Abstract

A new approach to alias-free wide-field fluorescence lifetime imaging in the frequency domain is demonstrated using a supercontinuum source for fluorescence excitation and a phase-modulated image intensifier for detection. This technique is referred to as phi-squared fluorescence lifetime imaging (ϕ2FLIM). The phase modulation and square-wave gating of the image intensifier eliminate aliasing by the effective suppression of higher harmonics. The ability to use picosecond excitation pulses without aliasing expands the range of excitation sources available for frequency-domain fluorescence lifetime imaging (fd-FLIM) and improves the modulation depth of conventional homodyne fd-FLIM measurements, which use sinusoidal intensity modulation of the excitation source. The ϕ2FLIM results are analyzed using AB-plots, which facilitate the identification of mono-exponential and multi-exponential fluorescence decays and provide measurements of the fluorophore fractions in two component mixtures. The rapid acquisition speed of the technique enables lifetime measurements in dynamic systems, such as temporally evolving samples and samples that are sensitive to photo-bleaching. Rapid ϕ2FLIM measurements are demonstrated by imaging the dynamic mixing of two different dye solutions at 5.5 Hz. The tunability of supercontinuum radiation enables excitation wavelength resolved FLIM measurements, which facilitates analysis of samples containing multiple fluorophores with different absorption spectra.

© 2009 OSA

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    [CrossRef]
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    [CrossRef] [PubMed]
  3. X. W. Dai, M. E. Eccleston, Z. L. Yue, N. K. H. Slater, and C. F. Kaminski, “A spectroscopic study of the self-association and inter-molecular aggregation behaviour of pH-responsive poly(L-lysine iso-phthalamide),” Polymer (Guildf.) 47(8), 2689–2698 (2006).
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    [CrossRef] [PubMed]
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  7. W. Becker, H. Hickl, C. Zander, K. H. Drexhage, S. S. M. Sauer, and J. Wolfrum, “Time-resolved detection and identification of single analyte molecules in microcapillaries by timecorrelated single-photon counting (TCSPC),” Rev. Sci. Instrum. 70(3), 1835–1841 (1999).
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    [CrossRef]
  10. J. R. Lakowicz, H. Szmacinski, K. Nowaczyk, K. W. Berndt, and M. Johnson, “Fluorescence lifetime imaging,” Anal. Biochem. 202(2), 316–330 (1992).
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    [CrossRef]
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    [CrossRef]
  23. J. H. Frank, A. D. Elder, J. Swartling, A. R. Venkitaraman, A. D. Jeyasekharan, and C. F. Kaminski, “A white light confocal microscope for spectrally resolved multidimensional imaging,” J. Microsc. 227(3), 203–215 (2007).
    [CrossRef] [PubMed]
  24. Q. S. Hanley, V. Subramaniam, D. J. Arndt-Jovin, and T. M. Jovin, “Fluorescence lifetime imaging: Multi-point calibration, minimum resolvable differences, and artifact suppression,” Cytometry 43(4), 248–260 (2001).
    [CrossRef] [PubMed]
  25. T. B. Settersten, A. Dreizler, and R. L. Farrow, “Temperature- and species-dependent quenching of CO B probed by two-photon laser-induced fluorescence using a picosecond laser,” J. Chem. Phys. 117(7), 3173–3179 (2002).
    [CrossRef]
  26. S. E. D. Webb, Y. Gu, S. Leveque-Fort, J. Siegel, M. J. Cole, K. Dowling, R. Jones, P. M. W. French, M. A. A. Neil, R. Juskaitis, L. O. D. Sucharov, T. Wilson, and M. J. Lever, “A wide-field time-domain fluorescence lifetime imaging microscope with optical sectioning,” Rev. Sci. Instrum. 73(4), 1898–1907 (2002).
    [CrossRef]
  27. C. Dunsby, P. M. P. Lanigan, J. McGinty, D. S. Elson, J. Requejo-Isidro, I. Munro, N. Galletly, F. McCann, B. Treanor, B. Onfelt, D. M. Davis, M. A. A. Neil, and P. M. W. French, “An electronically tunable ultrafast laser source applied to fluorescence imaging and fluorescence lifetime imaging microscopy,” J. Phys. D Appl. Phys. 37(23), 3296–3303 (2004).
    [CrossRef]
  28. R. K. P. Benninger, O. Hofmann, J. McGinty, J. Requejo-Isidro, I. Munro, M. A. A. Neil, A. J. Demello, and P. M. W. French, “Time-resolved fluorescence imaging of solvent interactions in microfluidic devices,” Opt. Express 13(16), 6275–6285 (2005).
    [CrossRef] [PubMed]
  29. 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. Kellet, A. Dymoke-Bradshaw, J. Hares, and P. M. W. French, “Real-time time-domain fluorescence lifetime imaging including single-shot acquisition with a segmented optical image intensifier,” N. J. Phys. 6, 180–192 (2004).
    [CrossRef]
  30. K. K. Sharman, A. Periasamy, H. Ashworth, J. N. Demas, and N. H. Snow, “Error analysis of the rapid lifetime determination method for double-exponential decays and new windowing schemes,” Anal. Chem. 71(5), 947–952 (1999).
    [CrossRef] [PubMed]
  31. O. Holub, M. J. Seufferheld, C. Gohlke, Govindjee, and R. M. Clegg, “Fluorescence lifetime imaging in real-time - a new technique in photosynthesis research,” Photosynthetica 38(4), 581–599 (2000).
    [CrossRef]

2009 (2)

A. D. Elder, A. Domin, G. S. Kaminski-Schierle, C. Lindon, J. Pines, A. Esposito, and C. F. Kaminski, “A quantitative protocol for dynamic measurements of protein interactions by FRET-sensitized fluorescence emission,” J. R. Soc. Interface 6, S59–S81 (2009).
[CrossRef]

S. Schlachter, A. D. Elder, A. Esposito, G. S. Kaminski, J. H. Frank, L. K. van Geest, and C. F. Kaminski, “mhFLIM: resolution of heterogeneous fluorescence decays in widefield lifetime microscopy,” Opt. Express 17(3), 1557–1570 (2009).
[CrossRef] [PubMed]

2008 (5)

A. D. Elder, S. Schlachter, and 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]

M. A. Digman, V. R. Caiolfa, M. Zamai, and E. Gratton, “The phasor approach to fluorescence lifetime imaging analysis,” Biophys. J. 94(2), 14–16 (2008).
[CrossRef] [PubMed]

G. J. Kremers, E. B. van Munster, J. Goedhart, and T. W. J. Gadella., “Quantitative lifetime unmixing of multiexponentially decaying fluorophores using single-frequency fluorescence lifetime imaging microscopy,” Biophys. J. 95(1), 378–389 (2008).
[CrossRef] [PubMed]

A. Esposito, T. Tiffert, J. M. A. Mauritz, S. Schlachter, L. H. Bannister, C. F. Kaminski, V. L. Lew, and J. M. Schnur, “FRET imaging of hemoglobin concentration in Plasmodium falciparum-infected red cells,” PLoS ONE 3(11), e3780 (2008).
[CrossRef] [PubMed]

C. F. Kaminski, R. S. Watt, A. D. Elder, J. H. Frank, and J. Hult, “Supercontinuum radiation for applications in chemical sensing and microscopy,” Appl. Phys. B 92(3), 367–378 (2008).
[CrossRef]

2007 (2)

J. H. Frank, A. D. Elder, J. Swartling, A. R. Venkitaraman, A. D. Jeyasekharan, and C. F. Kaminski, “A white light confocal microscope for spectrally resolved multidimensional imaging,” J. Microsc. 227(3), 203–215 (2007).
[CrossRef] [PubMed]

S. M. Matthews, A. D. Elder, K. Yunus, C. F. Kaminski, C. M. Brennan, and A. C. Fisher, “Quantitative kinetic analysis in a microfluidic device using frequency-domain fluorescence lifetime imaging,” Anal. Chem. 79(11), 4101–4109 (2007).
[CrossRef] [PubMed]

2006 (2)

X. W. Dai, M. E. Eccleston, Z. L. Yue, N. K. H. Slater, and C. F. Kaminski, “A spectroscopic study of the self-association and inter-molecular aggregation behaviour of pH-responsive poly(L-lysine iso-phthalamide),” Polymer (Guildf.) 47(8), 2689–2698 (2006).
[CrossRef]

A. D. Elder, J. H. Frank, J. Swartling, X. Dai, and C. F. Kaminski, “Calibration of a wide-field frequency-domain fluorescence lifetime microscopy system using light emitting diodes as light sources,” J. Microsc. 224(2), 166–180 (2006).
[CrossRef] [PubMed]

2005 (3)

Q. S. Hanley and A. H. A. Clayton, “AB-plot assisted determination of fluorophore mixtures in a fluorescence lifetime microscope using spectra or quenchers,” J. Microsc. 218(1), 62–67 (2005).
[CrossRef] [PubMed]

G. I. Redford and R. M. Clegg, “Polar plot representation for frequency-domain analysis of fluorescence lifetimes,” J. Fluoresc. 15(5), 805–815 (2005).
[CrossRef] [PubMed]

R. K. P. Benninger, O. Hofmann, J. McGinty, J. Requejo-Isidro, I. Munro, M. A. A. Neil, A. J. Demello, and P. M. W. French, “Time-resolved fluorescence imaging of solvent interactions in microfluidic devices,” Opt. Express 13(16), 6275–6285 (2005).
[CrossRef] [PubMed]

2004 (3)

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. Kellet, A. Dymoke-Bradshaw, J. Hares, and P. M. W. French, “Real-time time-domain fluorescence lifetime imaging including single-shot acquisition with a segmented optical image intensifier,” N. J. Phys. 6, 180–192 (2004).
[CrossRef]

E. B. Van Munster and T. W. J. Gadella., “phiFLIM: a new method to avoid aliasing in frequency-domain fluorescence lifetime imaging microscopy,” J. Microsc. 213(1), 29–38 (2004).
[CrossRef] [PubMed]

C. Dunsby, P. M. P. Lanigan, J. McGinty, D. S. Elson, J. Requejo-Isidro, I. Munro, N. Galletly, F. McCann, B. Treanor, B. Onfelt, D. M. Davis, M. A. A. Neil, and P. M. W. French, “An electronically tunable ultrafast laser source applied to fluorescence imaging and fluorescence lifetime imaging microscopy,” J. Phys. D Appl. Phys. 37(23), 3296–3303 (2004).
[CrossRef]

2003 (1)

2002 (2)

T. B. Settersten, A. Dreizler, and R. L. Farrow, “Temperature- and species-dependent quenching of CO B probed by two-photon laser-induced fluorescence using a picosecond laser,” J. Chem. Phys. 117(7), 3173–3179 (2002).
[CrossRef]

S. E. D. Webb, Y. Gu, S. Leveque-Fort, J. Siegel, M. J. Cole, K. Dowling, R. Jones, P. M. W. French, M. A. A. Neil, R. Juskaitis, L. O. D. Sucharov, T. Wilson, and M. J. Lever, “A wide-field time-domain fluorescence lifetime imaging microscope with optical sectioning,” Rev. Sci. Instrum. 73(4), 1898–1907 (2002).
[CrossRef]

2001 (1)

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

2000 (2)

O. Holub, M. J. Seufferheld, C. Gohlke, Govindjee, and R. M. Clegg, “Fluorescence lifetime imaging in real-time - a new technique in photosynthesis research,” Photosynthetica 38(4), 581–599 (2000).
[CrossRef]

A. Squire, P. J. Verveer, and P. I. H. Bastiaens, “Multiple frequency fluorescence lifetime imaging microscopy,” J. Microsc. 197(2), 136–149 (2000).
[CrossRef] [PubMed]

1999 (2)

W. Becker, H. Hickl, C. Zander, K. H. Drexhage, S. S. M. Sauer, and J. Wolfrum, “Time-resolved detection and identification of single analyte molecules in microcapillaries by timecorrelated single-photon counting (TCSPC),” Rev. Sci. Instrum. 70(3), 1835–1841 (1999).
[CrossRef]

K. K. Sharman, A. Periasamy, H. Ashworth, J. N. Demas, and N. H. Snow, “Error analysis of the rapid lifetime determination method for double-exponential decays and new windowing schemes,” Anal. Chem. 71(5), 947–952 (1999).
[CrossRef] [PubMed]

1997 (1)

P. Schneider and R. M. Clegg, “Rapid acquisition, analysis, and display of fuorescence lifetime-resolved images for real-time applications,” Rev. Sci. Instrum. 68(11), 4107 (1997).
[CrossRef]

1996 (1)

A. Periasamy, P. Wodnicki, X. Wang, S. Kwon, G. Gordon, and B. Herman, “Time-resolved fluorescence lifetime imaging microscopy using a picosecond pulsed tunable dye laser system,” Rev. Sci. Instrum. 67(10), 3722–3731 (1996).
[CrossRef]

1993 (1)

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

1992 (1)

J. R. Lakowicz, H. Szmacinski, K. Nowaczyk, K. W. Berndt, and M. Johnson, “Fluorescence lifetime imaging,” Anal. Biochem. 202(2), 316–330 (1992).
[CrossRef] [PubMed]

1991 (1)

J. R. Lakowicz and K. W. Berndt, “Lifetime-selective fluorescence imaging using an rf phase-sensitive camera,” Rev. Sci. Instrum. 62(7), 1727–1734 (1991).
[CrossRef]

Arndt-Jovin, D. J.

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

Ashworth, H.

K. K. Sharman, A. Periasamy, H. Ashworth, J. N. Demas, and N. H. Snow, “Error analysis of the rapid lifetime determination method for double-exponential decays and new windowing schemes,” Anal. Chem. 71(5), 947–952 (1999).
[CrossRef] [PubMed]

Bannister, L. H.

A. Esposito, T. Tiffert, J. M. A. Mauritz, S. Schlachter, L. H. Bannister, C. F. Kaminski, V. L. Lew, and J. M. Schnur, “FRET imaging of hemoglobin concentration in Plasmodium falciparum-infected red cells,” PLoS ONE 3(11), e3780 (2008).
[CrossRef] [PubMed]

Bastiaens, P. I. H.

A. Squire, P. J. Verveer, and P. I. H. Bastiaens, “Multiple frequency fluorescence lifetime imaging microscopy,” J. Microsc. 197(2), 136–149 (2000).
[CrossRef] [PubMed]

Becker, W.

W. Becker, H. Hickl, C. Zander, K. H. Drexhage, S. S. M. Sauer, and J. Wolfrum, “Time-resolved detection and identification of single analyte molecules in microcapillaries by timecorrelated single-photon counting (TCSPC),” Rev. Sci. Instrum. 70(3), 1835–1841 (1999).
[CrossRef]

Benninger, R. K. P.

Berndt, K. W.

J. R. Lakowicz, H. Szmacinski, K. Nowaczyk, K. W. Berndt, and M. Johnson, “Fluorescence lifetime imaging,” Anal. Biochem. 202(2), 316–330 (1992).
[CrossRef] [PubMed]

J. R. Lakowicz and K. W. Berndt, “Lifetime-selective fluorescence imaging using an rf phase-sensitive camera,” Rev. Sci. Instrum. 62(7), 1727–1734 (1991).
[CrossRef]

Brennan, C. M.

S. M. Matthews, A. D. Elder, K. Yunus, C. F. Kaminski, C. M. Brennan, and A. C. Fisher, “Quantitative kinetic analysis in a microfluidic device using frequency-domain fluorescence lifetime imaging,” Anal. Chem. 79(11), 4101–4109 (2007).
[CrossRef] [PubMed]

Caiolfa, V. R.

M. A. Digman, V. R. Caiolfa, M. Zamai, and E. Gratton, “The phasor approach to fluorescence lifetime imaging analysis,” Biophys. J. 94(2), 14–16 (2008).
[CrossRef] [PubMed]

Carlsson, K.

Clayton, A. H. A.

Q. S. Hanley and A. H. A. Clayton, “AB-plot assisted determination of fluorophore mixtures in a fluorescence lifetime microscope using spectra or quenchers,” J. Microsc. 218(1), 62–67 (2005).
[CrossRef] [PubMed]

Clegg, R. M.

G. I. Redford and R. M. Clegg, “Polar plot representation for frequency-domain analysis of fluorescence lifetimes,” J. Fluoresc. 15(5), 805–815 (2005).
[CrossRef] [PubMed]

O. Holub, M. J. Seufferheld, C. Gohlke, Govindjee, and R. M. Clegg, “Fluorescence lifetime imaging in real-time - a new technique in photosynthesis research,” Photosynthetica 38(4), 581–599 (2000).
[CrossRef]

P. Schneider and R. M. Clegg, “Rapid acquisition, analysis, and display of fuorescence lifetime-resolved images for real-time applications,” Rev. Sci. Instrum. 68(11), 4107 (1997).
[CrossRef]

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

Cole, M. J.

S. E. D. Webb, Y. Gu, S. Leveque-Fort, J. Siegel, M. J. Cole, K. Dowling, R. Jones, P. M. W. French, M. A. A. Neil, R. Juskaitis, L. O. D. Sucharov, T. Wilson, and M. J. Lever, “A wide-field time-domain fluorescence lifetime imaging microscope with optical sectioning,” Rev. Sci. Instrum. 73(4), 1898–1907 (2002).
[CrossRef]

Dai, X.

A. D. Elder, J. H. Frank, J. Swartling, X. Dai, and C. F. Kaminski, “Calibration of a wide-field frequency-domain fluorescence lifetime microscopy system using light emitting diodes as light sources,” J. Microsc. 224(2), 166–180 (2006).
[CrossRef] [PubMed]

Dai, X. W.

X. W. Dai, M. E. Eccleston, Z. L. Yue, N. K. H. Slater, and C. F. Kaminski, “A spectroscopic study of the self-association and inter-molecular aggregation behaviour of pH-responsive poly(L-lysine iso-phthalamide),” Polymer (Guildf.) 47(8), 2689–2698 (2006).
[CrossRef]

Davis, D. M.

C. Dunsby, P. M. P. Lanigan, J. McGinty, D. S. Elson, J. Requejo-Isidro, I. Munro, N. Galletly, F. McCann, B. Treanor, B. Onfelt, D. M. Davis, M. A. A. Neil, and P. M. W. French, “An electronically tunable ultrafast laser source applied to fluorescence imaging and fluorescence lifetime imaging microscopy,” J. Phys. D Appl. Phys. 37(23), 3296–3303 (2004).
[CrossRef]

Demas, J. N.

K. K. Sharman, A. Periasamy, H. Ashworth, J. N. Demas, and N. H. Snow, “Error analysis of the rapid lifetime determination method for double-exponential decays and new windowing schemes,” Anal. Chem. 71(5), 947–952 (1999).
[CrossRef] [PubMed]

Demello, A. J.

Digman, M. A.

M. A. Digman, V. R. Caiolfa, M. Zamai, and E. Gratton, “The phasor approach to fluorescence lifetime imaging analysis,” Biophys. J. 94(2), 14–16 (2008).
[CrossRef] [PubMed]

Domin, A.

A. D. Elder, A. Domin, G. S. Kaminski-Schierle, C. Lindon, J. Pines, A. Esposito, and C. F. Kaminski, “A quantitative protocol for dynamic measurements of protein interactions by FRET-sensitized fluorescence emission,” J. R. Soc. Interface 6, S59–S81 (2009).
[CrossRef]

Dowling, K.

S. E. D. Webb, Y. Gu, S. Leveque-Fort, J. Siegel, M. J. Cole, K. Dowling, R. Jones, P. M. W. French, M. A. A. Neil, R. Juskaitis, L. O. D. Sucharov, T. Wilson, and M. J. Lever, “A wide-field time-domain fluorescence lifetime imaging microscope with optical sectioning,” Rev. Sci. Instrum. 73(4), 1898–1907 (2002).
[CrossRef]

Dreizler, A.

T. B. Settersten, A. Dreizler, and R. L. Farrow, “Temperature- and species-dependent quenching of CO B probed by two-photon laser-induced fluorescence using a picosecond laser,” J. Chem. Phys. 117(7), 3173–3179 (2002).
[CrossRef]

Drexhage, K. H.

W. Becker, H. Hickl, C. Zander, K. H. Drexhage, S. S. M. Sauer, and J. Wolfrum, “Time-resolved detection and identification of single analyte molecules in microcapillaries by timecorrelated single-photon counting (TCSPC),” Rev. Sci. Instrum. 70(3), 1835–1841 (1999).
[CrossRef]

Dunsby, C.

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. Kellet, A. Dymoke-Bradshaw, J. Hares, and P. M. W. French, “Real-time time-domain fluorescence lifetime imaging including single-shot acquisition with a segmented optical image intensifier,” N. J. Phys. 6, 180–192 (2004).
[CrossRef]

C. Dunsby, P. M. P. Lanigan, J. McGinty, D. S. Elson, J. Requejo-Isidro, I. Munro, N. Galletly, F. McCann, B. Treanor, B. Onfelt, D. M. Davis, M. A. A. Neil, and P. M. W. French, “An electronically tunable ultrafast laser source applied to fluorescence imaging and fluorescence lifetime imaging microscopy,” J. Phys. D Appl. Phys. 37(23), 3296–3303 (2004).
[CrossRef]

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. Kellet, A. Dymoke-Bradshaw, J. Hares, and P. M. W. French, “Real-time time-domain fluorescence lifetime imaging including single-shot acquisition with a segmented optical image intensifier,” N. J. Phys. 6, 180–192 (2004).
[CrossRef]

Eccleston, M. E.

X. W. Dai, M. E. Eccleston, Z. L. Yue, N. K. H. Slater, and C. F. Kaminski, “A spectroscopic study of the self-association and inter-molecular aggregation behaviour of pH-responsive poly(L-lysine iso-phthalamide),” Polymer (Guildf.) 47(8), 2689–2698 (2006).
[CrossRef]

Elder, A. D.

A. D. Elder, A. Domin, G. S. Kaminski-Schierle, C. Lindon, J. Pines, A. Esposito, and C. F. Kaminski, “A quantitative protocol for dynamic measurements of protein interactions by FRET-sensitized fluorescence emission,” J. R. Soc. Interface 6, S59–S81 (2009).
[CrossRef]

S. Schlachter, A. D. Elder, A. Esposito, G. S. Kaminski, J. H. Frank, L. K. van Geest, and C. F. Kaminski, “mhFLIM: resolution of heterogeneous fluorescence decays in widefield lifetime microscopy,” Opt. Express 17(3), 1557–1570 (2009).
[CrossRef] [PubMed]

A. D. Elder, S. Schlachter, and 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]

C. F. Kaminski, R. S. Watt, A. D. Elder, J. H. Frank, and J. Hult, “Supercontinuum radiation for applications in chemical sensing and microscopy,” Appl. Phys. B 92(3), 367–378 (2008).
[CrossRef]

J. H. Frank, A. D. Elder, J. Swartling, A. R. Venkitaraman, A. D. Jeyasekharan, and C. F. Kaminski, “A white light confocal microscope for spectrally resolved multidimensional imaging,” J. Microsc. 227(3), 203–215 (2007).
[CrossRef] [PubMed]

S. M. Matthews, A. D. Elder, K. Yunus, C. F. Kaminski, C. M. Brennan, and A. C. Fisher, “Quantitative kinetic analysis in a microfluidic device using frequency-domain fluorescence lifetime imaging,” Anal. Chem. 79(11), 4101–4109 (2007).
[CrossRef] [PubMed]

A. D. Elder, J. H. Frank, J. Swartling, X. Dai, and C. F. Kaminski, “Calibration of a wide-field frequency-domain fluorescence lifetime microscopy system using light emitting diodes as light sources,” J. Microsc. 224(2), 166–180 (2006).
[CrossRef] [PubMed]

Elson, D. S.

C. Dunsby, P. M. P. Lanigan, J. McGinty, D. S. Elson, J. Requejo-Isidro, I. Munro, N. Galletly, F. McCann, B. Treanor, B. Onfelt, D. M. Davis, M. A. A. Neil, and P. M. W. French, “An electronically tunable ultrafast laser source applied to fluorescence imaging and fluorescence lifetime imaging microscopy,” J. Phys. D Appl. Phys. 37(23), 3296–3303 (2004).
[CrossRef]

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. Kellet, A. Dymoke-Bradshaw, J. Hares, and P. M. W. French, “Real-time time-domain fluorescence lifetime imaging including single-shot acquisition with a segmented optical image intensifier,” N. J. Phys. 6, 180–192 (2004).
[CrossRef]

Esposito, A.

S. Schlachter, A. D. Elder, A. Esposito, G. S. Kaminski, J. H. Frank, L. K. van Geest, and C. F. Kaminski, “mhFLIM: resolution of heterogeneous fluorescence decays in widefield lifetime microscopy,” Opt. Express 17(3), 1557–1570 (2009).
[CrossRef] [PubMed]

A. D. Elder, A. Domin, G. S. Kaminski-Schierle, C. Lindon, J. Pines, A. Esposito, and C. F. Kaminski, “A quantitative protocol for dynamic measurements of protein interactions by FRET-sensitized fluorescence emission,” J. R. Soc. Interface 6, S59–S81 (2009).
[CrossRef]

A. Esposito, T. Tiffert, J. M. A. Mauritz, S. Schlachter, L. H. Bannister, C. F. Kaminski, V. L. Lew, and J. M. Schnur, “FRET imaging of hemoglobin concentration in Plasmodium falciparum-infected red cells,” PLoS ONE 3(11), e3780 (2008).
[CrossRef] [PubMed]

Farrow, R. L.

T. B. Settersten, A. Dreizler, and R. L. Farrow, “Temperature- and species-dependent quenching of CO B probed by two-photon laser-induced fluorescence using a picosecond laser,” J. Chem. Phys. 117(7), 3173–3179 (2002).
[CrossRef]

Fisher, A. C.

S. M. Matthews, A. D. Elder, K. Yunus, C. F. Kaminski, C. M. Brennan, and A. C. Fisher, “Quantitative kinetic analysis in a microfluidic device using frequency-domain fluorescence lifetime imaging,” Anal. Chem. 79(11), 4101–4109 (2007).
[CrossRef] [PubMed]

Frank, J. H.

S. Schlachter, A. D. Elder, A. Esposito, G. S. Kaminski, J. H. Frank, L. K. van Geest, and C. F. Kaminski, “mhFLIM: resolution of heterogeneous fluorescence decays in widefield lifetime microscopy,” Opt. Express 17(3), 1557–1570 (2009).
[CrossRef] [PubMed]

C. F. Kaminski, R. S. Watt, A. D. Elder, J. H. Frank, and J. Hult, “Supercontinuum radiation for applications in chemical sensing and microscopy,” Appl. Phys. B 92(3), 367–378 (2008).
[CrossRef]

J. H. Frank, A. D. Elder, J. Swartling, A. R. Venkitaraman, A. D. Jeyasekharan, and C. F. Kaminski, “A white light confocal microscope for spectrally resolved multidimensional imaging,” J. Microsc. 227(3), 203–215 (2007).
[CrossRef] [PubMed]

A. D. Elder, J. H. Frank, J. Swartling, X. Dai, and C. F. Kaminski, “Calibration of a wide-field frequency-domain fluorescence lifetime microscopy system using light emitting diodes as light sources,” J. Microsc. 224(2), 166–180 (2006).
[CrossRef] [PubMed]

French, P. M. W.

R. K. P. Benninger, O. Hofmann, J. McGinty, J. Requejo-Isidro, I. Munro, M. A. A. Neil, A. J. Demello, and P. M. W. French, “Time-resolved fluorescence imaging of solvent interactions in microfluidic devices,” Opt. Express 13(16), 6275–6285 (2005).
[CrossRef] [PubMed]

C. Dunsby, P. M. P. Lanigan, J. McGinty, D. S. Elson, J. Requejo-Isidro, I. Munro, N. Galletly, F. McCann, B. Treanor, B. Onfelt, D. M. Davis, M. A. A. Neil, and P. M. W. French, “An electronically tunable ultrafast laser source applied to fluorescence imaging and fluorescence lifetime imaging microscopy,” J. Phys. D Appl. Phys. 37(23), 3296–3303 (2004).
[CrossRef]

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. Kellet, A. Dymoke-Bradshaw, J. Hares, and P. M. W. French, “Real-time time-domain fluorescence lifetime imaging including single-shot acquisition with a segmented optical image intensifier,” N. J. Phys. 6, 180–192 (2004).
[CrossRef]

S. E. D. Webb, Y. Gu, S. Leveque-Fort, J. Siegel, M. J. Cole, K. Dowling, R. Jones, P. M. W. French, M. A. A. Neil, R. Juskaitis, L. O. D. Sucharov, T. Wilson, and M. J. Lever, “A wide-field time-domain fluorescence lifetime imaging microscope with optical sectioning,” Rev. Sci. Instrum. 73(4), 1898–1907 (2002).
[CrossRef]

Gadella, T. W. J.

G. J. Kremers, E. B. van Munster, J. Goedhart, and T. W. J. Gadella., “Quantitative lifetime unmixing of multiexponentially decaying fluorophores using single-frequency fluorescence lifetime imaging microscopy,” Biophys. J. 95(1), 378–389 (2008).
[CrossRef] [PubMed]

E. B. Van Munster and T. W. J. Gadella., “phiFLIM: a new method to avoid aliasing in frequency-domain fluorescence lifetime imaging microscopy,” J. Microsc. 213(1), 29–38 (2004).
[CrossRef] [PubMed]

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

Galletly, N.

C. Dunsby, P. M. P. Lanigan, J. McGinty, D. S. Elson, J. Requejo-Isidro, I. Munro, N. Galletly, F. McCann, B. Treanor, B. Onfelt, D. M. Davis, M. A. A. Neil, and P. M. W. French, “An electronically tunable ultrafast laser source applied to fluorescence imaging and fluorescence lifetime imaging microscopy,” J. Phys. D Appl. Phys. 37(23), 3296–3303 (2004).
[CrossRef]

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. Kellet, A. Dymoke-Bradshaw, J. Hares, and P. M. W. French, “Real-time time-domain fluorescence lifetime imaging including single-shot acquisition with a segmented optical image intensifier,” N. J. Phys. 6, 180–192 (2004).
[CrossRef]

Goedhart, J.

G. J. Kremers, E. B. van Munster, J. Goedhart, and T. W. J. Gadella., “Quantitative lifetime unmixing of multiexponentially decaying fluorophores using single-frequency fluorescence lifetime imaging microscopy,” Biophys. J. 95(1), 378–389 (2008).
[CrossRef] [PubMed]

Gohlke, C.

O. Holub, M. J. Seufferheld, C. Gohlke, Govindjee, and R. M. Clegg, “Fluorescence lifetime imaging in real-time - a new technique in photosynthesis research,” Photosynthetica 38(4), 581–599 (2000).
[CrossRef]

Gordon, G.

A. Periasamy, P. Wodnicki, X. Wang, S. Kwon, G. Gordon, and B. Herman, “Time-resolved fluorescence lifetime imaging microscopy using a picosecond pulsed tunable dye laser system,” Rev. Sci. Instrum. 67(10), 3722–3731 (1996).
[CrossRef]

Govindjee,

O. Holub, M. J. Seufferheld, C. Gohlke, Govindjee, and R. M. Clegg, “Fluorescence lifetime imaging in real-time - a new technique in photosynthesis research,” Photosynthetica 38(4), 581–599 (2000).
[CrossRef]

Gratton, E.

M. A. Digman, V. R. Caiolfa, M. Zamai, and E. Gratton, “The phasor approach to fluorescence lifetime imaging analysis,” Biophys. J. 94(2), 14–16 (2008).
[CrossRef] [PubMed]

Gu, Y.

S. E. D. Webb, Y. Gu, S. Leveque-Fort, J. Siegel, M. J. Cole, K. Dowling, R. Jones, P. M. W. French, M. A. A. Neil, R. Juskaitis, L. O. D. Sucharov, T. Wilson, and M. J. Lever, “A wide-field time-domain fluorescence lifetime imaging microscope with optical sectioning,” Rev. Sci. Instrum. 73(4), 1898–1907 (2002).
[CrossRef]

Hanley, Q. S.

Q. S. Hanley and A. H. A. Clayton, “AB-plot assisted determination of fluorophore mixtures in a fluorescence lifetime microscope using spectra or quenchers,” J. Microsc. 218(1), 62–67 (2005).
[CrossRef] [PubMed]

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

Hares, 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. Kellet, A. Dymoke-Bradshaw, J. Hares, and P. M. W. French, “Real-time time-domain fluorescence lifetime imaging including single-shot acquisition with a segmented optical image intensifier,” N. J. Phys. 6, 180–192 (2004).
[CrossRef]

Herman, B.

A. Periasamy, P. Wodnicki, X. Wang, S. Kwon, G. Gordon, and B. Herman, “Time-resolved fluorescence lifetime imaging microscopy using a picosecond pulsed tunable dye laser system,” Rev. Sci. Instrum. 67(10), 3722–3731 (1996).
[CrossRef]

Hickl, H.

W. Becker, H. Hickl, C. Zander, K. H. Drexhage, S. S. M. Sauer, and J. Wolfrum, “Time-resolved detection and identification of single analyte molecules in microcapillaries by timecorrelated single-photon counting (TCSPC),” Rev. Sci. Instrum. 70(3), 1835–1841 (1999).
[CrossRef]

Hofmann, O.

Holub, O.

O. Holub, M. J. Seufferheld, C. Gohlke, Govindjee, and R. M. Clegg, “Fluorescence lifetime imaging in real-time - a new technique in photosynthesis research,” Photosynthetica 38(4), 581–599 (2000).
[CrossRef]

Hult, J.

C. F. Kaminski, R. S. Watt, A. D. Elder, J. H. Frank, and J. Hult, “Supercontinuum radiation for applications in chemical sensing and microscopy,” Appl. Phys. B 92(3), 367–378 (2008).
[CrossRef]

Jeyasekharan, A. D.

J. H. Frank, A. D. Elder, J. Swartling, A. R. Venkitaraman, A. D. Jeyasekharan, and C. F. Kaminski, “A white light confocal microscope for spectrally resolved multidimensional imaging,” J. Microsc. 227(3), 203–215 (2007).
[CrossRef] [PubMed]

Johnson, M.

J. R. Lakowicz, H. Szmacinski, K. Nowaczyk, K. W. Berndt, and M. Johnson, “Fluorescence lifetime imaging,” Anal. Biochem. 202(2), 316–330 (1992).
[CrossRef] [PubMed]

Jones, R.

S. E. D. Webb, Y. Gu, S. Leveque-Fort, J. Siegel, M. J. Cole, K. Dowling, R. Jones, P. M. W. French, M. A. A. Neil, R. Juskaitis, L. O. D. Sucharov, T. Wilson, and M. J. Lever, “A wide-field time-domain fluorescence lifetime imaging microscope with optical sectioning,” Rev. Sci. Instrum. 73(4), 1898–1907 (2002).
[CrossRef]

Jovin, T. M.

Q. S. Hanley, V. Subramaniam, D. J. Arndt-Jovin, and 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, and R. M. Clegg, “Fluorescence lifetime imaging microscopy (FLIM) - spatial resolution of microstructures on the nanosecond time-scale,” Biophys. Chem. 48(2), 221–239 (1993).
[CrossRef]

Juskaitis, R.

S. E. D. Webb, Y. Gu, S. Leveque-Fort, J. Siegel, M. J. Cole, K. Dowling, R. Jones, P. M. W. French, M. A. A. Neil, R. Juskaitis, L. O. D. Sucharov, T. Wilson, and M. J. Lever, “A wide-field time-domain fluorescence lifetime imaging microscope with optical sectioning,” Rev. Sci. Instrum. 73(4), 1898–1907 (2002).
[CrossRef]

Kaminski, C. F.

S. Schlachter, A. D. Elder, A. Esposito, G. S. Kaminski, J. H. Frank, L. K. van Geest, and C. F. Kaminski, “mhFLIM: resolution of heterogeneous fluorescence decays in widefield lifetime microscopy,” Opt. Express 17(3), 1557–1570 (2009).
[CrossRef] [PubMed]

A. D. Elder, A. Domin, G. S. Kaminski-Schierle, C. Lindon, J. Pines, A. Esposito, and C. F. Kaminski, “A quantitative protocol for dynamic measurements of protein interactions by FRET-sensitized fluorescence emission,” J. R. Soc. Interface 6, S59–S81 (2009).
[CrossRef]

A. Esposito, T. Tiffert, J. M. A. Mauritz, S. Schlachter, L. H. Bannister, C. F. Kaminski, V. L. Lew, and J. M. Schnur, “FRET imaging of hemoglobin concentration in Plasmodium falciparum-infected red cells,” PLoS ONE 3(11), e3780 (2008).
[CrossRef] [PubMed]

C. F. Kaminski, R. S. Watt, A. D. Elder, J. H. Frank, and J. Hult, “Supercontinuum radiation for applications in chemical sensing and microscopy,” Appl. Phys. B 92(3), 367–378 (2008).
[CrossRef]

A. D. Elder, S. Schlachter, and 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]

J. H. Frank, A. D. Elder, J. Swartling, A. R. Venkitaraman, A. D. Jeyasekharan, and C. F. Kaminski, “A white light confocal microscope for spectrally resolved multidimensional imaging,” J. Microsc. 227(3), 203–215 (2007).
[CrossRef] [PubMed]

S. M. Matthews, A. D. Elder, K. Yunus, C. F. Kaminski, C. M. Brennan, and A. C. Fisher, “Quantitative kinetic analysis in a microfluidic device using frequency-domain fluorescence lifetime imaging,” Anal. Chem. 79(11), 4101–4109 (2007).
[CrossRef] [PubMed]

X. W. Dai, M. E. Eccleston, Z. L. Yue, N. K. H. Slater, and C. F. Kaminski, “A spectroscopic study of the self-association and inter-molecular aggregation behaviour of pH-responsive poly(L-lysine iso-phthalamide),” Polymer (Guildf.) 47(8), 2689–2698 (2006).
[CrossRef]

A. D. Elder, J. H. Frank, J. Swartling, X. Dai, and C. F. Kaminski, “Calibration of a wide-field frequency-domain fluorescence lifetime microscopy system using light emitting diodes as light sources,” J. Microsc. 224(2), 166–180 (2006).
[CrossRef] [PubMed]

Kaminski, G. S.

Kaminski-Schierle, G. S.

A. D. Elder, A. Domin, G. S. Kaminski-Schierle, C. Lindon, J. Pines, A. Esposito, and C. F. Kaminski, “A quantitative protocol for dynamic measurements of protein interactions by FRET-sensitized fluorescence emission,” J. R. Soc. Interface 6, S59–S81 (2009).
[CrossRef]

Kellet, 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. Kellet, A. Dymoke-Bradshaw, J. Hares, and P. M. W. French, “Real-time time-domain fluorescence lifetime imaging including single-shot acquisition with a segmented optical image intensifier,” N. J. Phys. 6, 180–192 (2004).
[CrossRef]

Kremers, G. J.

G. J. Kremers, E. B. van Munster, J. Goedhart, and T. W. J. Gadella., “Quantitative lifetime unmixing of multiexponentially decaying fluorophores using single-frequency fluorescence lifetime imaging microscopy,” Biophys. J. 95(1), 378–389 (2008).
[CrossRef] [PubMed]

Kwon, S.

A. Periasamy, P. Wodnicki, X. Wang, S. Kwon, G. Gordon, and B. Herman, “Time-resolved fluorescence lifetime imaging microscopy using a picosecond pulsed tunable dye laser system,” Rev. Sci. Instrum. 67(10), 3722–3731 (1996).
[CrossRef]

Lakowicz, J. R.

J. R. Lakowicz, H. Szmacinski, K. Nowaczyk, K. W. Berndt, and M. Johnson, “Fluorescence lifetime imaging,” Anal. Biochem. 202(2), 316–330 (1992).
[CrossRef] [PubMed]

J. R. Lakowicz and K. W. Berndt, “Lifetime-selective fluorescence imaging using an rf phase-sensitive camera,” Rev. Sci. Instrum. 62(7), 1727–1734 (1991).
[CrossRef]

Lanigan, P. M. P.

C. Dunsby, P. M. P. Lanigan, J. McGinty, D. S. Elson, J. Requejo-Isidro, I. Munro, N. Galletly, F. McCann, B. Treanor, B. Onfelt, D. M. Davis, M. A. A. Neil, and P. M. W. French, “An electronically tunable ultrafast laser source applied to fluorescence imaging and fluorescence lifetime imaging microscopy,” J. Phys. D Appl. Phys. 37(23), 3296–3303 (2004).
[CrossRef]

Leveque-Fort, S.

S. E. D. Webb, Y. Gu, S. Leveque-Fort, J. Siegel, M. J. Cole, K. Dowling, R. Jones, P. M. W. French, M. A. A. Neil, R. Juskaitis, L. O. D. Sucharov, T. Wilson, and M. J. Lever, “A wide-field time-domain fluorescence lifetime imaging microscope with optical sectioning,” Rev. Sci. Instrum. 73(4), 1898–1907 (2002).
[CrossRef]

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. Kellet, A. Dymoke-Bradshaw, J. Hares, and P. M. W. French, “Real-time time-domain fluorescence lifetime imaging including single-shot acquisition with a segmented optical image intensifier,” N. J. Phys. 6, 180–192 (2004).
[CrossRef]

S. E. D. Webb, Y. Gu, S. Leveque-Fort, J. Siegel, M. J. Cole, K. Dowling, R. Jones, P. M. W. French, M. A. A. Neil, R. Juskaitis, L. O. D. Sucharov, T. Wilson, and M. J. Lever, “A wide-field time-domain fluorescence lifetime imaging microscope with optical sectioning,” Rev. Sci. Instrum. 73(4), 1898–1907 (2002).
[CrossRef]

Lew, V. L.

A. Esposito, T. Tiffert, J. M. A. Mauritz, S. Schlachter, L. H. Bannister, C. F. Kaminski, V. L. Lew, and J. M. Schnur, “FRET imaging of hemoglobin concentration in Plasmodium falciparum-infected red cells,” PLoS ONE 3(11), e3780 (2008).
[CrossRef] [PubMed]

Lindon, C.

A. D. Elder, A. Domin, G. S. Kaminski-Schierle, C. Lindon, J. Pines, A. Esposito, and C. F. Kaminski, “A quantitative protocol for dynamic measurements of protein interactions by FRET-sensitized fluorescence emission,” J. R. Soc. Interface 6, S59–S81 (2009).
[CrossRef]

Matthews, S. M.

S. M. Matthews, A. D. Elder, K. Yunus, C. F. Kaminski, C. M. Brennan, and A. C. Fisher, “Quantitative kinetic analysis in a microfluidic device using frequency-domain fluorescence lifetime imaging,” Anal. Chem. 79(11), 4101–4109 (2007).
[CrossRef] [PubMed]

Mauritz, J. M. A.

A. Esposito, T. Tiffert, J. M. A. Mauritz, S. Schlachter, L. H. Bannister, C. F. Kaminski, V. L. Lew, and J. M. Schnur, “FRET imaging of hemoglobin concentration in Plasmodium falciparum-infected red cells,” PLoS ONE 3(11), e3780 (2008).
[CrossRef] [PubMed]

McCann, F.

C. Dunsby, P. M. P. Lanigan, J. McGinty, D. S. Elson, J. Requejo-Isidro, I. Munro, N. Galletly, F. McCann, B. Treanor, B. Onfelt, D. M. Davis, M. A. A. Neil, and P. M. W. French, “An electronically tunable ultrafast laser source applied to fluorescence imaging and fluorescence lifetime imaging microscopy,” J. Phys. D Appl. Phys. 37(23), 3296–3303 (2004).
[CrossRef]

McGinty, J.

R. K. P. Benninger, O. Hofmann, J. McGinty, J. Requejo-Isidro, I. Munro, M. A. A. Neil, A. J. Demello, and P. M. W. French, “Time-resolved fluorescence imaging of solvent interactions in microfluidic devices,” Opt. Express 13(16), 6275–6285 (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. Kellet, A. Dymoke-Bradshaw, J. Hares, and P. M. W. French, “Real-time time-domain fluorescence lifetime imaging including single-shot acquisition with a segmented optical image intensifier,” N. J. Phys. 6, 180–192 (2004).
[CrossRef]

C. Dunsby, P. M. P. Lanigan, J. McGinty, D. S. Elson, J. Requejo-Isidro, I. Munro, N. Galletly, F. McCann, B. Treanor, B. Onfelt, D. M. Davis, M. A. A. Neil, and P. M. W. French, “An electronically tunable ultrafast laser source applied to fluorescence imaging and fluorescence lifetime imaging microscopy,” J. Phys. D Appl. Phys. 37(23), 3296–3303 (2004).
[CrossRef]

Munro, I.

R. K. P. Benninger, O. Hofmann, J. McGinty, J. Requejo-Isidro, I. Munro, M. A. A. Neil, A. J. Demello, and P. M. W. French, “Time-resolved fluorescence imaging of solvent interactions in microfluidic devices,” Opt. Express 13(16), 6275–6285 (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. Kellet, A. Dymoke-Bradshaw, J. Hares, and P. M. W. French, “Real-time time-domain fluorescence lifetime imaging including single-shot acquisition with a segmented optical image intensifier,” N. J. Phys. 6, 180–192 (2004).
[CrossRef]

C. Dunsby, P. M. P. Lanigan, J. McGinty, D. S. Elson, J. Requejo-Isidro, I. Munro, N. Galletly, F. McCann, B. Treanor, B. Onfelt, D. M. Davis, M. A. A. Neil, and P. M. W. French, “An electronically tunable ultrafast laser source applied to fluorescence imaging and fluorescence lifetime imaging microscopy,” J. Phys. D Appl. Phys. 37(23), 3296–3303 (2004).
[CrossRef]

Neil, M. A. A.

R. K. P. Benninger, O. Hofmann, J. McGinty, J. Requejo-Isidro, I. Munro, M. A. A. Neil, A. J. Demello, and P. M. W. French, “Time-resolved fluorescence imaging of solvent interactions in microfluidic devices,” Opt. Express 13(16), 6275–6285 (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. Kellet, A. Dymoke-Bradshaw, J. Hares, and P. M. W. French, “Real-time time-domain fluorescence lifetime imaging including single-shot acquisition with a segmented optical image intensifier,” N. J. Phys. 6, 180–192 (2004).
[CrossRef]

C. Dunsby, P. M. P. Lanigan, J. McGinty, D. S. Elson, J. Requejo-Isidro, I. Munro, N. Galletly, F. McCann, B. Treanor, B. Onfelt, D. M. Davis, M. A. A. Neil, and P. M. W. French, “An electronically tunable ultrafast laser source applied to fluorescence imaging and fluorescence lifetime imaging microscopy,” J. Phys. D Appl. Phys. 37(23), 3296–3303 (2004).
[CrossRef]

S. E. D. Webb, Y. Gu, S. Leveque-Fort, J. Siegel, M. J. Cole, K. Dowling, R. Jones, P. M. W. French, M. A. A. Neil, R. Juskaitis, L. O. D. Sucharov, T. Wilson, and M. J. Lever, “A wide-field time-domain fluorescence lifetime imaging microscope with optical sectioning,” Rev. Sci. Instrum. 73(4), 1898–1907 (2002).
[CrossRef]

Nowaczyk, K.

J. R. Lakowicz, H. Szmacinski, K. Nowaczyk, K. W. Berndt, and M. Johnson, “Fluorescence lifetime imaging,” Anal. Biochem. 202(2), 316–330 (1992).
[CrossRef] [PubMed]

Onfelt, B.

C. Dunsby, P. M. P. Lanigan, J. McGinty, D. S. Elson, J. Requejo-Isidro, I. Munro, N. Galletly, F. McCann, B. Treanor, B. Onfelt, D. M. Davis, M. A. A. Neil, and P. M. W. French, “An electronically tunable ultrafast laser source applied to fluorescence imaging and fluorescence lifetime imaging microscopy,” J. Phys. D Appl. Phys. 37(23), 3296–3303 (2004).
[CrossRef]

Periasamy, A.

K. K. Sharman, A. Periasamy, H. Ashworth, J. N. Demas, and N. H. Snow, “Error analysis of the rapid lifetime determination method for double-exponential decays and new windowing schemes,” Anal. Chem. 71(5), 947–952 (1999).
[CrossRef] [PubMed]

A. Periasamy, P. Wodnicki, X. Wang, S. Kwon, G. Gordon, and B. Herman, “Time-resolved fluorescence lifetime imaging microscopy using a picosecond pulsed tunable dye laser system,” Rev. Sci. Instrum. 67(10), 3722–3731 (1996).
[CrossRef]

Philip, J.

Pines, J.

A. D. Elder, A. Domin, G. S. Kaminski-Schierle, C. Lindon, J. Pines, A. Esposito, and C. F. Kaminski, “A quantitative protocol for dynamic measurements of protein interactions by FRET-sensitized fluorescence emission,” J. R. Soc. Interface 6, S59–S81 (2009).
[CrossRef]

Redford, G. I.

G. I. Redford and R. M. Clegg, “Polar plot representation for frequency-domain analysis of fluorescence lifetimes,” J. Fluoresc. 15(5), 805–815 (2005).
[CrossRef] [PubMed]

Requejo-Isidro, J.

R. K. P. Benninger, O. Hofmann, J. McGinty, J. Requejo-Isidro, I. Munro, M. A. A. Neil, A. J. Demello, and P. M. W. French, “Time-resolved fluorescence imaging of solvent interactions in microfluidic devices,” Opt. Express 13(16), 6275–6285 (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. Kellet, A. Dymoke-Bradshaw, J. Hares, and P. M. W. French, “Real-time time-domain fluorescence lifetime imaging including single-shot acquisition with a segmented optical image intensifier,” N. J. Phys. 6, 180–192 (2004).
[CrossRef]

C. Dunsby, P. M. P. Lanigan, J. McGinty, D. S. Elson, J. Requejo-Isidro, I. Munro, N. Galletly, F. McCann, B. Treanor, B. Onfelt, D. M. Davis, M. A. A. Neil, and P. M. W. French, “An electronically tunable ultrafast laser source applied to fluorescence imaging and fluorescence lifetime imaging microscopy,” J. Phys. D Appl. Phys. 37(23), 3296–3303 (2004).
[CrossRef]

Sauer, S. S. M.

W. Becker, H. Hickl, C. Zander, K. H. Drexhage, S. S. M. Sauer, and J. Wolfrum, “Time-resolved detection and identification of single analyte molecules in microcapillaries by timecorrelated single-photon counting (TCSPC),” Rev. Sci. Instrum. 70(3), 1835–1841 (1999).
[CrossRef]

Schlachter, S.

Schneider, P.

P. Schneider and R. M. Clegg, “Rapid acquisition, analysis, and display of fuorescence lifetime-resolved images for real-time applications,” Rev. Sci. Instrum. 68(11), 4107 (1997).
[CrossRef]

Schnur, J. M.

A. Esposito, T. Tiffert, J. M. A. Mauritz, S. Schlachter, L. H. Bannister, C. F. Kaminski, V. L. Lew, and J. M. Schnur, “FRET imaging of hemoglobin concentration in Plasmodium falciparum-infected red cells,” PLoS ONE 3(11), e3780 (2008).
[CrossRef] [PubMed]

Settersten, T. B.

T. B. Settersten, A. Dreizler, and R. L. Farrow, “Temperature- and species-dependent quenching of CO B probed by two-photon laser-induced fluorescence using a picosecond laser,” J. Chem. Phys. 117(7), 3173–3179 (2002).
[CrossRef]

Seufferheld, M. J.

O. Holub, M. J. Seufferheld, C. Gohlke, Govindjee, and R. M. Clegg, “Fluorescence lifetime imaging in real-time - a new technique in photosynthesis research,” Photosynthetica 38(4), 581–599 (2000).
[CrossRef]

Sharman, K. K.

K. K. Sharman, A. Periasamy, H. Ashworth, J. N. Demas, and N. H. Snow, “Error analysis of the rapid lifetime determination method for double-exponential decays and new windowing schemes,” Anal. Chem. 71(5), 947–952 (1999).
[CrossRef] [PubMed]

Siegel, J.

S. E. D. Webb, Y. Gu, S. Leveque-Fort, J. Siegel, M. J. Cole, K. Dowling, R. Jones, P. M. W. French, M. A. A. Neil, R. Juskaitis, L. O. D. Sucharov, T. Wilson, and M. J. Lever, “A wide-field time-domain fluorescence lifetime imaging microscope with optical sectioning,” Rev. Sci. Instrum. 73(4), 1898–1907 (2002).
[CrossRef]

Slater, N. K. H.

X. W. Dai, M. E. Eccleston, Z. L. Yue, N. K. H. Slater, and C. F. Kaminski, “A spectroscopic study of the self-association and inter-molecular aggregation behaviour of pH-responsive poly(L-lysine iso-phthalamide),” Polymer (Guildf.) 47(8), 2689–2698 (2006).
[CrossRef]

Snow, N. H.

K. K. Sharman, A. Periasamy, H. Ashworth, J. N. Demas, and N. H. Snow, “Error analysis of the rapid lifetime determination method for double-exponential decays and new windowing schemes,” Anal. Chem. 71(5), 947–952 (1999).
[CrossRef] [PubMed]

Squire, A.

A. Squire, P. J. Verveer, and P. I. H. Bastiaens, “Multiple frequency fluorescence lifetime imaging microscopy,” J. Microsc. 197(2), 136–149 (2000).
[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. Kellet, A. Dymoke-Bradshaw, J. Hares, and P. M. W. French, “Real-time time-domain fluorescence lifetime imaging including single-shot acquisition with a segmented optical image intensifier,” N. J. Phys. 6, 180–192 (2004).
[CrossRef]

Subramaniam, V.

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

Sucharov, L. O. D.

S. E. D. Webb, Y. Gu, S. Leveque-Fort, J. Siegel, M. J. Cole, K. Dowling, R. Jones, P. M. W. French, M. A. A. Neil, R. Juskaitis, L. O. D. Sucharov, T. Wilson, and M. J. Lever, “A wide-field time-domain fluorescence lifetime imaging microscope with optical sectioning,” Rev. Sci. Instrum. 73(4), 1898–1907 (2002).
[CrossRef]

Swartling, J.

J. H. Frank, A. D. Elder, J. Swartling, A. R. Venkitaraman, A. D. Jeyasekharan, and C. F. Kaminski, “A white light confocal microscope for spectrally resolved multidimensional imaging,” J. Microsc. 227(3), 203–215 (2007).
[CrossRef] [PubMed]

A. D. Elder, J. H. Frank, J. Swartling, X. Dai, and C. F. Kaminski, “Calibration of a wide-field frequency-domain fluorescence lifetime microscopy system using light emitting diodes as light sources,” J. Microsc. 224(2), 166–180 (2006).
[CrossRef] [PubMed]

Szmacinski, H.

J. R. Lakowicz, H. Szmacinski, K. Nowaczyk, K. W. Berndt, and M. Johnson, “Fluorescence lifetime imaging,” Anal. Biochem. 202(2), 316–330 (1992).
[CrossRef] [PubMed]

Tiffert, T.

A. Esposito, T. Tiffert, J. M. A. Mauritz, S. Schlachter, L. H. Bannister, C. F. Kaminski, V. L. Lew, and J. M. Schnur, “FRET imaging of hemoglobin concentration in Plasmodium falciparum-infected red cells,” PLoS ONE 3(11), e3780 (2008).
[CrossRef] [PubMed]

Treanor, B.

C. Dunsby, P. M. P. Lanigan, J. McGinty, D. S. Elson, J. Requejo-Isidro, I. Munro, N. Galletly, F. McCann, B. Treanor, B. Onfelt, D. M. Davis, M. A. A. Neil, and P. M. W. French, “An electronically tunable ultrafast laser source applied to fluorescence imaging and fluorescence lifetime imaging microscopy,” J. Phys. D Appl. Phys. 37(23), 3296–3303 (2004).
[CrossRef]

van Geest, L. K.

van Munster, E. B.

G. J. Kremers, E. B. van Munster, J. Goedhart, and T. W. J. Gadella., “Quantitative lifetime unmixing of multiexponentially decaying fluorophores using single-frequency fluorescence lifetime imaging microscopy,” Biophys. J. 95(1), 378–389 (2008).
[CrossRef] [PubMed]

E. B. Van Munster and T. W. J. Gadella., “phiFLIM: a new method to avoid aliasing in frequency-domain fluorescence lifetime imaging microscopy,” J. Microsc. 213(1), 29–38 (2004).
[CrossRef] [PubMed]

Venkitaraman, A. R.

J. H. Frank, A. D. Elder, J. Swartling, A. R. Venkitaraman, A. D. Jeyasekharan, and C. F. Kaminski, “A white light confocal microscope for spectrally resolved multidimensional imaging,” J. Microsc. 227(3), 203–215 (2007).
[CrossRef] [PubMed]

Verveer, P. J.

A. Squire, P. J. Verveer, and P. I. H. Bastiaens, “Multiple frequency fluorescence lifetime imaging microscopy,” J. Microsc. 197(2), 136–149 (2000).
[CrossRef] [PubMed]

Wang, X.

A. Periasamy, P. Wodnicki, X. Wang, S. Kwon, G. Gordon, and B. Herman, “Time-resolved fluorescence lifetime imaging microscopy using a picosecond pulsed tunable dye laser system,” Rev. Sci. Instrum. 67(10), 3722–3731 (1996).
[CrossRef]

Watt, R. S.

C. F. Kaminski, R. S. Watt, A. D. Elder, J. H. Frank, and J. Hult, “Supercontinuum radiation for applications in chemical sensing and microscopy,” Appl. Phys. B 92(3), 367–378 (2008).
[CrossRef]

Webb, S. E. D.

S. E. D. Webb, Y. Gu, S. Leveque-Fort, J. Siegel, M. J. Cole, K. Dowling, R. Jones, P. M. W. French, M. A. A. Neil, R. Juskaitis, L. O. D. Sucharov, T. Wilson, and M. J. Lever, “A wide-field time-domain fluorescence lifetime imaging microscope with optical sectioning,” Rev. Sci. Instrum. 73(4), 1898–1907 (2002).
[CrossRef]

Wilson, T.

S. E. D. Webb, Y. Gu, S. Leveque-Fort, J. Siegel, M. J. Cole, K. Dowling, R. Jones, P. M. W. French, M. A. A. Neil, R. Juskaitis, L. O. D. Sucharov, T. Wilson, and M. J. Lever, “A wide-field time-domain fluorescence lifetime imaging microscope with optical sectioning,” Rev. Sci. Instrum. 73(4), 1898–1907 (2002).
[CrossRef]

Wodnicki, P.

A. Periasamy, P. Wodnicki, X. Wang, S. Kwon, G. Gordon, and B. Herman, “Time-resolved fluorescence lifetime imaging microscopy using a picosecond pulsed tunable dye laser system,” Rev. Sci. Instrum. 67(10), 3722–3731 (1996).
[CrossRef]

Wolfrum, J.

W. Becker, H. Hickl, C. Zander, K. H. Drexhage, S. S. M. Sauer, and J. Wolfrum, “Time-resolved detection and identification of single analyte molecules in microcapillaries by timecorrelated single-photon counting (TCSPC),” Rev. Sci. Instrum. 70(3), 1835–1841 (1999).
[CrossRef]

Yue, Z. L.

X. W. Dai, M. E. Eccleston, Z. L. Yue, N. K. H. Slater, and C. F. Kaminski, “A spectroscopic study of the self-association and inter-molecular aggregation behaviour of pH-responsive poly(L-lysine iso-phthalamide),” Polymer (Guildf.) 47(8), 2689–2698 (2006).
[CrossRef]

Yunus, K.

S. M. Matthews, A. D. Elder, K. Yunus, C. F. Kaminski, C. M. Brennan, and A. C. Fisher, “Quantitative kinetic analysis in a microfluidic device using frequency-domain fluorescence lifetime imaging,” Anal. Chem. 79(11), 4101–4109 (2007).
[CrossRef] [PubMed]

Zamai, M.

M. A. Digman, V. R. Caiolfa, M. Zamai, and E. Gratton, “The phasor approach to fluorescence lifetime imaging analysis,” Biophys. J. 94(2), 14–16 (2008).
[CrossRef] [PubMed]

Zander, C.

W. Becker, H. Hickl, C. Zander, K. H. Drexhage, S. S. M. Sauer, and J. Wolfrum, “Time-resolved detection and identification of single analyte molecules in microcapillaries by timecorrelated single-photon counting (TCSPC),” Rev. Sci. Instrum. 70(3), 1835–1841 (1999).
[CrossRef]

Anal. Biochem. (1)

J. R. Lakowicz, H. Szmacinski, K. Nowaczyk, K. W. Berndt, and M. Johnson, “Fluorescence lifetime imaging,” Anal. Biochem. 202(2), 316–330 (1992).
[CrossRef] [PubMed]

Anal. Chem. (2)

S. M. Matthews, A. D. Elder, K. Yunus, C. F. Kaminski, C. M. Brennan, and A. C. Fisher, “Quantitative kinetic analysis in a microfluidic device using frequency-domain fluorescence lifetime imaging,” Anal. Chem. 79(11), 4101–4109 (2007).
[CrossRef] [PubMed]

K. K. Sharman, A. Periasamy, H. Ashworth, J. N. Demas, and N. H. Snow, “Error analysis of the rapid lifetime determination method for double-exponential decays and new windowing schemes,” Anal. Chem. 71(5), 947–952 (1999).
[CrossRef] [PubMed]

Appl. Phys. B (1)

C. F. Kaminski, R. S. Watt, A. D. Elder, J. H. Frank, and J. Hult, “Supercontinuum radiation for applications in chemical sensing and microscopy,” Appl. Phys. B 92(3), 367–378 (2008).
[CrossRef]

Biophys. Chem. (1)

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

Biophys. J. (2)

M. A. Digman, V. R. Caiolfa, M. Zamai, and E. Gratton, “The phasor approach to fluorescence lifetime imaging analysis,” Biophys. J. 94(2), 14–16 (2008).
[CrossRef] [PubMed]

G. J. Kremers, E. B. van Munster, J. Goedhart, and T. W. J. Gadella., “Quantitative lifetime unmixing of multiexponentially decaying fluorophores using single-frequency fluorescence lifetime imaging microscopy,” Biophys. J. 95(1), 378–389 (2008).
[CrossRef] [PubMed]

Cytometry (1)

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

J. Chem. Phys. (1)

T. B. Settersten, A. Dreizler, and R. L. Farrow, “Temperature- and species-dependent quenching of CO B probed by two-photon laser-induced fluorescence using a picosecond laser,” J. Chem. Phys. 117(7), 3173–3179 (2002).
[CrossRef]

J. Fluoresc. (1)

G. I. Redford and R. M. Clegg, “Polar plot representation for frequency-domain analysis of fluorescence lifetimes,” J. Fluoresc. 15(5), 805–815 (2005).
[CrossRef] [PubMed]

J. Microsc. (5)

E. B. Van Munster and T. W. J. Gadella., “phiFLIM: a new method to avoid aliasing in frequency-domain fluorescence lifetime imaging microscopy,” J. Microsc. 213(1), 29–38 (2004).
[CrossRef] [PubMed]

J. H. Frank, A. D. Elder, J. Swartling, A. R. Venkitaraman, A. D. Jeyasekharan, and C. F. Kaminski, “A white light confocal microscope for spectrally resolved multidimensional imaging,” J. Microsc. 227(3), 203–215 (2007).
[CrossRef] [PubMed]

A. D. Elder, J. H. Frank, J. Swartling, X. Dai, and C. F. Kaminski, “Calibration of a wide-field frequency-domain fluorescence lifetime microscopy system using light emitting diodes as light sources,” J. Microsc. 224(2), 166–180 (2006).
[CrossRef] [PubMed]

A. Squire, P. J. Verveer, and P. I. H. Bastiaens, “Multiple frequency fluorescence lifetime imaging microscopy,” J. Microsc. 197(2), 136–149 (2000).
[CrossRef] [PubMed]

Q. S. Hanley and A. H. A. Clayton, “AB-plot assisted determination of fluorophore mixtures in a fluorescence lifetime microscope using spectra or quenchers,” J. Microsc. 218(1), 62–67 (2005).
[CrossRef] [PubMed]

J. Opt. Soc. Am. A (2)

J. Phys. D Appl. Phys. (1)

C. Dunsby, P. M. P. Lanigan, J. McGinty, D. S. Elson, J. Requejo-Isidro, I. Munro, N. Galletly, F. McCann, B. Treanor, B. Onfelt, D. M. Davis, M. A. A. Neil, and P. M. W. French, “An electronically tunable ultrafast laser source applied to fluorescence imaging and fluorescence lifetime imaging microscopy,” J. Phys. D Appl. Phys. 37(23), 3296–3303 (2004).
[CrossRef]

J. R. Soc. Interface (1)

A. D. Elder, A. Domin, G. S. Kaminski-Schierle, C. Lindon, J. Pines, A. Esposito, and C. F. Kaminski, “A quantitative protocol for dynamic measurements of protein interactions by FRET-sensitized fluorescence emission,” J. R. Soc. Interface 6, S59–S81 (2009).
[CrossRef]

N. J. Phys. (1)

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. Kellet, A. Dymoke-Bradshaw, J. Hares, and P. M. W. French, “Real-time time-domain fluorescence lifetime imaging including single-shot acquisition with a segmented optical image intensifier,” N. J. Phys. 6, 180–192 (2004).
[CrossRef]

Opt. Express (2)

Photosynthetica (1)

O. Holub, M. J. Seufferheld, C. Gohlke, Govindjee, and R. M. Clegg, “Fluorescence lifetime imaging in real-time - a new technique in photosynthesis research,” Photosynthetica 38(4), 581–599 (2000).
[CrossRef]

PLoS ONE (1)

A. Esposito, T. Tiffert, J. M. A. Mauritz, S. Schlachter, L. H. Bannister, C. F. Kaminski, V. L. Lew, and J. M. Schnur, “FRET imaging of hemoglobin concentration in Plasmodium falciparum-infected red cells,” PLoS ONE 3(11), e3780 (2008).
[CrossRef] [PubMed]

Polymer (Guildf.) (1)

X. W. Dai, M. E. Eccleston, Z. L. Yue, N. K. H. Slater, and C. F. Kaminski, “A spectroscopic study of the self-association and inter-molecular aggregation behaviour of pH-responsive poly(L-lysine iso-phthalamide),” Polymer (Guildf.) 47(8), 2689–2698 (2006).
[CrossRef]

Rev. Sci. Instrum. (5)

A. Periasamy, P. Wodnicki, X. Wang, S. Kwon, G. Gordon, and B. Herman, “Time-resolved fluorescence lifetime imaging microscopy using a picosecond pulsed tunable dye laser system,” Rev. Sci. Instrum. 67(10), 3722–3731 (1996).
[CrossRef]

W. Becker, H. Hickl, C. Zander, K. H. Drexhage, S. S. M. Sauer, and J. Wolfrum, “Time-resolved detection and identification of single analyte molecules in microcapillaries by timecorrelated single-photon counting (TCSPC),” Rev. Sci. Instrum. 70(3), 1835–1841 (1999).
[CrossRef]

J. R. Lakowicz and K. W. Berndt, “Lifetime-selective fluorescence imaging using an rf phase-sensitive camera,” Rev. Sci. Instrum. 62(7), 1727–1734 (1991).
[CrossRef]

P. Schneider and R. M. Clegg, “Rapid acquisition, analysis, and display of fuorescence lifetime-resolved images for real-time applications,” Rev. Sci. Instrum. 68(11), 4107 (1997).
[CrossRef]

S. E. D. Webb, Y. Gu, S. Leveque-Fort, J. Siegel, M. J. Cole, K. Dowling, R. Jones, P. M. W. French, M. A. A. Neil, R. Juskaitis, L. O. D. Sucharov, T. Wilson, and M. J. Lever, “A wide-field time-domain fluorescence lifetime imaging microscope with optical sectioning,” Rev. Sci. Instrum. 73(4), 1898–1907 (2002).
[CrossRef]

Other (1)

J. R. Lakowicz, Principles of Fluorescence Spectroscopy, 2nd ed. (Plenum, New York, 1999).

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Figures (16)

Fig. 1
Fig. 1

AB-plot demonstrating the relationship between a data point in AB space and the phase and modulation lifetimes.

Fig. 2
Fig. 2

AB-plot showing the possible AB coordinates for a two component mixture.

Fig. 3
Fig. 3

The phase modulation applied to the detector gain waveform for ϕ2FLIM measurements.

Fig. 4
Fig. 4

Experimental configuration of the ϕ2FLIM system. SC450: supercontinuum source; AOTF: acousto-optic tunable filter; AWG: arbitrary waveform generator; ICCD: intensified charge-coupled device camera; DEL150: computer-controlled delay generator.

Fig. 5
Fig. 5

Harmonic content of the intensifier gain waveform used for ϕ2FLIM. The data was obtained by measuring elastic scattering from a piece of ground glass at 72 different phase offsets.

Fig. 6
Fig. 6

Diagram illustrating the calculation of the closest approach mono-exponential lifetime and associated uncertainties. The closest point on the semi-circle to a given AB coordinate lies along a straight line between that coordinate and the point (0.5,0.0), as indicated by the dotted lines. The closest approach lifetime is calculated as the closest point on the semi-circle to the mean AB coordinate. The uncertainties are determined from the intersection of the one standard deviation region and the semi-circle.

Fig. 7
Fig. 7

The measured signals and fitted waveforms for R6G dye in 0.0045 M KI (triangles) and 0.18 M KI (diamonds). These samples correspond to lifetimes of approximately 3.6 ns and 0.7 ns respectively. a) Shows the average data from a 10x10 pixel region for the case of conventional homodyne detection with 5 harmonics fitted, whilst b) shows the same for the ϕ2FLIM technique, with only one harmonic fitted. With the ϕ2FLIM technique a single harmonic produces a good fit to the data, indicating that higher harmonics are not present.

Fig. 8
Fig. 8

AB plots for solutions of R6G dye in water with five different concentrations of KI (0.0045 M, 0.027 M, 0.045 M, 0.09 M and 0.18 M) as measured using ϕ2FLIM.

Fig. 9
Fig. 9

Stern-Volmer plot comparing time-domain and ϕ2FLIM lifetime measurements from a series of KI quenched R6G solutions in water.

Fig. 10
Fig. 10

AB plots from conventional homodyne detection FLIM measurements, showing the mean and one standard deviation confidence region for samples of R6G dye in water with different KI concentrations. a) Two 36-step data sets with the first phase offset at 0 deg and 5 deg. b) Six 12-step data sets with the first phase offset at 0 deg., 5... 25 deg. If no aliasing is present, the results are independent of the choice of the first phase offset.

Fig. 11
Fig. 11

AB plots from ϕ2FLIM measurements, showing the mean values and one standard deviation confidence regions for samples of R6G dye in water with different concentrations of KI, as indicated on the diagrams. a) Six 12-step data sets with the first phase offset at 0 deg., 5...25 deg. b) Twenty-four 3-step data sets with the first phase offset at 0 deg., 5...115 deg.

Fig. 12
Fig. 12

Dependence of fluorescence lifetime on the initial phase offset for 3 phase-step ϕ2FLIM measurements. a) R6G solution with a fluorescence lifetime of 0.71 ns b) R6G solution with a fluorescence lifetime of 3.59 ns. The error bars represent one standard deviation of all pixels in an image.

Fig. 13
Fig. 13

Comparison of the relative noise, σ / τ , in the phase and modulation lifetimes as a function of ω τ for conventional homodyne detection and ϕ2FLIM measurements. The noise is calculated using the full 72 phase-step data sets.

Fig. 14
Fig. 14

a) Normalized fluorescence excitation spectra of Rhodamine B (dashed line) and Rhodamine 6G (dotted line). The expected fractional contribution of Rhodamine B to the fluorescence signal (solid line) based on the excitation spectra is plotted as a function of the excitation wavelength. The fractional contribution of RB measured with ϕ2FLIM is plotted for wavelengths between 525 nm and 550 nm (diamonds). b) Contour plot of the normalized distributions of AB coordinates for a mixture of 0.5 μM Rhodamine B and 0.5 μM Rhodamine 6G excited at a series of wavelengths from 525 to 550 nm in 5 nm increments. The mean AB position for each excitation wavelength is indicated by a cross. The curved solid line is the semi-circle for mono-exponential decays, and the straight solid line is the mixing line for Rhodamine B and Rhodamine 6G.

Fig. 15
Fig. 15

a) AB-plot for samples of pure R6G and pure RB in glycerol-water solutions. The mixing line indicates the range of AB coordinates for all possible mixtures of the R6G and RB solutions. The curve is the mono-exponential decay semi-circle. b) Distribution of AB coordinates from a ϕ2FLIM image of a droplet of RB solution mixing with a bath of R6G solution, c) Image showing measured fraction of RB dye solution.

Fig. 16
Fig. 16

Sequences of ϕ2FLIM images show drops of RB in a glycerol/water solution mixing into a bath of R6G in glycerol/water. Sample frames are extracted from ϕ2FLIM measurements acquired at 5.5 Hz. Frames (a)-(c) show the fraction of RB solution at three times separated by 1.45 s. Frames (d)-(f) show the fraction of RB solution for a second drop with 2.18 s between frames. For each frame, the corresponding AB-plot shows the distribution of AB-coordinates along the mixing line.

Equations (19)

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y i = a 1 + a 2 cos ( ϕ i g a 3 )
θ 1 = 1 N i = 1 N y i
θ 2 = 2 N i = 1 N y i cos ( ϕ i g )
θ 3 = 2 N i = 1 N y i sin ( ϕ i g )
θ 1 = a 1
θ 2 = a 2 cos ( a 3 )
θ 3 = a 2 sin ( a 3 )
τ ϕ = 1 ω tan ( arc tan ( θ 3 θ 2 ) arc tan ( θ 3 , r e f θ 2 , r e f ) + arc tan ( ω τ r e f ) )
τ m = 1 ω ( ( θ 2 , r e f 2 + θ 3 , r e f 2 θ 1 , r e f 2 ) ( θ 1 2 θ 2 2 + θ 3 2 ) ( 1 + ω 2 τ r e f 2 ) 1 ) 1 2
A = θ 3 θ 1
B = θ 2 θ 1
A = 1 θ 1 ( a 1 , r e f sin ( a 3 , r e f arc tan ( ω τ r e f ) ) a 2 , r e f ( 1 + ω 2 τ r e f 2 ) 0.5 θ 2 +     a 1 , r e f cos ( a 3 , r e f arc tan ( ω τ r e f ) ) a 2 , r e f ( 1 + ω 2 τ r e f 2 ) 0.5 θ 3 )
B = 1 θ 1 ( a 1 , r e f cos ( a 3 , r e f arc tan ( ω τ r e f ) ) a 2 , r e f ( 1 + ω 2 τ r e f 2 ) 0.5 θ 2 +     a 1 , r e f sin ( a 3 , r e f arc tan ( ω τ r e f ) ) a 2 , r e f ( 1 + ω 2 τ r e f 2 ) 0.5 θ 3 )
X i = a + j = 1 J b j cos ( j ϕ i g ξ j ) ( 1 + ( j ω τ ) 2 ) 1 2
X i = 0 2 ( a + j = 1 J b j cos ( j ( ϕ i g + arc cos ( 1 ψ ) ) ξ j ) ( 1 + ( j ω τ ) 2 ) 1 2 ) d ψ
X i = 0 π ( a + j = 1 J b j cos ( j γ + ( j ϕ i g ξ j ) ) ( 1 + ( j ω τ ) 2 ) 1 2 ) sin ( γ ) d γ
X i = 2 a [ ( b 1 π 2 ) ( 1 1 + ( ω τ ) 2 ) 1 2 sin ( ϕ i g ξ 1 ) ] +     e v e n j J [ ( 2 b j j 2 1 ) ( 1 1 + ( j ω τ ) 2 ) 1 2 cos ( j ϕ i g ξ j ) ]
X i = 2 a ( b 1 π 2 ) ( 1 1 + ( ω τ ) 2 ) 1 2 sin ( ϕ i g ξ 1 )
X i = 2 a ( b 1 π 2 ) ( 1 1 + ( ω τ ) 2 ) 1 2 cos ( ϕ i g arc tan ( ω τ ) )

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