Abstract

A novel method is described for the measurement and analysis of fluorescence decays of individual cells and particles in flow. It combines the rapid measurement capabilities of a flow cytometer and the robust measurement and analysis procedures of time-domain fluorescence-lifetime spectroscopy. For excitation we use a cw laser that is pulse modulated by an electro-optic modulator. The characteristics and the repetition rate of the excitation pulses can be easily adjusted to accommodate fluorescence decays with a wide range of lifetimes.

© 1996 Optical Society of America

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  1. M. R. Melamed, T. Lindmo, M. L. Mendelsohn, eds., Flow Cytometry and Sorting, 2nd ed. (Wiley-Liss, New York, 1990).
  2. H. A. Crissman, J. A. Steinkamp, “Cytochemical techniques for multivariate analysis of DNA and other cellular constituents,” in Ref. 1, pp. 227–248.
  3. J. W. Gray, F. Dolbeare, M. G. Pallavicini, “Quantitative cell cycle analysis,” in Ref. 1, pp. 445–468.
  4. J. W. Gray, L. S. Cram, “Flow karyotyping and chromosome sorting,” in Ref. 1, pp. 503–530.
  5. M. R. Loken, “Immunofluorescene techniques,” in Ref. 1, pp. 341–353.
  6. J. W. M. Visser, “Analysis and sorting of blood and bone marrow cells,” in Ref. 1, pp. 669–684.
  7. H. B. Steen, E. Boye, K. Starstad, B. Bloom, T. Godal, A. Mustafa, “Application of flow cytometry on bacteria: cell cycle kinetic, drug effects and quantitation of antibody binding,” Cytometry 2, 249–257 (1982).
    [CrossRef] [PubMed]
  8. J. R. Lakowicz: Principles of Fluorescence Spectroscopy (Plenum, New York, 1983).
    [CrossRef]
  9. F. Dorr, “Mechanisms of energy transfer,” in Biophysics, W. Hoppe, W. Lohmann, H. Markl, H. Ziegler, eds. (Springer-Verlag, Berlin, 1983), pp. 265–288.
    [CrossRef]
  10. R. F. Chen, J. R. Knutson, “Mechanism of fluorescence concentration quenching of carboxyfluorescein in liposomes—energy transfer to nonfluorescent dimers,” Anal. Biochem. 172, 61–77 (1988).
    [CrossRef] [PubMed]
  11. D. P. Heller, C. L. Greenstock, “Fluorescence lifetime analysis of DNA intercalated ethidium bromide and quenching by free dye,” Biophys. Chem. 50, 305–312 (1994).
    [CrossRef] [PubMed]
  12. H. Zeng, G. Durocher, “Analysis of fluorescence quenching in some antioxidants from nonlinear Stern–Volmer plots,” J. Lumin. 63, 75–84 (1995).
    [CrossRef]
  13. S. M. Keating, T. G. Wensel, “Nanosecond fluorescence microscopy,” Biophys. J. 59, 186–202 (1991).
    [CrossRef] [PubMed]
  14. A. Kusumi, A. Tsuji, M. Murata, Y. Sako, A. C. Yoshizawa, S. Kagiwada, T. Hayakawa, S. Ohnishi, “Development of a streak-camera based time-resolved microscope fluorimeter and its application to studies of membrane fusion in single cells,” Biochemistry 30, 6517–6527 (1991).
    [CrossRef] [PubMed]
  15. J. A. Steinkamp, T. M. Yoshida, J. C. Martin, “Flow cytometer for resolving signals from heterogeneous fluorescence emissions and quantifying lifetime in fluorochrome labeled cells and particles by phase-sensitive detection,” Rev. Sci. Instrum. 64, 3440–3450 (1993).
    [CrossRef]
  16. C. Deka, L. A. Sklar, J. A. Steinkamp, “Fluorescence lifetime measurements in a flow cytometer by amplitude demodulation using digital data acquisition technique,” Cytometry 17, 94–101 (1994).
    [CrossRef] [PubMed]
  17. B. G. Pinsky, J. J. Ladasky, J. R. Lakowicz, K. Berndt, R. A. Hoffman, “Phase-resolved fluorescence lifetime measurements for flow cytometry,” Cytometry 14, 123–135 (1993).
    [CrossRef] [PubMed]
  18. J. R. Lakowicz, H. Cherek, “Phase-sensitive fluorescence spectroscopy: a new method to resolve fluorescence lifetimes and emission spectra of components in a mixture of fluorophores,” J. Biochem. Biophys. Methods. 5, 19–35 (1981).
    [CrossRef] [PubMed]
  19. J. R. Lakowicz, G. Laczko, I. Gryczynski, “Picosecond resolution of tryosine fluorescence and anisotropy decays by 2-GHz frequency-domain fluorometry,” Biochemistry 26, 82–90 (1987).
    [CrossRef] [PubMed]
  20. E. Gratton, R. Lopez-Delgado, “Measuring fluorescence decay times by phase-shift and modulation using the high harmonic content of pulsed light sources,” Nuovo Cimento B 15, 110–124 (1980).
    [CrossRef]
  21. W. R. Ware, L. J. Doemeny, T. L. Nemzek, “Deconvolution of fluorescence and phosphorescence decay curves—least squares method,” J. Phys. Chem. 77, 2038–2048 (1973).
    [CrossRef]
  22. D. M. Reyner, A. E. McKinnon, A. G. Szabo, “Correction of instrumental time response variation with wavelength in fluorescence lifetime determinations in the ultraviolet region,” Rev. Sci. Instrum. 48, 1050–1054 (1977).
    [CrossRef]
  23. A. Grinvald, I. Z. Steinberg, “On the analysis of fluorescence decay kinetics by the method of least squares,” Anal. Biochem. 59, 583–598 (1974).
    [CrossRef] [PubMed]
  24. P. R. Bevington, Data Reduction and Error Analysis for the Physical Sciences (McGraw-Hill, New York, 1969).
  25. C. W. Wilkerson, P. M. Goodwin, W. P. Ambrose, J. C. Martin, R. A. Keller, “Detection and lifetime measurement of single molecules in flowing sample streams by laser-induced fluorescence,” Appl. Phys. Lett. 62 (17), 2030–2032 (1993).
    [CrossRef]
  26. S. A. Soper, B. L. Legendre, “Error analysis of simple algorithms for determining fluorescence lifetimes of ultradilute dye solutions,” Appl. Spectrosc. 48, 400–405 (1994).
    [CrossRef]
  27. H. A. Crissman, J. A. Steinkamp, “Rapid one step staining procedures for analysis of cellular DNA and protein by single and dual laser flow cytometry,” Cytometry 3, 84–90 (1982).
    [CrossRef] [PubMed]

1995 (1)

H. Zeng, G. Durocher, “Analysis of fluorescence quenching in some antioxidants from nonlinear Stern–Volmer plots,” J. Lumin. 63, 75–84 (1995).
[CrossRef]

1994 (3)

D. P. Heller, C. L. Greenstock, “Fluorescence lifetime analysis of DNA intercalated ethidium bromide and quenching by free dye,” Biophys. Chem. 50, 305–312 (1994).
[CrossRef] [PubMed]

C. Deka, L. A. Sklar, J. A. Steinkamp, “Fluorescence lifetime measurements in a flow cytometer by amplitude demodulation using digital data acquisition technique,” Cytometry 17, 94–101 (1994).
[CrossRef] [PubMed]

S. A. Soper, B. L. Legendre, “Error analysis of simple algorithms for determining fluorescence lifetimes of ultradilute dye solutions,” Appl. Spectrosc. 48, 400–405 (1994).
[CrossRef]

1993 (3)

C. W. Wilkerson, P. M. Goodwin, W. P. Ambrose, J. C. Martin, R. A. Keller, “Detection and lifetime measurement of single molecules in flowing sample streams by laser-induced fluorescence,” Appl. Phys. Lett. 62 (17), 2030–2032 (1993).
[CrossRef]

B. G. Pinsky, J. J. Ladasky, J. R. Lakowicz, K. Berndt, R. A. Hoffman, “Phase-resolved fluorescence lifetime measurements for flow cytometry,” Cytometry 14, 123–135 (1993).
[CrossRef] [PubMed]

J. A. Steinkamp, T. M. Yoshida, J. C. Martin, “Flow cytometer for resolving signals from heterogeneous fluorescence emissions and quantifying lifetime in fluorochrome labeled cells and particles by phase-sensitive detection,” Rev. Sci. Instrum. 64, 3440–3450 (1993).
[CrossRef]

1991 (2)

S. M. Keating, T. G. Wensel, “Nanosecond fluorescence microscopy,” Biophys. J. 59, 186–202 (1991).
[CrossRef] [PubMed]

A. Kusumi, A. Tsuji, M. Murata, Y. Sako, A. C. Yoshizawa, S. Kagiwada, T. Hayakawa, S. Ohnishi, “Development of a streak-camera based time-resolved microscope fluorimeter and its application to studies of membrane fusion in single cells,” Biochemistry 30, 6517–6527 (1991).
[CrossRef] [PubMed]

1988 (1)

R. F. Chen, J. R. Knutson, “Mechanism of fluorescence concentration quenching of carboxyfluorescein in liposomes—energy transfer to nonfluorescent dimers,” Anal. Biochem. 172, 61–77 (1988).
[CrossRef] [PubMed]

1987 (1)

J. R. Lakowicz, G. Laczko, I. Gryczynski, “Picosecond resolution of tryosine fluorescence and anisotropy decays by 2-GHz frequency-domain fluorometry,” Biochemistry 26, 82–90 (1987).
[CrossRef] [PubMed]

1982 (2)

H. A. Crissman, J. A. Steinkamp, “Rapid one step staining procedures for analysis of cellular DNA and protein by single and dual laser flow cytometry,” Cytometry 3, 84–90 (1982).
[CrossRef] [PubMed]

H. B. Steen, E. Boye, K. Starstad, B. Bloom, T. Godal, A. Mustafa, “Application of flow cytometry on bacteria: cell cycle kinetic, drug effects and quantitation of antibody binding,” Cytometry 2, 249–257 (1982).
[CrossRef] [PubMed]

1981 (1)

J. R. Lakowicz, H. Cherek, “Phase-sensitive fluorescence spectroscopy: a new method to resolve fluorescence lifetimes and emission spectra of components in a mixture of fluorophores,” J. Biochem. Biophys. Methods. 5, 19–35 (1981).
[CrossRef] [PubMed]

1980 (1)

E. Gratton, R. Lopez-Delgado, “Measuring fluorescence decay times by phase-shift and modulation using the high harmonic content of pulsed light sources,” Nuovo Cimento B 15, 110–124 (1980).
[CrossRef]

1977 (1)

D. M. Reyner, A. E. McKinnon, A. G. Szabo, “Correction of instrumental time response variation with wavelength in fluorescence lifetime determinations in the ultraviolet region,” Rev. Sci. Instrum. 48, 1050–1054 (1977).
[CrossRef]

1974 (1)

A. Grinvald, I. Z. Steinberg, “On the analysis of fluorescence decay kinetics by the method of least squares,” Anal. Biochem. 59, 583–598 (1974).
[CrossRef] [PubMed]

1973 (1)

W. R. Ware, L. J. Doemeny, T. L. Nemzek, “Deconvolution of fluorescence and phosphorescence decay curves—least squares method,” J. Phys. Chem. 77, 2038–2048 (1973).
[CrossRef]

Ambrose, W. P.

C. W. Wilkerson, P. M. Goodwin, W. P. Ambrose, J. C. Martin, R. A. Keller, “Detection and lifetime measurement of single molecules in flowing sample streams by laser-induced fluorescence,” Appl. Phys. Lett. 62 (17), 2030–2032 (1993).
[CrossRef]

Berndt, K.

B. G. Pinsky, J. J. Ladasky, J. R. Lakowicz, K. Berndt, R. A. Hoffman, “Phase-resolved fluorescence lifetime measurements for flow cytometry,” Cytometry 14, 123–135 (1993).
[CrossRef] [PubMed]

Bevington, P. R.

P. R. Bevington, Data Reduction and Error Analysis for the Physical Sciences (McGraw-Hill, New York, 1969).

Bloom, B.

H. B. Steen, E. Boye, K. Starstad, B. Bloom, T. Godal, A. Mustafa, “Application of flow cytometry on bacteria: cell cycle kinetic, drug effects and quantitation of antibody binding,” Cytometry 2, 249–257 (1982).
[CrossRef] [PubMed]

Boye, E.

H. B. Steen, E. Boye, K. Starstad, B. Bloom, T. Godal, A. Mustafa, “Application of flow cytometry on bacteria: cell cycle kinetic, drug effects and quantitation of antibody binding,” Cytometry 2, 249–257 (1982).
[CrossRef] [PubMed]

Chen, R. F.

R. F. Chen, J. R. Knutson, “Mechanism of fluorescence concentration quenching of carboxyfluorescein in liposomes—energy transfer to nonfluorescent dimers,” Anal. Biochem. 172, 61–77 (1988).
[CrossRef] [PubMed]

Cherek, H.

J. R. Lakowicz, H. Cherek, “Phase-sensitive fluorescence spectroscopy: a new method to resolve fluorescence lifetimes and emission spectra of components in a mixture of fluorophores,” J. Biochem. Biophys. Methods. 5, 19–35 (1981).
[CrossRef] [PubMed]

Cram, L. S.

J. W. Gray, L. S. Cram, “Flow karyotyping and chromosome sorting,” in Ref. 1, pp. 503–530.

Crissman, H. A.

H. A. Crissman, J. A. Steinkamp, “Rapid one step staining procedures for analysis of cellular DNA and protein by single and dual laser flow cytometry,” Cytometry 3, 84–90 (1982).
[CrossRef] [PubMed]

H. A. Crissman, J. A. Steinkamp, “Cytochemical techniques for multivariate analysis of DNA and other cellular constituents,” in Ref. 1, pp. 227–248.

Deka, C.

C. Deka, L. A. Sklar, J. A. Steinkamp, “Fluorescence lifetime measurements in a flow cytometer by amplitude demodulation using digital data acquisition technique,” Cytometry 17, 94–101 (1994).
[CrossRef] [PubMed]

Doemeny, L. J.

W. R. Ware, L. J. Doemeny, T. L. Nemzek, “Deconvolution of fluorescence and phosphorescence decay curves—least squares method,” J. Phys. Chem. 77, 2038–2048 (1973).
[CrossRef]

Dolbeare, F.

J. W. Gray, F. Dolbeare, M. G. Pallavicini, “Quantitative cell cycle analysis,” in Ref. 1, pp. 445–468.

Dorr, F.

F. Dorr, “Mechanisms of energy transfer,” in Biophysics, W. Hoppe, W. Lohmann, H. Markl, H. Ziegler, eds. (Springer-Verlag, Berlin, 1983), pp. 265–288.
[CrossRef]

Durocher, G.

H. Zeng, G. Durocher, “Analysis of fluorescence quenching in some antioxidants from nonlinear Stern–Volmer plots,” J. Lumin. 63, 75–84 (1995).
[CrossRef]

Godal, T.

H. B. Steen, E. Boye, K. Starstad, B. Bloom, T. Godal, A. Mustafa, “Application of flow cytometry on bacteria: cell cycle kinetic, drug effects and quantitation of antibody binding,” Cytometry 2, 249–257 (1982).
[CrossRef] [PubMed]

Goodwin, P. M.

C. W. Wilkerson, P. M. Goodwin, W. P. Ambrose, J. C. Martin, R. A. Keller, “Detection and lifetime measurement of single molecules in flowing sample streams by laser-induced fluorescence,” Appl. Phys. Lett. 62 (17), 2030–2032 (1993).
[CrossRef]

Gratton, E.

E. Gratton, R. Lopez-Delgado, “Measuring fluorescence decay times by phase-shift and modulation using the high harmonic content of pulsed light sources,” Nuovo Cimento B 15, 110–124 (1980).
[CrossRef]

Gray, J. W.

J. W. Gray, F. Dolbeare, M. G. Pallavicini, “Quantitative cell cycle analysis,” in Ref. 1, pp. 445–468.

J. W. Gray, L. S. Cram, “Flow karyotyping and chromosome sorting,” in Ref. 1, pp. 503–530.

Greenstock, C. L.

D. P. Heller, C. L. Greenstock, “Fluorescence lifetime analysis of DNA intercalated ethidium bromide and quenching by free dye,” Biophys. Chem. 50, 305–312 (1994).
[CrossRef] [PubMed]

Grinvald, A.

A. Grinvald, I. Z. Steinberg, “On the analysis of fluorescence decay kinetics by the method of least squares,” Anal. Biochem. 59, 583–598 (1974).
[CrossRef] [PubMed]

Gryczynski, I.

J. R. Lakowicz, G. Laczko, I. Gryczynski, “Picosecond resolution of tryosine fluorescence and anisotropy decays by 2-GHz frequency-domain fluorometry,” Biochemistry 26, 82–90 (1987).
[CrossRef] [PubMed]

Hayakawa, T.

A. Kusumi, A. Tsuji, M. Murata, Y. Sako, A. C. Yoshizawa, S. Kagiwada, T. Hayakawa, S. Ohnishi, “Development of a streak-camera based time-resolved microscope fluorimeter and its application to studies of membrane fusion in single cells,” Biochemistry 30, 6517–6527 (1991).
[CrossRef] [PubMed]

Heller, D. P.

D. P. Heller, C. L. Greenstock, “Fluorescence lifetime analysis of DNA intercalated ethidium bromide and quenching by free dye,” Biophys. Chem. 50, 305–312 (1994).
[CrossRef] [PubMed]

Hoffman, R. A.

B. G. Pinsky, J. J. Ladasky, J. R. Lakowicz, K. Berndt, R. A. Hoffman, “Phase-resolved fluorescence lifetime measurements for flow cytometry,” Cytometry 14, 123–135 (1993).
[CrossRef] [PubMed]

Kagiwada, S.

A. Kusumi, A. Tsuji, M. Murata, Y. Sako, A. C. Yoshizawa, S. Kagiwada, T. Hayakawa, S. Ohnishi, “Development of a streak-camera based time-resolved microscope fluorimeter and its application to studies of membrane fusion in single cells,” Biochemistry 30, 6517–6527 (1991).
[CrossRef] [PubMed]

Keating, S. M.

S. M. Keating, T. G. Wensel, “Nanosecond fluorescence microscopy,” Biophys. J. 59, 186–202 (1991).
[CrossRef] [PubMed]

Keller, R. A.

C. W. Wilkerson, P. M. Goodwin, W. P. Ambrose, J. C. Martin, R. A. Keller, “Detection and lifetime measurement of single molecules in flowing sample streams by laser-induced fluorescence,” Appl. Phys. Lett. 62 (17), 2030–2032 (1993).
[CrossRef]

Knutson, J. R.

R. F. Chen, J. R. Knutson, “Mechanism of fluorescence concentration quenching of carboxyfluorescein in liposomes—energy transfer to nonfluorescent dimers,” Anal. Biochem. 172, 61–77 (1988).
[CrossRef] [PubMed]

Kusumi, A.

A. Kusumi, A. Tsuji, M. Murata, Y. Sako, A. C. Yoshizawa, S. Kagiwada, T. Hayakawa, S. Ohnishi, “Development of a streak-camera based time-resolved microscope fluorimeter and its application to studies of membrane fusion in single cells,” Biochemistry 30, 6517–6527 (1991).
[CrossRef] [PubMed]

Laczko, G.

J. R. Lakowicz, G. Laczko, I. Gryczynski, “Picosecond resolution of tryosine fluorescence and anisotropy decays by 2-GHz frequency-domain fluorometry,” Biochemistry 26, 82–90 (1987).
[CrossRef] [PubMed]

Ladasky, J. J.

B. G. Pinsky, J. J. Ladasky, J. R. Lakowicz, K. Berndt, R. A. Hoffman, “Phase-resolved fluorescence lifetime measurements for flow cytometry,” Cytometry 14, 123–135 (1993).
[CrossRef] [PubMed]

Lakowicz, J. R.

B. G. Pinsky, J. J. Ladasky, J. R. Lakowicz, K. Berndt, R. A. Hoffman, “Phase-resolved fluorescence lifetime measurements for flow cytometry,” Cytometry 14, 123–135 (1993).
[CrossRef] [PubMed]

J. R. Lakowicz, G. Laczko, I. Gryczynski, “Picosecond resolution of tryosine fluorescence and anisotropy decays by 2-GHz frequency-domain fluorometry,” Biochemistry 26, 82–90 (1987).
[CrossRef] [PubMed]

J. R. Lakowicz, H. Cherek, “Phase-sensitive fluorescence spectroscopy: a new method to resolve fluorescence lifetimes and emission spectra of components in a mixture of fluorophores,” J. Biochem. Biophys. Methods. 5, 19–35 (1981).
[CrossRef] [PubMed]

J. R. Lakowicz: Principles of Fluorescence Spectroscopy (Plenum, New York, 1983).
[CrossRef]

Legendre, B. L.

Loken, M. R.

M. R. Loken, “Immunofluorescene techniques,” in Ref. 1, pp. 341–353.

Lopez-Delgado, R.

E. Gratton, R. Lopez-Delgado, “Measuring fluorescence decay times by phase-shift and modulation using the high harmonic content of pulsed light sources,” Nuovo Cimento B 15, 110–124 (1980).
[CrossRef]

Martin, J. C.

C. W. Wilkerson, P. M. Goodwin, W. P. Ambrose, J. C. Martin, R. A. Keller, “Detection and lifetime measurement of single molecules in flowing sample streams by laser-induced fluorescence,” Appl. Phys. Lett. 62 (17), 2030–2032 (1993).
[CrossRef]

J. A. Steinkamp, T. M. Yoshida, J. C. Martin, “Flow cytometer for resolving signals from heterogeneous fluorescence emissions and quantifying lifetime in fluorochrome labeled cells and particles by phase-sensitive detection,” Rev. Sci. Instrum. 64, 3440–3450 (1993).
[CrossRef]

McKinnon, A. E.

D. M. Reyner, A. E. McKinnon, A. G. Szabo, “Correction of instrumental time response variation with wavelength in fluorescence lifetime determinations in the ultraviolet region,” Rev. Sci. Instrum. 48, 1050–1054 (1977).
[CrossRef]

Murata, M.

A. Kusumi, A. Tsuji, M. Murata, Y. Sako, A. C. Yoshizawa, S. Kagiwada, T. Hayakawa, S. Ohnishi, “Development of a streak-camera based time-resolved microscope fluorimeter and its application to studies of membrane fusion in single cells,” Biochemistry 30, 6517–6527 (1991).
[CrossRef] [PubMed]

Mustafa, A.

H. B. Steen, E. Boye, K. Starstad, B. Bloom, T. Godal, A. Mustafa, “Application of flow cytometry on bacteria: cell cycle kinetic, drug effects and quantitation of antibody binding,” Cytometry 2, 249–257 (1982).
[CrossRef] [PubMed]

Nemzek, T. L.

W. R. Ware, L. J. Doemeny, T. L. Nemzek, “Deconvolution of fluorescence and phosphorescence decay curves—least squares method,” J. Phys. Chem. 77, 2038–2048 (1973).
[CrossRef]

Ohnishi, S.

A. Kusumi, A. Tsuji, M. Murata, Y. Sako, A. C. Yoshizawa, S. Kagiwada, T. Hayakawa, S. Ohnishi, “Development of a streak-camera based time-resolved microscope fluorimeter and its application to studies of membrane fusion in single cells,” Biochemistry 30, 6517–6527 (1991).
[CrossRef] [PubMed]

Pallavicini, M. G.

J. W. Gray, F. Dolbeare, M. G. Pallavicini, “Quantitative cell cycle analysis,” in Ref. 1, pp. 445–468.

Pinsky, B. G.

B. G. Pinsky, J. J. Ladasky, J. R. Lakowicz, K. Berndt, R. A. Hoffman, “Phase-resolved fluorescence lifetime measurements for flow cytometry,” Cytometry 14, 123–135 (1993).
[CrossRef] [PubMed]

Reyner, D. M.

D. M. Reyner, A. E. McKinnon, A. G. Szabo, “Correction of instrumental time response variation with wavelength in fluorescence lifetime determinations in the ultraviolet region,” Rev. Sci. Instrum. 48, 1050–1054 (1977).
[CrossRef]

Sako, Y.

A. Kusumi, A. Tsuji, M. Murata, Y. Sako, A. C. Yoshizawa, S. Kagiwada, T. Hayakawa, S. Ohnishi, “Development of a streak-camera based time-resolved microscope fluorimeter and its application to studies of membrane fusion in single cells,” Biochemistry 30, 6517–6527 (1991).
[CrossRef] [PubMed]

Sklar, L. A.

C. Deka, L. A. Sklar, J. A. Steinkamp, “Fluorescence lifetime measurements in a flow cytometer by amplitude demodulation using digital data acquisition technique,” Cytometry 17, 94–101 (1994).
[CrossRef] [PubMed]

Soper, S. A.

Starstad, K.

H. B. Steen, E. Boye, K. Starstad, B. Bloom, T. Godal, A. Mustafa, “Application of flow cytometry on bacteria: cell cycle kinetic, drug effects and quantitation of antibody binding,” Cytometry 2, 249–257 (1982).
[CrossRef] [PubMed]

Steen, H. B.

H. B. Steen, E. Boye, K. Starstad, B. Bloom, T. Godal, A. Mustafa, “Application of flow cytometry on bacteria: cell cycle kinetic, drug effects and quantitation of antibody binding,” Cytometry 2, 249–257 (1982).
[CrossRef] [PubMed]

Steinberg, I. Z.

A. Grinvald, I. Z. Steinberg, “On the analysis of fluorescence decay kinetics by the method of least squares,” Anal. Biochem. 59, 583–598 (1974).
[CrossRef] [PubMed]

Steinkamp, J. A.

C. Deka, L. A. Sklar, J. A. Steinkamp, “Fluorescence lifetime measurements in a flow cytometer by amplitude demodulation using digital data acquisition technique,” Cytometry 17, 94–101 (1994).
[CrossRef] [PubMed]

J. A. Steinkamp, T. M. Yoshida, J. C. Martin, “Flow cytometer for resolving signals from heterogeneous fluorescence emissions and quantifying lifetime in fluorochrome labeled cells and particles by phase-sensitive detection,” Rev. Sci. Instrum. 64, 3440–3450 (1993).
[CrossRef]

H. A. Crissman, J. A. Steinkamp, “Rapid one step staining procedures for analysis of cellular DNA and protein by single and dual laser flow cytometry,” Cytometry 3, 84–90 (1982).
[CrossRef] [PubMed]

H. A. Crissman, J. A. Steinkamp, “Cytochemical techniques for multivariate analysis of DNA and other cellular constituents,” in Ref. 1, pp. 227–248.

Szabo, A. G.

D. M. Reyner, A. E. McKinnon, A. G. Szabo, “Correction of instrumental time response variation with wavelength in fluorescence lifetime determinations in the ultraviolet region,” Rev. Sci. Instrum. 48, 1050–1054 (1977).
[CrossRef]

Tsuji, A.

A. Kusumi, A. Tsuji, M. Murata, Y. Sako, A. C. Yoshizawa, S. Kagiwada, T. Hayakawa, S. Ohnishi, “Development of a streak-camera based time-resolved microscope fluorimeter and its application to studies of membrane fusion in single cells,” Biochemistry 30, 6517–6527 (1991).
[CrossRef] [PubMed]

Visser, J. W. M.

J. W. M. Visser, “Analysis and sorting of blood and bone marrow cells,” in Ref. 1, pp. 669–684.

Ware, W. R.

W. R. Ware, L. J. Doemeny, T. L. Nemzek, “Deconvolution of fluorescence and phosphorescence decay curves—least squares method,” J. Phys. Chem. 77, 2038–2048 (1973).
[CrossRef]

Wensel, T. G.

S. M. Keating, T. G. Wensel, “Nanosecond fluorescence microscopy,” Biophys. J. 59, 186–202 (1991).
[CrossRef] [PubMed]

Wilkerson, C. W.

C. W. Wilkerson, P. M. Goodwin, W. P. Ambrose, J. C. Martin, R. A. Keller, “Detection and lifetime measurement of single molecules in flowing sample streams by laser-induced fluorescence,” Appl. Phys. Lett. 62 (17), 2030–2032 (1993).
[CrossRef]

Yoshida, T. M.

J. A. Steinkamp, T. M. Yoshida, J. C. Martin, “Flow cytometer for resolving signals from heterogeneous fluorescence emissions and quantifying lifetime in fluorochrome labeled cells and particles by phase-sensitive detection,” Rev. Sci. Instrum. 64, 3440–3450 (1993).
[CrossRef]

Yoshizawa, A. C.

A. Kusumi, A. Tsuji, M. Murata, Y. Sako, A. C. Yoshizawa, S. Kagiwada, T. Hayakawa, S. Ohnishi, “Development of a streak-camera based time-resolved microscope fluorimeter and its application to studies of membrane fusion in single cells,” Biochemistry 30, 6517–6527 (1991).
[CrossRef] [PubMed]

Zeng, H.

H. Zeng, G. Durocher, “Analysis of fluorescence quenching in some antioxidants from nonlinear Stern–Volmer plots,” J. Lumin. 63, 75–84 (1995).
[CrossRef]

Anal. Biochem. (2)

R. F. Chen, J. R. Knutson, “Mechanism of fluorescence concentration quenching of carboxyfluorescein in liposomes—energy transfer to nonfluorescent dimers,” Anal. Biochem. 172, 61–77 (1988).
[CrossRef] [PubMed]

A. Grinvald, I. Z. Steinberg, “On the analysis of fluorescence decay kinetics by the method of least squares,” Anal. Biochem. 59, 583–598 (1974).
[CrossRef] [PubMed]

Appl. Phys. Lett. (1)

C. W. Wilkerson, P. M. Goodwin, W. P. Ambrose, J. C. Martin, R. A. Keller, “Detection and lifetime measurement of single molecules in flowing sample streams by laser-induced fluorescence,” Appl. Phys. Lett. 62 (17), 2030–2032 (1993).
[CrossRef]

Appl. Spectrosc. (1)

Biochemistry (2)

A. Kusumi, A. Tsuji, M. Murata, Y. Sako, A. C. Yoshizawa, S. Kagiwada, T. Hayakawa, S. Ohnishi, “Development of a streak-camera based time-resolved microscope fluorimeter and its application to studies of membrane fusion in single cells,” Biochemistry 30, 6517–6527 (1991).
[CrossRef] [PubMed]

J. R. Lakowicz, G. Laczko, I. Gryczynski, “Picosecond resolution of tryosine fluorescence and anisotropy decays by 2-GHz frequency-domain fluorometry,” Biochemistry 26, 82–90 (1987).
[CrossRef] [PubMed]

Biophys. Chem. (1)

D. P. Heller, C. L. Greenstock, “Fluorescence lifetime analysis of DNA intercalated ethidium bromide and quenching by free dye,” Biophys. Chem. 50, 305–312 (1994).
[CrossRef] [PubMed]

Biophys. J. (1)

S. M. Keating, T. G. Wensel, “Nanosecond fluorescence microscopy,” Biophys. J. 59, 186–202 (1991).
[CrossRef] [PubMed]

Cytometry (4)

H. A. Crissman, J. A. Steinkamp, “Rapid one step staining procedures for analysis of cellular DNA and protein by single and dual laser flow cytometry,” Cytometry 3, 84–90 (1982).
[CrossRef] [PubMed]

H. B. Steen, E. Boye, K. Starstad, B. Bloom, T. Godal, A. Mustafa, “Application of flow cytometry on bacteria: cell cycle kinetic, drug effects and quantitation of antibody binding,” Cytometry 2, 249–257 (1982).
[CrossRef] [PubMed]

C. Deka, L. A. Sklar, J. A. Steinkamp, “Fluorescence lifetime measurements in a flow cytometer by amplitude demodulation using digital data acquisition technique,” Cytometry 17, 94–101 (1994).
[CrossRef] [PubMed]

B. G. Pinsky, J. J. Ladasky, J. R. Lakowicz, K. Berndt, R. A. Hoffman, “Phase-resolved fluorescence lifetime measurements for flow cytometry,” Cytometry 14, 123–135 (1993).
[CrossRef] [PubMed]

J. Biochem. Biophys. Methods. (1)

J. R. Lakowicz, H. Cherek, “Phase-sensitive fluorescence spectroscopy: a new method to resolve fluorescence lifetimes and emission spectra of components in a mixture of fluorophores,” J. Biochem. Biophys. Methods. 5, 19–35 (1981).
[CrossRef] [PubMed]

J. Lumin. (1)

H. Zeng, G. Durocher, “Analysis of fluorescence quenching in some antioxidants from nonlinear Stern–Volmer plots,” J. Lumin. 63, 75–84 (1995).
[CrossRef]

J. Phys. Chem. (1)

W. R. Ware, L. J. Doemeny, T. L. Nemzek, “Deconvolution of fluorescence and phosphorescence decay curves—least squares method,” J. Phys. Chem. 77, 2038–2048 (1973).
[CrossRef]

Nuovo Cimento B (1)

E. Gratton, R. Lopez-Delgado, “Measuring fluorescence decay times by phase-shift and modulation using the high harmonic content of pulsed light sources,” Nuovo Cimento B 15, 110–124 (1980).
[CrossRef]

Rev. Sci. Instrum. (2)

D. M. Reyner, A. E. McKinnon, A. G. Szabo, “Correction of instrumental time response variation with wavelength in fluorescence lifetime determinations in the ultraviolet region,” Rev. Sci. Instrum. 48, 1050–1054 (1977).
[CrossRef]

J. A. Steinkamp, T. M. Yoshida, J. C. Martin, “Flow cytometer for resolving signals from heterogeneous fluorescence emissions and quantifying lifetime in fluorochrome labeled cells and particles by phase-sensitive detection,” Rev. Sci. Instrum. 64, 3440–3450 (1993).
[CrossRef]

Other (9)

J. R. Lakowicz: Principles of Fluorescence Spectroscopy (Plenum, New York, 1983).
[CrossRef]

F. Dorr, “Mechanisms of energy transfer,” in Biophysics, W. Hoppe, W. Lohmann, H. Markl, H. Ziegler, eds. (Springer-Verlag, Berlin, 1983), pp. 265–288.
[CrossRef]

M. R. Melamed, T. Lindmo, M. L. Mendelsohn, eds., Flow Cytometry and Sorting, 2nd ed. (Wiley-Liss, New York, 1990).

H. A. Crissman, J. A. Steinkamp, “Cytochemical techniques for multivariate analysis of DNA and other cellular constituents,” in Ref. 1, pp. 227–248.

J. W. Gray, F. Dolbeare, M. G. Pallavicini, “Quantitative cell cycle analysis,” in Ref. 1, pp. 445–468.

J. W. Gray, L. S. Cram, “Flow karyotyping and chromosome sorting,” in Ref. 1, pp. 503–530.

M. R. Loken, “Immunofluorescene techniques,” in Ref. 1, pp. 341–353.

J. W. M. Visser, “Analysis and sorting of blood and bone marrow cells,” in Ref. 1, pp. 669–684.

P. R. Bevington, Data Reduction and Error Analysis for the Physical Sciences (McGraw-Hill, New York, 1969).

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Figures (5)

Fig. 1
Fig. 1

(a) Pair of simulated excitation (dashed curve) and fluorescence (open circles) pulses. The fluorescence pulse was calculated by convolution of a single-exponential decay, which has a lifetime of 7 ns, with the excitation pulse. The simulated excitation pulse had a cw baseline of K = 0.05. The calculations were made with a sampling interval of 100 ps. (b) Theoretical fit obtained by application of the iterative-reconvolution analysis to the pair of data sets from (a) after their respective baselines were subtracted. For clarity, only the data points at half-nanosecond intervals are shown for the fluorescence signal, and the theoretical fit is shown by a solid line.

Fig. 2
Fig. 2

Schematic diagram of the flow cytometric system for fluorescence decay measurement. FL1, photomultiplier tube for fluorescence and scattered signal measurement; LM, photomultiplier tube for measuring laser pulses; EOM, electro-optic modulator; BS, beam splitter; FCO, fluorescence collection optics; L1 and L2, cylindrical lenses; FLS, forward light scatter detector (not used in the time-resolved experiment).

Fig. 3
Fig. 3

(a) Oscilloscope traces of the electrical pulses from the LeCroy 9210/9211 pulse generator (top) and the pulsed laser output from the EOM (bottom). (b) Pulse shape of the electrical signal (top) and the laser output from the EOM (bottom). (c) Flow cytometric signal obtained from a nonfluorescent particle passing through the pulsed laser beam.

Fig. 4
Fig. 4

(a) Fluorescence pulse from an Immuno-Check microsphere (circles) and a scatter pulse from a nonfluorescent microsphere (beaded curve). Each pulse was acquired in channel 2 of the digital oscilloscope and is overlaid in this figure for comparison. The pulse parameters of the electrical driver signal were w = 10 ns, lead 1 ns, and trail 6 ns. (b) Laser pulses measured by the laser-monitor PMT (LM), corresponding to the scatter signal from the nonfluorescent microsphere (curve with dots) and the fluorescence signal from the fluorescent microsphere (curve with circles), respectively. Each of these pulses was acquired in channel 1 of the oscilloscope. The oscilloscope was triggered by the signal in channel 2. (c) Scatter pulse from Fig. 4(a) after being shifted relative to the fluorescence pulse by an amount equal to the apparent shift between the corresponding laser pulses and corrected for the respective cw baselines. Iterative reconvolution analysis of the pair of time-corrected and baseline-subtracted fluorescence and scatter signals resulted in the theoretical fit shown by the solid curve passing through the fluorescence data points (circles).

Fig. 5
Fig. 5

Iterative reconvolution analysis of the fluorescence signals from CHO cells stained with (a) FITC, (b) PI, and (c) both FITC and PI. The pulse parameters of the electrical driver signal for the EOM were w = 10 ns, lead 1 ns, and trail 6 ns for (a) and (b). For the measurements shown in (c) the electrical driver signal parameters were w = 8 ns, lead 2 ns, and trail 1 ns. For each case a pair of fluorescence and scattered signals are shown after the trigger-time and base-line corrections. The baseline-subtracted fluorescence data points are represented by the circles, the time-corrected and base-line-subtracted scatter signal is represented by the jagged curve with dots, and the theoretical best fit obtained by the iterative reconvolution is represented by the smooth curve.

Equations (6)

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e ( t ) = [ L ( t ) S ( t ) ] ,
F ( t ) = e ( t ) D ( t ) ,
F ( t ) = 0 t D ( t ) e ( t t ) d t .
E ( t ) = K + e ( t ) .
F ( t ) = 0 t D ( t ) [ K + e ( t t ) ] d t , F ( t ) = K 0 t D ( t ) d t + 0 t D ( t ) e ( t t ) d t , F ( t ) = K + 0 t D ( t ) e ( t t ) d t .
D ( t ) = i α i exp ( t / τ i )

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