Abstract

The signal-to-noise ratio of a measurement is determined by the photon economy of the detection technique and the available photons that are emitted by the sample. We investigate the efficiency of various frequency-domain lifetime detection techniques also in relation to time-domain detection. Nonlinear effects are discussed that are introduced by the use of image intensifiers and by fluorophore saturation. The efficiency of fluorescence lifetime imaging microscopy setups is connected to the speed of acquisition and thus to the imaging throughput. We report on the optimal conditions for balancing signal-to-noise ratio and acquisition speed in fluorescence lifetime sensing.

© 2007 Optical Society of America

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    [CrossRef]
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    [CrossRef]
  3. M. Kollner and J. Wolfrum, "How many photons are necessary for fluorescence-lifetime measurements," Chem. Phys. Lett. 200, 199-204 (1992).
    [CrossRef]
  4. J. Sytsma, J. M. Vroom, C. J. de Grauw, and H. C. Gerritsen, "Time-gated fluorescence lifetime imaging and microvolume spectroscopy using two-photon excitation," J. Microsc. 191, 39-51 (1998).
    [CrossRef]
  5. S. P. Chan, Z. J. Fuller, J. N. Demas, and B. A. DeGraff, "Optimized gating scheme for rapid lifetime determinations of single-exponential luminescence lifetimes," Anal. Chem. 73, 4486-4490 (2001).
    [CrossRef] [PubMed]
  6. C. J. de Grauw and H. C. Gerritsen, "Multiple time-gate module for fluorescence lifetime imaging," Appl. Spectrosc. 55, 670-678 (2001).
    [CrossRef]
  7. K. Carlsson and J. P. Philip, "Theoretical investigation of the signal-to-noise ratio for different fluorescence lifetime imaging techniques," Proc. SPIE 4622, 70-74 (2002).
    [CrossRef]
  8. J. Philip and K. Carlsson, "Theoretical investigation of the signal-to-noise ratio in fluorescence lifetime imaging," J. Opt. Soc. Am. A 20, 368-379 (2003).
    [CrossRef]
  9. H. C. Gerritsen, M. A. Asselbergs, A. V. Agronskaia, and W. G. Van Sark, "Fluorescence lifetime imaging in scanning microscopes: acquisition speed, photon economy and lifetime resolution," J. Microsc. 206, 218-224 (2002).
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    [CrossRef]
  12. R. M. Clegg and P. C. Schneider, "Fluorescence lifetime-resolved imaging microscopy: a general description of lifetime-resolved imaging measurements," in Fluorescence Microscopy and Fluorescent Probes, J.Slavik, ed. (Plenum, 1996).
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  14. D. S. Elson, I. Munro, J. Requejo-Isidro, J. McGinty, C. Dunsby, N. Galletly, G. W. Stamp, M. A. A. Neil, M. J. Lever, P. A. Kellett, A. Dymoke-Bradshaw, J. Hares, and P. M. W. French, "Real-time time-domain fluorescence lifetime imaging including single-shot acquisition with a segmented optical image intensifier," New J. Phys. 6, 180-193 (2004).
    [CrossRef]
  15. A. Esposito, T. Oggier, H. C. Gerritsen, F. Lustenberger, and F. S. Wouters, "All-solid-state lock-in imaging for wide-field fluorescence lifetime sensing," Opt. Express 13, 9812-9821 (2005).
    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
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    [CrossRef]
  19. A. Esposito, H. C. Gerritsen, and F. S. Wouters, "Fluorescence lifetime heterogeneity resolution in the frequency-domain by lifetime moments analysis (LiMA)," Biophys. J. 89, 4286-4299 (2005).
    [CrossRef] [PubMed]
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    [CrossRef]
  21. J. R. Lakowicz and A. Balter, "Theory of phase-modulation fluorescence spectroscopy for excited-state processes," Biophys. Chem. 16, 99-115 (1982).
    [CrossRef] [PubMed]
  22. J. R. Lakowicz and K. W. Berndt, "Lifetime-selective fluorescence imaging using an rf phase-sensitive camera," Rev. Sci. Instrum. 62, 1727-1734 (1991).
    [CrossRef]
  23. E. B. van Munster and T. W. Gadella, Jr., "phiFLIM: a new method to avoid aliasing in frequency-domain fluorescence lifetime imaging microscopy," J. Microsc. 213, 29-38 (2004).
    [CrossRef]
  24. E. B. van Munster and T. W. Gadella, Jr., "Suppression of photobleaching-induced artifacts in frequency-domain FLIM by permutation of the recording order," Cytometry 58A, 185-194 (2004).
    [CrossRef]
  25. W. Becker, A. Bergmann, G. Biscotti, K. König, I. Riemann, L. Kelbauskas, and C. Biskup, "High-speed FLIM data acquisition by time-correlated single photon counting," Proc. SPIE 5223, 1-9 (2004).
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    [CrossRef]
  28. A. Esposito, H. C. Gerritsen, and F. S. Wouters, "Fluorescence lifetime imaging microscopy: quality assessment and standards" in Standardization in Fluorometry: State of the Art and Future Challenges, U.Resch-Genger, ed. (Springer, to be published).
  29. K. Carlsson and A. Liljeborg, "Simultaneous confocal lifetime imaging of multiple fluorophores using the intensity-modulated multiple-wavelength scanning (IMS) technique," J. Microsc. 191, 119-127 (1998).
    [CrossRef] [PubMed]
  30. M. J. Booth and T. Wilson, "Low-cost, frequency-domain, fluorescence lifetime confocal microscopy," J. Microsc. 214, 36-42 (2004).
    [CrossRef] [PubMed]
  31. K. Kemnitz, L. Pfeifer, and M. R. Ainbund, "Detector for multichannel spectroscopy and fluorescence lifetime imaging on the picosecond timescale," Nucl. Instrum. Methods Phys. Res. Sect. A 387, 86-87 (1997).
    [CrossRef]
  32. J. R. Lakowicz, H. Szmacinski, K. Nowaczyk, K. W. Berndt, and M. Johnson, "Fluorescence lifetime imaging," Anal. Biochem. 202, 316-330 (1992).
    [CrossRef] [PubMed]

2006

A. Esposito, H. C. Gerritsen, F. Lustenberger, T. Oggier, and F. S. Wouters, "Innovating lifetime microscopy: a compact and simple tool for the life sciences, screening and diagnostics," J. Biomed. Opt. 11, 34016-34023 (2006).
[CrossRef] [PubMed]

2005

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

A. Esposito, T. Oggier, H. C. Gerritsen, F. Lustenberger, and F. S. Wouters, "All-solid-state lock-in imaging for wide-field fluorescence lifetime sensing," Opt. Express 13, 9812-9821 (2005).
[CrossRef] [PubMed]

2004

D. S. Elson, I. Munro, J. Requejo-Isidro, J. McGinty, C. Dunsby, N. Galletly, G. W. Stamp, M. A. A. Neil, M. J. Lever, P. A. Kellett, A. Dymoke-Bradshaw, J. Hares, and P. M. W. French, "Real-time time-domain fluorescence lifetime imaging including single-shot acquisition with a segmented optical image intensifier," New J. Phys. 6, 180-193 (2004).
[CrossRef]

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

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

E. B. van Munster and T. W. Gadella, Jr., "Suppression of photobleaching-induced artifacts in frequency-domain FLIM by permutation of the recording order," Cytometry 58A, 185-194 (2004).
[CrossRef]

W. Becker, A. Bergmann, G. Biscotti, K. König, I. Riemann, L. Kelbauskas, and C. Biskup, "High-speed FLIM data acquisition by time-correlated single photon counting," Proc. SPIE 5223, 1-9 (2004).
[CrossRef]

2003

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]

A. V. Agronskaia, L. Tertoolen, and H. C. Gerritsen, "High frame rate fluorescence lifetime imaging," J. Phys. D 36, 1655-1662 (2003).
[CrossRef]

J. Philip and K. Carlsson, "Theoretical investigation of the signal-to-noise ratio in fluorescence lifetime imaging," J. Opt. Soc. Am. A 20, 368-379 (2003).
[CrossRef]

2002

K. Carlsson and J. P. Philip, "Theoretical investigation of the signal-to-noise ratio for different fluorescence lifetime imaging techniques," Proc. SPIE 4622, 70-74 (2002).
[CrossRef]

H. C. Gerritsen, M. A. Asselbergs, A. V. Agronskaia, and W. G. Van Sark, "Fluorescence lifetime imaging in scanning microscopes: acquisition speed, photon economy and lifetime resolution," J. Microsc. 206, 218-224 (2002).
[CrossRef] [PubMed]

2001

S. P. Chan, Z. J. Fuller, J. N. Demas, and B. A. DeGraff, "Optimized gating scheme for rapid lifetime determinations of single-exponential luminescence lifetimes," Anal. Chem. 73, 4486-4490 (2001).
[CrossRef] [PubMed]

C. J. de Grauw and H. C. Gerritsen, "Multiple time-gate module for fluorescence lifetime imaging," Appl. Spectrosc. 55, 670-678 (2001).
[CrossRef]

1998

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

J. Sytsma, J. M. Vroom, C. J. de Grauw, and H. C. Gerritsen, "Time-gated fluorescence lifetime imaging and microvolume spectroscopy using two-photon excitation," J. Microsc. 191, 39-51 (1998).
[CrossRef]

1997

P. C. Schneider and R. M. Clegg, "Rapid acquisition, analysis, and display of fluorescence lifetime-resolved images for real-time applications," Rev. Sci. Instrum. 68, 4107-4119 (1997).
[CrossRef]

K. Kemnitz, L. Pfeifer, and M. R. Ainbund, "Detector for multichannel spectroscopy and fluorescence lifetime imaging on the picosecond timescale," Nucl. Instrum. Methods Phys. Res. Sect. A 387, 86-87 (1997).
[CrossRef]

1994

T. W. Gadella, Jr., R. M. Clegg, and T. M. Jovin, "Fluorescence lifetime imaging microscopy: pixel-by-pixel analysis of phase-modulation data," Bioimaging 2, 139-159 (1994).
[CrossRef]

1993

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

1992

M. Kollner and J. Wolfrum, "How many photons are necessary for fluorescence-lifetime measurements," Chem. Phys. Lett. 200, 199-204 (1992).
[CrossRef]

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

1991

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

R. M. Ballew and J. N. Demas, "Error analysis of the rapid lifetime determination method for single exponential decays with a nonzero base-line," Anal. Chim. Acta 245, 121-127 (1991).
[CrossRef]

1989

R. M. Ballew and J. N. Demas, "An error analysis of the rapid lifetime determination method for the evaluation of single exponential decays," Anal. Chem. 61, 30-33 (1989).
[CrossRef]

1982

J. R. Lakowicz and A. Balter, "Theory of phase-modulation fluorescence spectroscopy for excited-state processes," Biophys. Chem. 16, 99-115 (1982).
[CrossRef] [PubMed]

Agronskaia, A. V.

A. V. Agronskaia, L. Tertoolen, and H. C. Gerritsen, "High frame rate fluorescence lifetime imaging," J. Phys. D 36, 1655-1662 (2003).
[CrossRef]

H. C. Gerritsen, M. A. Asselbergs, A. V. Agronskaia, and W. G. Van Sark, "Fluorescence lifetime imaging in scanning microscopes: acquisition speed, photon economy and lifetime resolution," J. Microsc. 206, 218-224 (2002).
[CrossRef] [PubMed]

H. C. Gerritsen, A. Draaijer, D. J. van den Heuvel, and A. V. Agronskaia, "Fluorescence lifetime imaging in scanning microscopy," in Handbook of Biological Confocal Microscopy, 2nd ed., J.Pawley, ed. (Plenum, 2006), pp. 516-534.
[CrossRef]

Ainbund, M. R.

K. Kemnitz, L. Pfeifer, and M. R. Ainbund, "Detector for multichannel spectroscopy and fluorescence lifetime imaging on the picosecond timescale," Nucl. Instrum. Methods Phys. Res. Sect. A 387, 86-87 (1997).
[CrossRef]

Asselbergs, M. A.

H. C. Gerritsen, M. A. Asselbergs, A. V. Agronskaia, and W. G. Van Sark, "Fluorescence lifetime imaging in scanning microscopes: acquisition speed, photon economy and lifetime resolution," J. Microsc. 206, 218-224 (2002).
[CrossRef] [PubMed]

Ballew, R. M.

R. M. Ballew and J. N. Demas, "Error analysis of the rapid lifetime determination method for single exponential decays with a nonzero base-line," Anal. Chim. Acta 245, 121-127 (1991).
[CrossRef]

R. M. Ballew and J. N. Demas, "An error analysis of the rapid lifetime determination method for the evaluation of single exponential decays," Anal. Chem. 61, 30-33 (1989).
[CrossRef]

Balter, A.

J. R. Lakowicz and A. Balter, "Theory of phase-modulation fluorescence spectroscopy for excited-state processes," Biophys. Chem. 16, 99-115 (1982).
[CrossRef] [PubMed]

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]

Becker, W.

W. Becker, A. Bergmann, G. Biscotti, K. König, I. Riemann, L. Kelbauskas, and C. Biskup, "High-speed FLIM data acquisition by time-correlated single photon counting," Proc. SPIE 5223, 1-9 (2004).
[CrossRef]

Bergmann, A.

W. Becker, A. Bergmann, G. Biscotti, K. König, I. Riemann, L. Kelbauskas, and C. Biskup, "High-speed FLIM data acquisition by time-correlated single photon counting," Proc. SPIE 5223, 1-9 (2004).
[CrossRef]

Berndt, K. W.

J. R. Lakowicz, H. Szmacinski, K. Nowaczyk, K. W. Berndt, and M. Johnson, "Fluorescence lifetime imaging," Anal. Biochem. 202, 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, 1727-1734 (1991).
[CrossRef]

Biscotti, G.

W. Becker, A. Bergmann, G. Biscotti, K. König, I. Riemann, L. Kelbauskas, and C. Biskup, "High-speed FLIM data acquisition by time-correlated single photon counting," Proc. SPIE 5223, 1-9 (2004).
[CrossRef]

Biskup, C.

W. Becker, A. Bergmann, G. Biscotti, K. König, I. Riemann, L. Kelbauskas, and C. Biskup, "High-speed FLIM data acquisition by time-correlated single photon counting," Proc. SPIE 5223, 1-9 (2004).
[CrossRef]

Booth, M. J.

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

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]

Carlsson, K.

J. Philip and K. Carlsson, "Theoretical investigation of the signal-to-noise ratio in fluorescence lifetime imaging," J. Opt. Soc. Am. A 20, 368-379 (2003).
[CrossRef]

K. Carlsson and J. P. Philip, "Theoretical investigation of the signal-to-noise ratio for different fluorescence lifetime imaging techniques," Proc. SPIE 4622, 70-74 (2002).
[CrossRef]

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

Chan, S. P.

S. P. Chan, Z. J. Fuller, J. N. Demas, and B. A. DeGraff, "Optimized gating scheme for rapid lifetime determinations of single-exponential luminescence lifetimes," Anal. Chem. 73, 4486-4490 (2001).
[CrossRef] [PubMed]

Clegg, R. M.

P. C. Schneider and R. M. Clegg, "Rapid acquisition, analysis, and display of fluorescence lifetime-resolved images for real-time applications," Rev. Sci. Instrum. 68, 4107-4119 (1997).
[CrossRef]

T. W. Gadella, Jr., R. M. Clegg, and T. M. Jovin, "Fluorescence lifetime imaging microscopy: pixel-by-pixel analysis of phase-modulation data," Bioimaging 2, 139-159 (1994).
[CrossRef]

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

R. M. Clegg and P. C. Schneider, "Fluorescence lifetime-resolved imaging microscopy: a general description of lifetime-resolved imaging measurements," in Fluorescence Microscopy and Fluorescent Probes, J.Slavik, ed. (Plenum, 1996).

de Grauw, C. J.

C. J. de Grauw and H. C. Gerritsen, "Multiple time-gate module for fluorescence lifetime imaging," Appl. Spectrosc. 55, 670-678 (2001).
[CrossRef]

J. Sytsma, J. M. Vroom, C. J. de Grauw, and H. C. Gerritsen, "Time-gated fluorescence lifetime imaging and microvolume spectroscopy using two-photon excitation," J. Microsc. 191, 39-51 (1998).
[CrossRef]

DeGraff, B. A.

S. P. Chan, Z. J. Fuller, J. N. Demas, and B. A. DeGraff, "Optimized gating scheme for rapid lifetime determinations of single-exponential luminescence lifetimes," Anal. Chem. 73, 4486-4490 (2001).
[CrossRef] [PubMed]

Demas, J. N.

S. P. Chan, Z. J. Fuller, J. N. Demas, and B. A. DeGraff, "Optimized gating scheme for rapid lifetime determinations of single-exponential luminescence lifetimes," Anal. Chem. 73, 4486-4490 (2001).
[CrossRef] [PubMed]

R. M. Ballew and J. N. Demas, "Error analysis of the rapid lifetime determination method for single exponential decays with a nonzero base-line," Anal. Chim. Acta 245, 121-127 (1991).
[CrossRef]

R. M. Ballew and J. N. Demas, "An error analysis of the rapid lifetime determination method for the evaluation of single exponential decays," Anal. Chem. 61, 30-33 (1989).
[CrossRef]

Draaijer, A.

H. C. Gerritsen, A. Draaijer, D. J. van den Heuvel, and A. V. Agronskaia, "Fluorescence lifetime imaging in scanning microscopy," in Handbook of Biological Confocal Microscopy, 2nd ed., J.Pawley, ed. (Plenum, 2006), pp. 516-534.
[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. Kellett, 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," New J. Phys. 6, 180-193 (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. Kellett, 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," New J. Phys. 6, 180-193 (2004).
[CrossRef]

Elson, D. S.

D. S. Elson, I. Munro, J. Requejo-Isidro, J. McGinty, C. Dunsby, N. Galletly, G. W. Stamp, M. A. A. Neil, M. J. Lever, P. A. Kellett, A. Dymoke-Bradshaw, J. Hares, and P. M. W. French, "Real-time time-domain fluorescence lifetime imaging including single-shot acquisition with a segmented optical image intensifier," New J. Phys. 6, 180-193 (2004).
[CrossRef]

Esposito, A.

A. Esposito, H. C. Gerritsen, F. Lustenberger, T. Oggier, and F. S. Wouters, "Innovating lifetime microscopy: a compact and simple tool for the life sciences, screening and diagnostics," J. Biomed. Opt. 11, 34016-34023 (2006).
[CrossRef] [PubMed]

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

A. Esposito, T. Oggier, H. C. Gerritsen, F. Lustenberger, and F. S. Wouters, "All-solid-state lock-in imaging for wide-field fluorescence lifetime sensing," Opt. Express 13, 9812-9821 (2005).
[CrossRef] [PubMed]

A. Esposito, H. C. Gerritsen, and F. S. Wouters, "Fluorescence lifetime imaging microscopy: quality assessment and standards" in Standardization in Fluorometry: State of the Art and Future Challenges, U.Resch-Genger, ed. (Springer, to be published).

French, P. M. W.

D. S. Elson, I. Munro, J. Requejo-Isidro, J. McGinty, C. Dunsby, N. Galletly, G. W. Stamp, M. A. A. Neil, M. J. Lever, P. A. Kellett, A. Dymoke-Bradshaw, J. Hares, and P. M. W. French, "Real-time time-domain fluorescence lifetime imaging including single-shot acquisition with a segmented optical image intensifier," New J. Phys. 6, 180-193 (2004).
[CrossRef]

Fuller, Z. J.

S. P. Chan, Z. J. Fuller, J. N. Demas, and B. A. DeGraff, "Optimized gating scheme for rapid lifetime determinations of single-exponential luminescence lifetimes," Anal. Chem. 73, 4486-4490 (2001).
[CrossRef] [PubMed]

Gadella, T. W.

E. B. van Munster and T. W. Gadella, Jr., "Suppression of photobleaching-induced artifacts in frequency-domain FLIM by permutation of the recording order," Cytometry 58A, 185-194 (2004).
[CrossRef]

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

T. W. Gadella, Jr., R. M. Clegg, and T. M. Jovin, "Fluorescence lifetime imaging microscopy: pixel-by-pixel analysis of phase-modulation data," Bioimaging 2, 139-159 (1994).
[CrossRef]

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

Galletly, N.

D. S. Elson, I. Munro, J. Requejo-Isidro, J. McGinty, C. Dunsby, N. Galletly, G. W. Stamp, M. A. A. Neil, M. J. Lever, P. A. Kellett, A. Dymoke-Bradshaw, J. Hares, and P. M. W. French, "Real-time time-domain fluorescence lifetime imaging including single-shot acquisition with a segmented optical image intensifier," New J. Phys. 6, 180-193 (2004).
[CrossRef]

Gerritsen, H. C.

A. Esposito, H. C. Gerritsen, F. Lustenberger, T. Oggier, and F. S. Wouters, "Innovating lifetime microscopy: a compact and simple tool for the life sciences, screening and diagnostics," J. Biomed. Opt. 11, 34016-34023 (2006).
[CrossRef] [PubMed]

A. Esposito, T. Oggier, H. C. Gerritsen, F. Lustenberger, and F. S. Wouters, "All-solid-state lock-in imaging for wide-field fluorescence lifetime sensing," Opt. Express 13, 9812-9821 (2005).
[CrossRef] [PubMed]

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

A. V. Agronskaia, L. Tertoolen, and H. C. Gerritsen, "High frame rate fluorescence lifetime imaging," J. Phys. D 36, 1655-1662 (2003).
[CrossRef]

H. C. Gerritsen, M. A. Asselbergs, A. V. Agronskaia, and W. G. Van Sark, "Fluorescence lifetime imaging in scanning microscopes: acquisition speed, photon economy and lifetime resolution," J. Microsc. 206, 218-224 (2002).
[CrossRef] [PubMed]

C. J. de Grauw and H. C. Gerritsen, "Multiple time-gate module for fluorescence lifetime imaging," Appl. Spectrosc. 55, 670-678 (2001).
[CrossRef]

J. Sytsma, J. M. Vroom, C. J. de Grauw, and H. C. Gerritsen, "Time-gated fluorescence lifetime imaging and microvolume spectroscopy using two-photon excitation," J. Microsc. 191, 39-51 (1998).
[CrossRef]

A. Esposito, H. C. Gerritsen, and F. S. Wouters, "Fluorescence lifetime imaging microscopy: quality assessment and standards" in Standardization in Fluorometry: State of the Art and Future Challenges, U.Resch-Genger, ed. (Springer, to be published).

H. C. Gerritsen, A. Draaijer, D. J. van den Heuvel, and A. V. Agronskaia, "Fluorescence lifetime imaging in scanning microscopy," in Handbook of Biological Confocal Microscopy, 2nd ed., J.Pawley, ed. (Plenum, 2006), pp. 516-534.
[CrossRef]

Gratton, E.

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]

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. Kellett, 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," New J. Phys. 6, 180-193 (2004).
[CrossRef]

Johnson, M.

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

Jovin, T. M.

T. W. Gadella, Jr., R. M. Clegg, and T. M. Jovin, "Fluorescence lifetime imaging microscopy: pixel-by-pixel analysis of phase-modulation data," Bioimaging 2, 139-159 (1994).
[CrossRef]

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

Kelbauskas, L.

W. Becker, A. Bergmann, G. Biscotti, K. König, I. Riemann, L. Kelbauskas, and C. Biskup, "High-speed FLIM data acquisition by time-correlated single photon counting," Proc. SPIE 5223, 1-9 (2004).
[CrossRef]

Kellett, P. A.

D. S. Elson, I. Munro, J. Requejo-Isidro, J. McGinty, C. Dunsby, N. Galletly, G. W. Stamp, M. A. A. Neil, M. J. Lever, P. A. Kellett, A. Dymoke-Bradshaw, J. Hares, and P. M. W. French, "Real-time time-domain fluorescence lifetime imaging including single-shot acquisition with a segmented optical image intensifier," New J. Phys. 6, 180-193 (2004).
[CrossRef]

Kemnitz, K.

K. Kemnitz, L. Pfeifer, and M. R. Ainbund, "Detector for multichannel spectroscopy and fluorescence lifetime imaging on the picosecond timescale," Nucl. Instrum. Methods Phys. Res. Sect. A 387, 86-87 (1997).
[CrossRef]

Kollner, M.

M. Kollner and J. Wolfrum, "How many photons are necessary for fluorescence-lifetime measurements," Chem. Phys. Lett. 200, 199-204 (1992).
[CrossRef]

König, K.

W. Becker, A. Bergmann, G. Biscotti, K. König, I. Riemann, L. Kelbauskas, and C. Biskup, "High-speed FLIM data acquisition by time-correlated single photon counting," Proc. SPIE 5223, 1-9 (2004).
[CrossRef]

Lakowicz, J. R.

J. R. Lakowicz, H. Szmacinski, K. Nowaczyk, K. W. Berndt, and M. Johnson, "Fluorescence lifetime imaging," Anal. Biochem. 202, 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, 1727-1734 (1991).
[CrossRef]

J. R. Lakowicz and A. Balter, "Theory of phase-modulation fluorescence spectroscopy for excited-state processes," Biophys. Chem. 16, 99-115 (1982).
[CrossRef] [PubMed]

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

Lever, M. J.

D. S. Elson, I. Munro, J. Requejo-Isidro, J. McGinty, C. Dunsby, N. Galletly, G. W. Stamp, M. A. A. Neil, M. J. Lever, P. A. Kellett, A. Dymoke-Bradshaw, J. Hares, and P. M. W. French, "Real-time time-domain fluorescence lifetime imaging including single-shot acquisition with a segmented optical image intensifier," New J. Phys. 6, 180-193 (2004).
[CrossRef]

Liljeborg, A.

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

Lustenberger, F.

A. Esposito, H. C. Gerritsen, F. Lustenberger, T. Oggier, and F. S. Wouters, "Innovating lifetime microscopy: a compact and simple tool for the life sciences, screening and diagnostics," J. Biomed. Opt. 11, 34016-34023 (2006).
[CrossRef] [PubMed]

A. Esposito, T. Oggier, H. C. Gerritsen, F. Lustenberger, and F. S. Wouters, "All-solid-state lock-in imaging for wide-field fluorescence lifetime sensing," Opt. Express 13, 9812-9821 (2005).
[CrossRef] [PubMed]

McGinty, J.

D. S. Elson, I. Munro, J. Requejo-Isidro, J. McGinty, C. Dunsby, N. Galletly, G. W. Stamp, M. A. A. Neil, M. J. Lever, P. A. Kellett, A. Dymoke-Bradshaw, J. Hares, and P. M. W. French, "Real-time time-domain fluorescence lifetime imaging including single-shot acquisition with a segmented optical image intensifier," New J. Phys. 6, 180-193 (2004).
[CrossRef]

Munro, I.

D. S. Elson, I. Munro, J. Requejo-Isidro, J. McGinty, C. Dunsby, N. Galletly, G. W. Stamp, M. A. A. Neil, M. J. Lever, P. A. Kellett, A. Dymoke-Bradshaw, J. Hares, and P. M. W. French, "Real-time time-domain fluorescence lifetime imaging including single-shot acquisition with a segmented optical image intensifier," New J. Phys. 6, 180-193 (2004).
[CrossRef]

Neil, M. A. A.

D. S. Elson, I. Munro, J. Requejo-Isidro, J. McGinty, C. Dunsby, N. Galletly, G. W. Stamp, M. A. A. Neil, M. J. Lever, P. A. Kellett, A. Dymoke-Bradshaw, J. Hares, and P. M. W. French, "Real-time time-domain fluorescence lifetime imaging including single-shot acquisition with a segmented optical image intensifier," New J. Phys. 6, 180-193 (2004).
[CrossRef]

Nowaczyk, K.

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

Oggier, T.

A. Esposito, H. C. Gerritsen, F. Lustenberger, T. Oggier, and F. S. Wouters, "Innovating lifetime microscopy: a compact and simple tool for the life sciences, screening and diagnostics," J. Biomed. Opt. 11, 34016-34023 (2006).
[CrossRef] [PubMed]

A. Esposito, T. Oggier, H. C. Gerritsen, F. Lustenberger, and F. S. Wouters, "All-solid-state lock-in imaging for wide-field fluorescence lifetime sensing," Opt. Express 13, 9812-9821 (2005).
[CrossRef] [PubMed]

Pawley, J.

J. Pawley, "Sources of noise in three-dimensional microscopical data sets," in "Three-Dimensional Confocal Microscopy: Volume Investigation of Biological Specimens, J.Stevens, L.Mills, and J.Trogadis, eds. (Academic, 1994), pp. 47-94.

Pfeifer, L.

K. Kemnitz, L. Pfeifer, and M. R. Ainbund, "Detector for multichannel spectroscopy and fluorescence lifetime imaging on the picosecond timescale," Nucl. Instrum. Methods Phys. Res. Sect. A 387, 86-87 (1997).
[CrossRef]

Philip, J.

Philip, J. P.

K. Carlsson and J. P. Philip, "Theoretical investigation of the signal-to-noise ratio for different fluorescence lifetime imaging techniques," Proc. SPIE 4622, 70-74 (2002).
[CrossRef]

Requejo-Isidro, J.

D. S. Elson, I. Munro, J. Requejo-Isidro, J. McGinty, C. Dunsby, N. Galletly, G. W. Stamp, M. A. A. Neil, M. J. Lever, P. A. Kellett, A. Dymoke-Bradshaw, J. Hares, and P. M. W. French, "Real-time time-domain fluorescence lifetime imaging including single-shot acquisition with a segmented optical image intensifier," New J. Phys. 6, 180-193 (2004).
[CrossRef]

Riemann, I.

W. Becker, A. Bergmann, G. Biscotti, K. König, I. Riemann, L. Kelbauskas, and C. Biskup, "High-speed FLIM data acquisition by time-correlated single photon counting," Proc. SPIE 5223, 1-9 (2004).
[CrossRef]

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]

Schneider, P. C.

P. C. Schneider and R. M. Clegg, "Rapid acquisition, analysis, and display of fluorescence lifetime-resolved images for real-time applications," Rev. Sci. Instrum. 68, 4107-4119 (1997).
[CrossRef]

R. M. Clegg and P. C. Schneider, "Fluorescence lifetime-resolved imaging microscopy: a general description of lifetime-resolved imaging measurements," in Fluorescence Microscopy and Fluorescent Probes, J.Slavik, ed. (Plenum, 1996).

Stamp, G. W.

D. S. Elson, I. Munro, J. Requejo-Isidro, J. McGinty, C. Dunsby, N. Galletly, G. W. Stamp, M. A. A. Neil, M. J. Lever, P. A. Kellett, A. Dymoke-Bradshaw, J. Hares, and P. M. W. French, "Real-time time-domain fluorescence lifetime imaging including single-shot acquisition with a segmented optical image intensifier," New J. Phys. 6, 180-193 (2004).
[CrossRef]

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]

Sytsma, J.

J. Sytsma, J. M. Vroom, C. J. de Grauw, and H. C. Gerritsen, "Time-gated fluorescence lifetime imaging and microvolume spectroscopy using two-photon excitation," J. Microsc. 191, 39-51 (1998).
[CrossRef]

Szmacinski, H.

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

Tertoolen, L.

A. V. Agronskaia, L. Tertoolen, and H. C. Gerritsen, "High frame rate fluorescence lifetime imaging," J. Phys. D 36, 1655-1662 (2003).
[CrossRef]

van den Heuvel, D. J.

H. C. Gerritsen, A. Draaijer, D. J. van den Heuvel, and A. V. Agronskaia, "Fluorescence lifetime imaging in scanning microscopy," in Handbook of Biological Confocal Microscopy, 2nd ed., J.Pawley, ed. (Plenum, 2006), pp. 516-534.
[CrossRef]

van Munster, E. B.

E. B. van Munster and T. W. Gadella, Jr., "Suppression of photobleaching-induced artifacts in frequency-domain FLIM by permutation of the recording order," Cytometry 58A, 185-194 (2004).
[CrossRef]

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

Van Sark, W. G.

H. C. Gerritsen, M. A. Asselbergs, A. V. Agronskaia, and W. G. Van Sark, "Fluorescence lifetime imaging in scanning microscopes: acquisition speed, photon economy and lifetime resolution," J. Microsc. 206, 218-224 (2002).
[CrossRef] [PubMed]

Vroom, J. M.

J. Sytsma, J. M. Vroom, C. J. de Grauw, and H. C. Gerritsen, "Time-gated fluorescence lifetime imaging and microvolume spectroscopy using two-photon excitation," J. Microsc. 191, 39-51 (1998).
[CrossRef]

Wilson, T.

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

Wolfrum, J.

M. Kollner and J. Wolfrum, "How many photons are necessary for fluorescence-lifetime measurements," Chem. Phys. Lett. 200, 199-204 (1992).
[CrossRef]

Wouters, F. S.

A. Esposito, H. C. Gerritsen, F. Lustenberger, T. Oggier, and F. S. Wouters, "Innovating lifetime microscopy: a compact and simple tool for the life sciences, screening and diagnostics," J. Biomed. Opt. 11, 34016-34023 (2006).
[CrossRef] [PubMed]

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

A. Esposito, T. Oggier, H. C. Gerritsen, F. Lustenberger, and F. S. Wouters, "All-solid-state lock-in imaging for wide-field fluorescence lifetime sensing," Opt. Express 13, 9812-9821 (2005).
[CrossRef] [PubMed]

A. Esposito, H. C. Gerritsen, and F. S. Wouters, "Fluorescence lifetime imaging microscopy: quality assessment and standards" in Standardization in Fluorometry: State of the Art and Future Challenges, U.Resch-Genger, ed. (Springer, to be published).

Anal. Biochem.

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

Anal. Chem.

R. M. Ballew and J. N. Demas, "An error analysis of the rapid lifetime determination method for the evaluation of single exponential decays," Anal. Chem. 61, 30-33 (1989).
[CrossRef]

S. P. Chan, Z. J. Fuller, J. N. Demas, and B. A. DeGraff, "Optimized gating scheme for rapid lifetime determinations of single-exponential luminescence lifetimes," Anal. Chem. 73, 4486-4490 (2001).
[CrossRef] [PubMed]

Anal. Chim. Acta

R. M. Ballew and J. N. Demas, "Error analysis of the rapid lifetime determination method for single exponential decays with a nonzero base-line," Anal. Chim. Acta 245, 121-127 (1991).
[CrossRef]

Appl. Spectrosc.

Bioimaging

T. W. Gadella, Jr., R. M. Clegg, and T. M. Jovin, "Fluorescence lifetime imaging microscopy: pixel-by-pixel analysis of phase-modulation data," Bioimaging 2, 139-159 (1994).
[CrossRef]

Biophys. Chem.

J. R. Lakowicz and A. Balter, "Theory of phase-modulation fluorescence spectroscopy for excited-state processes," Biophys. Chem. 16, 99-115 (1982).
[CrossRef] [PubMed]

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

Biophys. J.

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

Chem. Phys. Lett.

M. Kollner and J. Wolfrum, "How many photons are necessary for fluorescence-lifetime measurements," Chem. Phys. Lett. 200, 199-204 (1992).
[CrossRef]

Cytometry

E. B. van Munster and T. W. Gadella, Jr., "Suppression of photobleaching-induced artifacts in frequency-domain FLIM by permutation of the recording order," Cytometry 58A, 185-194 (2004).
[CrossRef]

J. Biomed. Opt.

A. Esposito, H. C. Gerritsen, F. Lustenberger, T. Oggier, and F. S. Wouters, "Innovating lifetime microscopy: a compact and simple tool for the life sciences, screening and diagnostics," J. Biomed. Opt. 11, 34016-34023 (2006).
[CrossRef] [PubMed]

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. Microsc.

J. Sytsma, J. M. Vroom, C. J. de Grauw, and H. C. Gerritsen, "Time-gated fluorescence lifetime imaging and microvolume spectroscopy using two-photon excitation," J. Microsc. 191, 39-51 (1998).
[CrossRef]

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

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

H. C. Gerritsen, M. A. Asselbergs, A. V. Agronskaia, and W. G. Van Sark, "Fluorescence lifetime imaging in scanning microscopes: acquisition speed, photon economy and lifetime resolution," J. Microsc. 206, 218-224 (2002).
[CrossRef] [PubMed]

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

J. Opt. Soc. Am. A

J. Phys. D

A. V. Agronskaia, L. Tertoolen, and H. C. Gerritsen, "High frame rate fluorescence lifetime imaging," J. Phys. D 36, 1655-1662 (2003).
[CrossRef]

New J. Phys.

D. S. Elson, I. Munro, J. Requejo-Isidro, J. McGinty, C. Dunsby, N. Galletly, G. W. Stamp, M. A. A. Neil, M. J. Lever, P. A. Kellett, A. Dymoke-Bradshaw, J. Hares, and P. M. W. French, "Real-time time-domain fluorescence lifetime imaging including single-shot acquisition with a segmented optical image intensifier," New J. Phys. 6, 180-193 (2004).
[CrossRef]

Nucl. Instrum. Methods Phys. Res. Sect. A

K. Kemnitz, L. Pfeifer, and M. R. Ainbund, "Detector for multichannel spectroscopy and fluorescence lifetime imaging on the picosecond timescale," Nucl. Instrum. Methods Phys. Res. Sect. A 387, 86-87 (1997).
[CrossRef]

Opt. Express

Proc. SPIE

K. Carlsson and J. P. Philip, "Theoretical investigation of the signal-to-noise ratio for different fluorescence lifetime imaging techniques," Proc. SPIE 4622, 70-74 (2002).
[CrossRef]

W. Becker, A. Bergmann, G. Biscotti, K. König, I. Riemann, L. Kelbauskas, and C. Biskup, "High-speed FLIM data acquisition by time-correlated single photon counting," Proc. SPIE 5223, 1-9 (2004).
[CrossRef]

Rev. Sci. Instrum.

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

P. C. Schneider and R. M. Clegg, "Rapid acquisition, analysis, and display of fluorescence lifetime-resolved images for real-time applications," Rev. Sci. Instrum. 68, 4107-4119 (1997).
[CrossRef]

Other

H. C. Gerritsen, A. Draaijer, D. J. van den Heuvel, and A. V. Agronskaia, "Fluorescence lifetime imaging in scanning microscopy," in Handbook of Biological Confocal Microscopy, 2nd ed., J.Pawley, ed. (Plenum, 2006), pp. 516-534.
[CrossRef]

J. Pawley, "Sources of noise in three-dimensional microscopical data sets," in "Three-Dimensional Confocal Microscopy: Volume Investigation of Biological Specimens, J.Stevens, L.Mills, and J.Trogadis, eds. (Academic, 1994), pp. 47-94.

A. Esposito, H. C. Gerritsen, and F. S. Wouters, "Fluorescence lifetime imaging microscopy: quality assessment and standards" in Standardization in Fluorometry: State of the Art and Future Challenges, U.Resch-Genger, ed. (Springer, to be published).

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

R. M. Clegg and P. C. Schneider, "Fluorescence lifetime-resolved imaging microscopy: a general description of lifetime-resolved imaging measurements," in Fluorescence Microscopy and Fluorescent Probes, J.Slavik, ed. (Plenum, 1996).

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Figures (8)

Fig. 1
Fig. 1

Dependence of the F value of FD detection on modulation frequency for sine and Dirac excitation. Different detection techniques and estimators exhibit different photon economies. Shown are the fitted Monte Carlo simulations. (a) Phase- (dash-dotted curve), modulation- (gray), and average- (solid black) lifetime estimators. (b) Phase lock in (gray), RLD algorithm (upper black curve and gray circles), and the analytical solution for the RLD (lower black curve). Results are shown for both sine and Dirac excitation. Note the better performance of Dirac excitation, the high efficiency of the RLD, and the different optimal modulation frequency for the various estimators.

Fig. 2
Fig. 2

Comparison between different excitation methods: sine, rectangular, and Dirac excitation. Rectangular excitation was simulated with 50% and 20% duty cycles. With simultaneous collection, lock-in detection ( J = 2 ) exhibits a photon economy similar to phase detection ( J = 3 ) . The optimal photon economy for the RLD estimator ( J = 2 ) and the average ( J = 3 ) estimator are similarly high.

Fig. 3
Fig. 3

Dependence of FD photon economy on the number of phase images. For gated detectors (gray bars), the photon economy depends on the number of phase images, even for simultaneous collection, shown here for sine (left column) and Dirac (right column) excitation. The photon economy of all of phase- (top row), modulation- (middle row) and average- (bottom row) lifetime estimators generally converge to that observed with sine-modulated (white bars) gain.

Fig. 4
Fig. 4

Dependence of FD photon economy on the number of phase images for rectangular excitation with 50% and 20% duty cycles (see also Fig. 3).

Fig. 5
Fig. 5

Minimal F values of the average-lifetime estimator versus number of phase images. The hatched and solid histograms show the photon economy for parallel and sequential acquisition, respectively. The beneficial effect of increasing the number of phase steps is outweighed by the consequent loss of photons for a number of phase steps higher than 5 or 6.

Fig. 6
Fig. 6

F values and relative throughput of the average-lifetime estimator versus excitation duty cycle. The photon economy of lifetime detection improves at shorter duty cycles (a) converging to the optimal value close to zero (i.e., Dirac excitation). A constant SNR is maintained by scaling the integration time to compensate for the reduction of emitted photons. This is shown for different excitation rates ( κ ) , with (b) a gated light source and (c) a pulsed source with constant average excitation intensity.

Fig. 7
Fig. 7

Artifacts caused by fluorophore saturation in FD detection. Inset of (a), significant saturation of the fluorophore (shown is 90%) introduces higher harmonics in the fluorescence emission (solid curve) even when a single harmonic is present in the excitation signal (dashed curve). (a) Modulation- (black curves) and phase- (gray curves) lifetime estimation as a function of fluorophore saturation level for x = 1 . The saturation-dependent errors are reduced at lower excitation duty cycles. (b) Saturation levels that cause a 5% relative error in modulation- (black curves) and phase- (gray curves) lifetime estimators. Saturation levels are shown as a function of duty cycle for rectangular excitation. Dirac excitation (duty cycle 0 % ) exhibits no systematic errors in any of the estimators.

Fig. 8
Fig. 8

Fluorescence emission (bold line and grayed area) and excitation (dashed line) time profiles for A, sine, B, rectangular, and, C, Dirac excitation. Here, low excitation rates are considered in order to allow an intuitive comparison between these three excitation regimes. The hatched areas in panels A and B show equivalent areas from which the emission rates [Eqs. (6, 7, 8, 9)] can be directly appreciated.

Tables (5)

Tables Icon

Table 1 Photon Economies of Rectangular-Wave Excitation for Different Duty Cycles Is Obtained by Quadratic Curve Fitting of Monte Carlo Simulation Data

Tables Icon

Table 2 Relative Throughput of FD Detection

Tables Icon

Table 3 Relative Throughput of TD Detection

Tables Icon

Table 4 Acquisition Speed of Lifetime Imaging–Scanning Systems a

Tables Icon

Table 5 Acquisition Speed of Lifetime Imaging: Wide-Field Systems a

Equations (15)

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

F = Δ τ τ ( Δ N N ) 1 = σ τ τ N .
τ ϕ = ω 1 tan ϕ ; τ m = ω 1 ( m 2 1 ) 1 2 .
m = { ( I 0 I 180 ) [ 2 ( I 0 + I 180 ) ] } 1 2 .
A = E F 2 k n τ 1 ,
F F D R L D D i r a c = 1 2 ( 1 + x 2 ) 5 + 2 x 2 1 + 4 x 2 ,
k G = κ κ + 1 α ( n τ 1 ) ,
k C = κ κ + α α ( n τ 1 ) .
A G 1 F 2 k G n τ 1 = α F ( α ) 2 κ κ + 1 ,
A C 1 F 2 k C n τ 1 = α F ( α ) 2 κ κ + α .
A D = 1 F ( x ) 2 κ κ + 1 τ T = 1 F ( x ) 2 κ κ + 1 x 2 π ,
A S = 1 F ( x ) 2 κ κ + 1 .
E = t d w e l l t i m e t f r a m e 1 N p i x e l s .
E 1 J .
k G = ( κ κ + 1 n τ 1 ) α ( 1 + κ ) + x 2 π κ { 1 exp [ 2 π α ( 1 + κ ) x 1 ] } 1 + κ ,
k C = ( κ κ + α n τ 1 ) α ( α + κ ) + x 2 π κ { 1 exp [ 2 π ( α + κ ) x 1 ] } α + κ .

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