Advertisement

Journal of Fluorescence

, Volume 8, Issue 1, pp 27–34 | Cite as

Quantification of fluorescent molecules in heterogeneous media by use of the fluorescence decay amplitude analysis

  • G. E. DobretsovEmail author
  • T. I. Syrejshchikova
  • Yu. A. Gryzunov
  • M. N. Yakimenko
Regular Papers

Abstract

In heterogeneous media, including biological objects, fluorescent molecules of one kind often exist as a mixture of species with different fluorescence parameters. Fractional concentrations of these species can be measured by analyzing their fluorescence decay amplitudes. The amplitudes are linear functions of concentrations of actually fluorescent molecules, i.e., molecules whose fluorescence decay can be measured. Other (quenched) molecules do not influence these amplitudes. The other parameter that has to be measured to calculate these concentrations is the radiative rate constant. The parameter can be excluded by comparison of decay amplitudes of the sample studied and a standard. The comparison should be made taking into account the dependence of the radiation rates on emision wavelength. The method has been tested in experiments with the fluorescent probe 3-methoxybenzanthrone (MBA) bound with phosphatidylcholine bilayer membranes. The probe has a complex fluorescence decay in these membranes. The decay can be described as two exponentials, with decay times of 2 and 12 ns and a blue-shifted fluorescence spectrum of the short-life component as compared with long-life one. The shift was used to correct calculated radiative rate values. After this, about 100% of the MBA molecules were found to be fluorescent in these membranes. Thus, this approach can be used to measure absolute concentrations of subpopulations of fluorescent molecules in heterogeneous biological objects.

Key words

Fluorescent probes fluorescence decay concentration measurement 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. 1.
    J. R. Lakowicz (Ed.) (1994)Probe Design and Chemical Sensing. Top. Fluoresc. Spectrosc. 4, Plenum, New York.Google Scholar
  2. 2.
    A. Grinvald and I. Z. Steinberg (1976)Biochim. Biophys. Acta 427(2), 663–678.PubMedGoogle Scholar
  3. 3.
    G. Hazan, E. Haas, and I. Z. Steinberg (1976)Biochim. Biophys. Acta 434(1), 144–153.PubMedGoogle Scholar
  4. 4.
    L. Brand and J. R. Gohlke (1971)J. Biol. Chem. 246(7), 2317–2319.PubMedGoogle Scholar
  5. 5.
    L. Davenport, J. R. Knutson, and L. Brand (1989)Suhcell. Biochem. 14, 145–188.CrossRefGoogle Scholar
  6. 6.
    M. J. Kronman and L. G. Holmes (1971)Photochem. Pholobiol. 14(2), 113–134.Google Scholar
  7. 7.
    A. Grinvald, J. Schlessinger, I. Pecht, and I. Z. Steinberg (1975)Biochemistry 14(9), 1921–1929.PubMedCrossRefGoogle Scholar
  8. 8.
    V. I. Sorokovoj, G. E. Dobretsov, V. E. Mishijev, G. I. Klebanov, and Yu. A. Vladimirov (1974)Biophysica 19(1), 30–33(Russian).Google Scholar
  9. 9.
    L. G. Korkina, G. E. Dobretsov, G. Walzel, E. M. Kogan, Yu. I. Zimin, and Yu. A. Vladimirov (1981)J. Immunol. Methods 45, 227–237.PubMedCrossRefGoogle Scholar
  10. 10.
    L. G. Korkina, G. E. Dobretsov, G. Walzel, E. M. Kogan, Yu. I. Zimin, and Yu. A. Vladimirov (1982)J. Immunol. Methods 46, 179–183.CrossRefGoogle Scholar
  11. 11.
    G. E. Dobretsov (1989)Fluorescent Probes in the Study of Cells, Membranes and Lipoproteins, Nauka, Moscow (Russian).Google Scholar
  12. 12.
    Yu. A. Gryzunov and G. E. Dobretsov (Eds.) (1994)Serum Albumin in Clinical Medicine, IRIUS, Moscow (Russian).Google Scholar
  13. 13.
    G. E. Dobretsov, Yu. A. Gryzunov, M. N. Komarova, T. I. Syrejshchikova, and M. N. Yakimenko (1996) Lebedev Physical Institute, Preprint No. 33 (Russian).Google Scholar
  14. 14.
    G. E. Dobretsov, Yu. A. Gryzunov, M. N. Komarova, T. I. Syrejshchikova, and M. N. Yakimenko (1998)Nucl. Instr. Methods Phys. Res. A 405, 344–347.CrossRefGoogle Scholar
  15. 15.
    G. E. Dobretsov, V. A. Petrov, V. E. Mishijev, G. I. Klebanov, and Yu. A. Vladimirov (1977)Studio Biophys. B65(H.2), S.91-S.98.Google Scholar
  16. 16.
    N. K. Kurek, G. E. Dobretsov, Yu. V. Makhota, and V. P. Zvolinsky (1985)J. Appl. Spectr. (Minsk) 43(4), 579–584 (Russian).Google Scholar
  17. 17.
    N. S. Proskuriakova and R. N. Nurmuchametov (1969)Opt. Spectrosc. 27(2), 224–227 (Russian).Google Scholar
  18. 18.
    B. M. Krasovitskii and B. M. Bolotin (1976)Organic Luminophores, Chimia, Leningrad, p. 327.Google Scholar
  19. 19.
    M. D. Galanin, A. A. Kutienkov, V. N. Smorchkov, Yu. P. Timofeev, and Z. A. Chijikova (1982)Opt. Spectrosc. 53(4), 683–690 (Russian).Google Scholar
  20. 20.
    L. Gati (1969)Acta Phys. Chim. 15(1-2), 5–17.Google Scholar
  21. 21.
    G. E. Dobretsov (1979) inProgress of Science and Technique. Biophysics, Vinity, Moscow, Vol. 11, p. 163.Google Scholar
  22. 22.
    A. V. Akimov, G. V. Demjanov, N. K. Kurek, S. S. Molchanov, G. S. Pashchenko, T. I. Syrejshikova, R. V. Fedorchuk, and M. N. Yakimenko (1995)Nucl. Instrum. Methods Phys. Res. A359, 345–347.Google Scholar
  23. 23.
    G. E. Dobretsov, N. K. Kurek, V. N. Machov, T. I. Syrejshchikova, and M. N. Yakimenko (1989)J. Biochem. Biophys. Methods 19, 259–274.PubMedCrossRefGoogle Scholar
  24. 24.
    J. Lakowicz and S. Keating (1983)J. Biol. Chem. 258(9), 5519–5524.PubMedGoogle Scholar
  25. 25.
    A. Einstein (1916)Verhandl. Dtsch. Phys. Ces. B18, 318–323.Google Scholar
  26. 26.
    S. J. Strickler and R. A. Berg (1962)J. Chem. Phys. 37(2), 814–822.CrossRefGoogle Scholar

Copyright information

© Plenum Publishing Corporation 1998

Authors and Affiliations

  • G. E. Dobretsov
    • 1
    Email author
  • T. I. Syrejshchikova
    • 2
  • Yu. A. Gryzunov
    • 1
  • M. N. Yakimenko
    • 2
  1. 1.Research Institute for Physical Chemical MedicineMoscowRussia
  2. 2.Lebedev Physical Institute of Russia Academy of SciencesMoscowRussia

Personalised recommendations