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Applied Optics

Applied Optics


  • Vol. 35, Iss. 22 — Aug. 1, 1996
  • pp: 4481–4489

Time-resolved fluorescence-decay measurement and analysis on single cells by flow cytometry

Chiranjit Deka and John A. Steinkamp  »View Author Affiliations

Applied Optics, Vol. 35, Issue 22, pp. 4481-4489 (1996)

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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

Original Manuscript: September 11, 1995
Revised Manuscript: January 2, 1996
Published: August 1, 1996

Chiranjit Deka and John A. Steinkamp, "Time-resolved fluorescence-decay measurement and analysis on single cells by flow cytometry," Appl. Opt. 35, 4481-4489 (1996)

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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]

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