).
[CrossRef] [PubMed]

P. J. Verveer, A. Squire, and P. I. H. Bastiaens, "Global analysis of fluorescence lifetime imaging microscopy data," Biophys. J. 78, 2127-2137 (2000).
[CrossRef] [PubMed]

P. I. H. Bastiaens and A. Squire, "Fluorescence lifetime imaging microscopy: spatial resolution of biochemical processes in the cell," Trends Cell Biol. 9, 48-52 (1999).
[CrossRef] [PubMed]

Sutin, J.

E. Gratton, S. Breusegem, J. Sutin, Q. Ruan, and N. Barry, "Fluorescence lifetime imaging for the two-photon microscope: time-domain and frequency-domain methods," J. Biomed. Opt. 8, 381-390 (2003).
[CrossRef] [PubMed]

Taraviras, S.

G. Xouri, A. Squire, M. Dimaki, B. Geverts, P. J. Verveer, S. Taraviras, H. Nishitani, A. B. Houtsmuller, P. I. H. Bastiaens, and Z. Lygerou, "Cdt1 associates dynamically with chromatin throughout G1 and recruits Geminin onto chromatin," EMBO J. 26, 1303-14 (2007).
[CrossRef] [PubMed]

Tischer, C.

A. R. Reynolds, C. Tischer, P. J. Verveer, O. Rocks, and P. I. H. Bastiaens, "EGFR activation coupled to inhibition of tyrosine phosphatases causes lateral signal propagation," Nat. Cell Biol. 5, 447-453 (2003).
[CrossRef] [PubMed]

Verveer, P. J.

G. Xouri, A. Squire, M. Dimaki, B. Geverts, P. J. Verveer, S. Taraviras, H. Nishitani, A. B. Houtsmuller, P. I. H. Bastiaens, and Z. Lygerou, "Cdt1 associates dynamically with chromatin throughout G1 and recruits Geminin onto chromatin," EMBO J. 26, 1303-14 (2007).
[CrossRef] [PubMed]

O. Rocks, A. Peyker, M. Kahms, P. J. Verveer, C. Koerner, M. Lumbierres, J. Kuhlmann, H. Waldmann, A. Wittinghofer, and P. I. H. Bastiaens, "An acylation cycle regulates localization and activity of palmitoylated Ras isoforms," Science 307, 1746-1752 (2005).
[CrossRef] [PubMed]

A. H. A. Clayton, Q. S. Hanley, and P. J. Verveer, "Graphical representation and multicomponent analysis of single-frequency fluorescence lifetime imaging microscopy data," J. Microsc. 213, 1-5 (2004).
[CrossRef]

A. R. Reynolds, C. Tischer, P. J. Verveer, O. Rocks, and P. I. H. Bastiaens, "EGFR activation coupled to inhibition of tyrosine phosphatases causes lateral signal propagation," Nat. Cell Biol. 5, 447-453 (2003).
[CrossRef] [PubMed]

P. J. Verveer and P. I. H. Bastiaens, "Evaluation of global analysis algorithms for single frequency fluorescence lifetime imaging microscopy data," J. Microsc. 209, 1-7 (2003).
[CrossRef] [PubMed]

T. Ng, M. Parsons, W. E. Hughes, J. Monypenny, D. Zicha, A. Gautreau, M. Arpin, S. Gschmeissner, P. J. Verveer, P. I. H. Bastiaens, and P. J. Parker, "Ezrin is a downstream effector of trafficking PKC-integrin complexes involved in the control of cell motility," EMBO J. 20, 2723-2741 (2001).
[CrossRef] [PubMed]

F. S. Wouters, P. J. Verveer, and P. I. H. Bastiaens, "Imaging biochemistry inside cells," Trends Cell Biol. 11, 203-211 (2001).
[CrossRef] [PubMed]

P. J. Verveer, A. Squire, and P. I. H. Bastiaens, "Global analysis of fluorescence lifetime imaging microscopy data," Biophys. J. 78, 2127-2137 (2000).
[CrossRef] [PubMed]

P. J. Verveer, F. S. Wouters, A. R. Reynolds, and P. I. H. Bastiaens, "Quantitative imaging of lateral ErbB1 receptor signal propagation in the plasma membrane," Science 290, 1567-1570 (2000).
[CrossRef] [PubMed]

Vojnovic, B.

P. Barber, S. Ameer-Beg, J. Gilbey, L. Carlin, M. Keppler, T. Ng, and B. Vojnovic, "Multiphoton time-domain fluorescence lifetime imaging microscopy: practical application to protein-protein interactions using global analysis," J. R. Soc. Interface 6, S93-S105 (2008).
[CrossRef]

P. Barber, S. Ameer-Beg, J. Gilbey, R. J. Edens, I. Ezike, and B. Vojnovic, "Global and pixel kinetic data analysis for FRET detection by multi-photon time-domain FLIM," Proc. SPIE 5700, 171-181 (2005).
[CrossRef]

Waldmann, H.

O. Rocks, A. Peyker, M. Kahms, P. J. Verveer, C. Koerner, M. Lumbierres, J. Kuhlmann, H. Waldmann, A. Wittinghofer, and P. I. H. Bastiaens, "An acylation cycle regulates localization and activity of palmitoylated Ras isoforms," Science 307, 1746-1752 (2005).
[CrossRef] [PubMed]

Wittinghofer, A.

O. Rocks, A. Peyker, M. Kahms, P. J. Verveer, C. Koerner, M. Lumbierres, J. Kuhlmann, H. Waldmann, A. Wittinghofer, and P. I. H. Bastiaens, "An acylation cycle regulates localization and activity of palmitoylated Ras isoforms," Science 307, 1746-1752 (2005).
[CrossRef] [PubMed]

Wouters, F. S.

A. Esposito, H. C. Gerritsen, and F. S. Wouters, "Fluorescence lifetime heterogeneity resolution in the frequency domain by lifetime moments analysis," Biophys. J. 89, 4286-4299 (2005).
[CrossRef] [PubMed]

F. S. Wouters, P. J. Verveer, and P. I. H. Bastiaens, "Imaging biochemistry inside cells," Trends Cell Biol. 11, 203-211 (2001).
[CrossRef] [PubMed]

P. J. Verveer, F. S. Wouters, A. R. Reynolds, and P. I. H. Bastiaens, "Quantitative imaging of lateral ErbB1 receptor signal propagation in the plasma membrane," Science 290, 1567-1570 (2000).
[CrossRef] [PubMed]

F. S. Wouters and P. I. H. Bastiaens, "Fluorescence lifetime imaging of receptor tyrosine kinase activity in cells," Curr. Biol. 9, 1127-1130 (1999).
[CrossRef] [PubMed]

Xouri, G.

G. Xouri, A. Squire, M. Dimaki, B. Geverts, P. J. Verveer, S. Taraviras, H. Nishitani, A. B. Houtsmuller, P. I. H. Bastiaens, and Z. Lygerou, "Cdt1 associates dynamically with chromatin throughout G1 and recruits Geminin onto chromatin," EMBO J. 26, 1303-14 (2007).
[CrossRef] [PubMed]

Zicha, D.

T. Ng, M. Parsons, W. E. Hughes, J. Monypenny, D. Zicha, A. Gautreau, M. Arpin, S. Gschmeissner, P. J. Verveer, P. I. H. Bastiaens, and P. J. Parker, "Ezrin is a downstream effector of trafficking PKC-integrin complexes involved in the control of cell motility," EMBO J. 20, 2723-2741 (2001).
[CrossRef] [PubMed]

Appl. Opt. (1)

Appl. Spec. Rev. (1)

D. M. Jameson, E. Gratton, and R. Hall, "The measurement and analysis of heterogeneous emissions by multifrequency phase and modulation fluorometry." Appl. Spec. Rev. 20, 55-106 (1984).
[CrossRef]

Biol. Cell (1)

M. Peter and S. M. Ameer-Beg, "Imaging molecular interactions by multiphoton FLIM," Biol. Cell 96, 231-236 (2004).
[CrossRef] [PubMed]

Biophys. J. (5)

S. Pelet, M. J. R. Previte, L. H. Laiho, and P. T. C. So, "A fast global fitting algorithm for fluorescence lifetime imaging microscopy based on image segmentation," Biophys. J. 87, 2807-2817 (2004).
[CrossRef] [PubMed]

P. J. Verveer, A. Squire, and P. I. H. Bastiaens, "Global analysis of fluorescence lifetime imaging microscopy data," Biophys. J. 78, 2127-2137 (2000).
[CrossRef] [PubMed]

S. Padilla-Parra, N. Auduge, M. Coppey-Moisan, and M. Tramier, "Quantitative FRET analysis by fast acquisition time domain FLIM at high spatial resolution in living cells," Biophys. J. 95, 2976-2988 (2008).
[CrossRef] [PubMed]

A. Esposito, H. C. Gerritsen, and F. S. Wouters, "Fluorescence lifetime heterogeneity resolution in the frequency domain by lifetime moments analysis," Biophys. J. 89, 4286-4299 (2005).
[CrossRef] [PubMed]

G. W. Gordon, G. Berry, X. H. Liang, B. Levine, and B. Herman, "Quantitative fluorescence resonance energy transfer measurements using fluorescence microscopy," Biophys. J. 74, 2702-2713 (1998).
[CrossRef] [PubMed]

Curr. Biol. (1)

F. S. Wouters and P. I. H. Bastiaens, "Fluorescence lifetime imaging of receptor tyrosine kinase activity in cells," Curr. Biol. 9, 1127-1130 (1999).
[CrossRef] [PubMed]

EMBO J. (2)

G. Xouri, A. Squire, M. Dimaki, B. Geverts, P. J. Verveer, S. Taraviras, H. Nishitani, A. B. Houtsmuller, P. I. H. Bastiaens, and Z. Lygerou, "Cdt1 associates dynamically with chromatin throughout G1 and recruits Geminin onto chromatin," EMBO J. 26, 1303-14 (2007).
[CrossRef] [PubMed]

T. Ng, M. Parsons, W. E. Hughes, J. Monypenny, D. Zicha, A. Gautreau, M. Arpin, S. Gschmeissner, P. J. Verveer, P. I. H. Bastiaens, and P. J. Parker, "Ezrin is a downstream effector of trafficking PKC-integrin complexes involved in the control of cell motility," EMBO J. 20, 2723-2741 (2001).
[CrossRef] [PubMed]

Fluorescence Imaging Spectroscopy and Microscopy (1)

R. M. Clegg, "Fluorescence resonance energy tranfer," Fluorescence Imaging Spectroscopy and Microscopy 137, 179-251 (1996).

J. Biomed. Opt. (1)

E. Gratton, S. Breusegem, J. Sutin, Q. Ruan, and N. Barry, "Fluorescence lifetime imaging for the two-photon microscope: time-domain and frequency-domain methods," J. Biomed. Opt. 8, 381-390 (2003).
[CrossRef] [PubMed]

J. Fluoresc. (1)

G. I. Redford and R. M. Clegg, "Polar plot representation for frequency-domain analysis of fluorescence lifetimes," J. Fluoresc. 15, 805-815 (2005).
[CrossRef] [PubMed]

J. Microsc. (3)

K. Carlsson, A. Liljeborg, R. M. Andersson, and H. Brismar, "Confocal pH imaging of microscopic specimens using fluorescence lifetimes and phase fluorometry: influence of parameter choice on system performance," J. Microsc. 199, 106-114 (2000).
[CrossRef] [PubMed]

P. J. Verveer and P. I. H. Bastiaens, "Evaluation of global analysis algorithms for single frequency fluorescence lifetime imaging microscopy data," J. Microsc. 209, 1-7 (2003).
[CrossRef] [PubMed]

A. H. A. Clayton, Q. S. Hanley, and P. J. Verveer, "Graphical representation and multicomponent analysis of single-frequency fluorescence lifetime imaging microscopy data," J. Microsc. 213, 1-5 (2004).
[CrossRef]

J. R. Soc. Interface (1)

P. Barber, S. Ameer-Beg, J. Gilbey, L. Carlin, M. Keppler, T. Ng, and B. Vojnovic, "Multiphoton time-domain fluorescence lifetime imaging microscopy: practical application to protein-protein interactions using global analysis," J. R. Soc. Interface 6, S93-S105 (2008).
[CrossRef]

Microsc. Res. Tech. (2)

W. Becker, A. Bergmann, M. A. Hink, K. Konig, K. Benndorf, and C. Biskup, "Fluorescence lifetime imaging by time-correlated single-photon counting," Microsc. Res. Tech. 63, 58-66 (2004).
[CrossRef]

R. A. Colyer, C. Lee, and E. Gratton, "A novel fluorescence lifetime imaging system that optimizes photon efficiency," Microsc. Res. Tech. 71, 201-213 (2008).
[CrossRef]

Nat. Cell Biol. (1)

A. R. Reynolds, C. Tischer, P. J. Verveer, O. Rocks, and P. I. H. Bastiaens, "EGFR activation coupled to inhibition of tyrosine phosphatases causes lateral signal propagation," Nat. Cell Biol. 5, 447-453 (2003).
[CrossRef] [PubMed]

Proc. SPIE (1)

P. Barber, S. Ameer-Beg, J. Gilbey, R. J. Edens, I. Ezike, and B. Vojnovic, "Global and pixel kinetic data analysis for FRET detection by multi-photon time-domain FLIM," Proc. SPIE 5700, 171-181 (2005).
[CrossRef]

Science (2)

P. J. Verveer, F. S. Wouters, A. R. Reynolds, and P. I. H. Bastiaens, "Quantitative imaging of lateral ErbB1 receptor signal propagation in the plasma membrane," Science 290, 1567-1570 (2000).
[CrossRef] [PubMed]

O. Rocks, A. Peyker, M. Kahms, P. J. Verveer, C. Koerner, M. Lumbierres, J. Kuhlmann, H. Waldmann, A. Wittinghofer, and P. I. H. Bastiaens, "An acylation cycle regulates localization and activity of palmitoylated Ras isoforms," Science 307, 1746-1752 (2005).
[CrossRef] [PubMed]

Trends Cell Biol. (2)

P. I. H. Bastiaens and A. Squire, "Fluorescence lifetime imaging microscopy: spatial resolution of biochemical processes in the cell," Trends Cell Biol. 9, 48-52 (1999).
[CrossRef] [PubMed]

F. S. Wouters, P. J. Verveer, and P. I. H. Bastiaens, "Imaging biochemistry inside cells," Trends Cell Biol. 11, 203-211 (2001).
[CrossRef] [PubMed]

Other (2)

J. R. Lakowicz, Principles of Fluorescence Spectroscopy, 3rd ed. (Springer, 2006).
[CrossRef]

M. Digman, V. R. Caiolfa, M. Zamai, and E. Gratton, "The Phasor approach to fluorescence lifetime imaging analysis," Biophys. J. (2007).
[PubMed]

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Figures (7)

Fig. 1.
Fig. 1.

Graphical representation of global analysis of FLIM data using a phasor plot [24]. The half-circle represents all possible values of mono- exponential lifetimes, whereas the straight-line segment represents all possible mixtures of two mono-exponential species. Global analysis proceeds by estimating the straight line from data and the intersections with the half-circle (R n,1, R n,2, open square and circle). For a noisy data point, (Ri n , filled circle), the fluorescence fraction is given by the projected distance to R n,1.

Fig. 2.
Fig. 2.

The argument and the absolute value of the Fourier coefficients of TCSPC FLIM data. The Fourier coefficients for a simulated FLIM decay curve (τ 1 = 3 ns, τ 2 = 1.5 ns, and α = 0.5), were calculated. The parameters of the Gaussian IRF were t 0 = 2.0 ns and σ= 0.1 ns. (a) Argument of the Fourier coefficient as a function of the harmonic number n. (b) Logarithm of the absolute value of the Fourier coefficient as a function of n 2. The straight lines are linear fits to the higher harmonics (n > 20, dotted lines).

Fig. 3.
Fig. 3.

Geometrical representation of the filtering of FRET-FLIM data. The imaginary parts of the first harmonic of the FLIM data are plotted against the corresponding real parts. From donor-only data, a Gaussian distribution of the donor Fourier components is estimated, plotted as a blue contour at a preset distance from its mean. Points outside the two red dotted straight lines are rejected. Points within the donor distribution contour are assumed to be donor-only points and are also not used. Black circles: rejected points, blue circles: donor points, red circles: accepted points.

Fig. 4.
Fig. 4.

Recovery of IRF parameters from simulated TCSPC FLIM data. (a) Mean value and standard deviation of the estimated value of the offset t 0 as a function of mean photon count per pixel, for different total numbers of pixels. (b) Mean value and standard deviation of the estimated value of the pulse width σ as a function of mean photon count per pixel, for different total numbers of pixels.

Fig. 5.
Fig. 5.

Global analysis of simulated TCSPC FRET-FLIM data. (a) Mean value and standard deviation of the short lifetime estimated by global analysis, as a function of mean photon count per pixel, for different total numbers of pixels. (b) Fractional fluorescence error as a function of mean photon count per pixel, for different total numbers of pixels. The fractional fluorescence error is defined as the mean of the absolute difference between the recovered and simulated α.

Fig. 6.
Fig. 6.

Recovery of IRF parameters from experimental TCSPC FLIM data. (a) Argument of the Fourier coefficient of the averaged FLIM data as a function of the harmonic number n. (b) Logarithm of the absolute value of the Fourier coefficients as a function of n 2. The red lines are linear fits to the higher harmonics (n > 23, blue dotted lines). (c) Plots of the measured IRF, the Gaussian pulse with parameters derived estimated from the estimated Fourier coefficients, and a Gaussian pulse with parameters derived from the first harmonic of the measured IRF.

Fig. 7.
Fig. 7.

Global analysis of experimental TCSPC FRET-FLIM data. (a) Fluorescence intensity of EGFR-YFP. The colorbar indicates the number of counts per pixel. (b) Relative concentration of phosphorylated EGFR-YFP estimated from a pixel-by-pixel bi-exponential fit. (c) Tail fit to the average of all FLIM curves for two different regions (5-20 ns and 6-21 ns). (d) Phasor plot of a random subset of all donor-only data, with the estimated Gaussian distribution indicated by a contour at one standard deviation from the mean. (e) Phasor plot of a random subset of all data from donor/acceptor samples, with points that are accepted by the filtering procedure indicated in red color. The straight line represents a linear fit to the accepted data from which the two lifetimes are calculated. (f) Relative concentration of phosphorylated EGFR-YFP estimated by global analysis.

Equations (23)

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D ( t ) = q = 1 Q α q τ q exp ( t / τ q ) ,
E ( t ) = E 0 [ 1 + n = n 0 E n exp ( jnωt ) ] ,
F ( t ) = F T t E ( u ) D ( t u ) d u ,
F N ( t ) = F ( t ) F T E 0 = 1 + n = n 0 q = 1 Q E n α q exp ( jnωt ) 1 jnω τ q .
R n , q = 1 1 jnω τ q = exp [ j arctan ( τ q ) ] ( 1 + n 2 ω 2 τ q 2 ) 1 / 2 ,
F N i ( t ) = 1 + n = n 0 E n R n i exp ( jnωt ) ,
R n i = q = 1 Q α q i R n , q ,
R n i = ( 1 α i ) R n , 1 + α i R n , 2 ,
Im R n i = u n + v n Re R n i ,
u n = Im ( R n , 2 * R n , 1 ) Re ( R n , 2 R n , 1 ) and v n = Im ( R n , 2 R n , 1 ) Re ( R n , 2 R n , 1 ) .
τ 1,2 = 1 ± [ 1 4 u n ( u n + v n ) ] 1 / 2 2 u n .
α i = ( τ 1 + τ 2 ) Re R n i + ( n 2 ω 2 τ 1 τ 2 1 ) Im R n i τ 2 ( τ 1 τ 2 ) .
G ( t ) = exp [ ( t t 0 ) 2 / 2 σ 2 ] σ 2 π .
E n = exp ( n 2 ω 2 σ 2 / 2 ) exp ( jnω t 0 ) .
arg ( E n R n ) = arctan ( nωτ ) + t 0 ,
E n R n = exp ( n 2 ω 2 σ 2 / 2 ) ( 1 + n 2 ω 2 τ 2 ) 1 / 2 .
arg ( E n R n ) arctan ( n 0 ωτ ) + n 0 ωτ 1 + n 0 1 ω 2 τ 2 + ( τ 1 + n 0 1 ω 2 τ 2 + t 0 ) ,
In E n R n n 0 2 ω 2 τ 2 2 ( 1 + n 0 2 ω 2 τ 2 ) 1 2 ln ( 1 + n 0 2 ω 2 τ 2 ) 1 2 ( τ 2 1 + n 0 2 ω 2 τ 2 + σ 2 ) n 2 ω 2 .
arg ( E n R n ) a + t 0 ,
In E n R n b 1 2 n 2 ω 2 σ 2 ,
E n R n i = k = 1 B exp ( 2 πn b k i / B )
u n = Im R ¯ n , 1 v Re R ¯ n , 1 ,
v n = S RI S R Im R ¯ n , 1 S I Re R ¯ n , 1 + S w Re R ¯ n , 1 Im R ¯ n , 1 S RR 2 S R Re R ¯ n , 1 + S w ( Re R ¯ n , 1 ) 2

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