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Deconvolution of single photon counting data with a reference method and global analysis

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Abstract

A method based on quenched references and global analysis was used to deconvolute timeresolved single photon counting data. The results from both computer simulated data and real experiments showed that highly accurate and reliable deconvolutions were possible. Fluorescence lifetimes and Stern-Volmer quenching constants for quenching with NaI were determined for the reference substances para-terphenyl, PPO (2,5-diphenyloxazol), POPOP (1,4-bis-(5-phenyl-2-oxazolyl)-benzene), and dimethyl-POPOP, all in ethanol. The fluorescence from a mixture of POPOP, anthracene, and diphenylanthracene in ethanol at different wavelengths was successfully resolved into the known relative contributions from the species at each wavelength. Fluorescence intensity decays of tryptophan in solution were studied at different wavelengths and globally analyzed with the method. Also, fluorescence anisotropy described by isotropic and anisotropic rotations in homogeneous and heterogeneous emitting systems were simulated and successfully deconvoluted. The method was applied to real fluorescence anisotropy data of diphenylanthracene and POPOP in paraffin oil, as well as to data from experiments on the blue copper-containing protein stellacyanin and its apo-form. In these cases, the method both corrected for errors due to, for example, the wavelength-dependent transit-times in the photomultiplier, and realized global deconvolutions of the total, parallel, and perpendicular components of the fluorescence. General algorithms for arbitrary fluorescence impulse responses are given.

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References

  1. Almgren M (1974) Analysis of pulse fluorometry data of complex systems. Ph.D. Thesis, Chalmers University of Technology, Göteborg

  2. Almgren M, Löfroth J-E (1982) Effects of polydispersity on fluorescence quenching in micelles. J Chem Phys 76:2734–2742

  3. Birch DJS, Imhof RE (1982) The origin of fluorescence from trans-trans diphenylbutadiene. Chem Phys Lett 88:243–247

  4. Dahlin S, Reinhammar B, Wilson M (1984) Direct measurements of the self-exchange rate of stellacyanin by a novel e.p.r. method. Biochem J 218:609–614

  5. Ehrenberg M, Rigler R, Wintermeyer W (1979) On the structure and conformational dynamics of yeast phenylalanine-accepting transfer ribonucleic acid in solution. Biochemistry 18:4588–4599

  6. Gaudochon P, Wahl Ph (1978) Pulsefluorimetry of tyrosyl peptides. Biophys Chem 8:87–104

  7. Gilbert CW (1983) A vector method for the non-linear least squares reconvolution-and-fitting analysis of polarized fluorescence decay data. In: Cundall RB, Dale RE (eds) Timeresolved fluorescence spectroscopy in biochemistry and biology. NATO ASI, St. Andrews, Scotland, Plenum Press, New York, pp 605–606

  8. Grinvald A, Steinberg IZ (1974) On the analysis of fluorescence decay kinetics by the method of least-squares. Anal Biochem 59:583–598

  9. Gudgin E, Lopez-Delgado R, Ware WR (1981) The trytophan fluorescence lifetime puzzle. a study of decay times in aqueous solution as function of pH and buffer composition. Can J Chem 59:1037–1044

  10. Helman WP (1971) Analysis of very fast transient luminescence behavior. Int J Radiat Phys Chem 3:283–294

  11. Irvin JA, Quickenden TI, Sangster DF (1981) Criterion of godness of fit for deconvolution calculations. Rev Sci Instrum 52:191–194

  12. Isenberg I (1975) Time decay fluorometry by photon counting. In: Chen RF, Edelhoch H (eds) Biochemical Fluorescence: concepts. Marcel Dekker, New York, pp 43–77

  13. Knight AEW, Selinger BK (1971) The deconvolution of fluoresence decay curves. A non-method for real data. Spectrochim Acta 27A:1223–1234

  14. Knight AEW, Selinger BK (1973) Single photon decay spectroscopy. Aust J Chem 26:1–27

  15. Knutson JR, Beechem J, Brand L (1983) Simultaneous analysis of multiple fluorescence decay curves: a global approach. Chem Phys Lett 102:501–507

  16. Lakowicz JR, Balter A (1982) Resolution of initially excited and relaxed states of tryptophan fluorescence by differential-wavelength deconvolution of time-resolved fluorescence decays. Biophys Chem 15:353–360

  17. Lakowicz JR, Cherek H, Balter A (1981) Correction of timing errors in photomultiplier tubes used in phase-modulation fluorometry. J Biochem Biophys Methods 5:131–146

  18. Lewis C, Ware WR, Doemeny LJ, Nemzek TL (1973) The measurement of short-lived fluorescence decay using the single photon counting method. Rev Sci Instrum 44:107–114

  19. Libertini LJ, Small EW (1984) F/F deconvolution of fluorescence decay data. Anal Biochem 138:314–318

  20. Löfroth JE (1982) Fluorescence quenching in micelle solutions. Ph.D. Thesis, University of Göteborg, Göteborg

  21. Löfroth JE (1985a) Deconvolution of single photon counting data with a reference method and global analysis. In: Szabo AG, Masotti L (eds) Excited state probes in biochemistry and biology. NATO ASI, Acireale, Italy. Plenum Press, New York (in press)

  22. Löfroth JE (1985b) TRES, DAS, and SAS (Time-resolved emission spectra, decay associated spectra, and species associated spectra). Application to excited state reactions. J Phys Chem (submitted for publication)

  23. Löfroth JE (1985c) Recent developments in the analysis of fluorescence intensity and anisotropy data. (submitted for publication in an upcoming special issue of Analytical Instrumentation on Time Resolved Fluorescence Spectroscopy)

  24. Löfroth JE, Almgren M (1982) Quenching of pyrene fluorescence by alkyl iodides in sodium dodecyl sulphate micelles. J Phys Chem 86:1636–1641

  25. McKinnon AE, Szabo AG, Miller DR (1977) The deconvolution of photoluminescence data. J Phys Chem 81:1564–1570

  26. Nemzek TL, Ware WR (1975) Kinetics of diffusion-controlled reactions: Transient effects in fluorescence quenching. J Chem Phys 62:477–489

  27. Petrich JW, Chang MC, McDonald DB, Fleming GR (1983) On the origin of nonexponential fluorescence decay in tryptophan and its derivatives. J Am Chem Soc 105:3824–3832

  28. Rayner DM, McKinnon AE, Szabo AG (1976) Confidence in fluorescence lifetime determinations: a ratio correction for the photomultiplier time response variation with wavelength. Can J Chem 54:3246–3259

  29. Rayner DM, McKinnon AE, Szabo AG (1977) Correction of instrumental time response variation with wavelength in fluorescence lifetime determinations in the ultraviolet region. Rev Sci Instrum 48:1050–1054

  30. Reinhammar B (1970) Purification and properties of laccase and stellacyanin from Rhus Vernicifera. Biochim Biophys Acta 205:35–47

  31. Selinger BK, Hinde AL (1983) Least squares methods of analysis. I. Confidence limits. In: Cundall RB, Dale RE (eds) Time-resolved fluorescence spectroscopy in biochemistry and biology. NATO ASI, St. Andrews, Scotland. Plenum Press, New York, pp 129–141

  32. Selinger BK, Harris C, Kallir K (1983) Least squares methods of analysis. II. Convolution and data optimization. In: Cundall RB, Dale RE (eds) Time-resolved fluorescence spectroscopy in biochemistry and biology. NATO ASI, St. Andrews, Scotland. Plenum Press, New York, pp. 143–153

  33. Szabo AG, Rayner DM (1980) Fluorescence decay of tryptophan conformers in aqueous solution. J Am Chem Soc 102:554–563

  34. Valeur B (1978) Analysis of time-dependent fluorescence experiments by the method of modulating functions with special attention to pulse fluorometry. Chem Phys 30:85–93

  35. Wahl Ph (1977) Statistical accuracy of rotational correlation times determined by the photocounting pulse fluorimetry. Chem Phys 22:245–256

  36. Wahl Ph (1979) Analysis of fluorescence anisotropy decays by a least square method. Biophys Chem 10:91–104

  37. Wahl Ph, Auchet JC, Donzel B (1974) The wavelength dependence of the response of a pulse fluorometer using the single photoelectron counting method. Rev Sci Instrum 45:28–32

  38. Ware WR (1971) Transient luminescence measurements. In: Lamoda AA (ed) Creation and detection of the excited state, Vol 1, part A. Marcel Dekker, New York, pp 213–302

  39. Ware WR, André JC (1983) The influence of diffusion on fluorescence quenching. In: Cundall RB, Dale RE (eds) Time-resolved fluorescence spectroscopy in biochemistry and biology. NATO ASI, St. Andrews, Scotland. Plenum Press, New York, pp 363–392

  40. Ware WR, Doemeny LJ, Nemzek TL (1973) Deconvolution of fluorescence and phosphorescence decay curves. A least square method. J Phys Chem 77:2038–2048

  41. Ware WR, Pratinidhi M, Bauer RK (1983) Performance characteristics of a small side-window photomultiplier in laser single-photon fluorescence decay measurements. Rev Sci Instrum 54:1148–1156

  42. Wijnaendts van Resandt RW, Vogel RH, Provencher SW (1982) Double beam fluorescence lifetime spectrometer with subnanosecond resolution: Application to aqueous tryptophan. Rev Sci Instrum 53:1392–1397

  43. Zuker M, Szabo AG, Krajcarski DT, Selinger BK (in press) Correction methods in TCPC & multicomponent analysis. In: Szabo AG, Masotti L (eds) Excited state probes in biochemistry and biology. NATO ASI, Acireale, Italy. Plenum Press New York

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Löfroth, J.-. Deconvolution of single photon counting data with a reference method and global analysis. Eur Biophys J 13, 45–58 (1985). https://doi.org/10.1007/BF00266309

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Key words

  • Fluorescence
  • anisotropy
  • deconvolution
  • reference
  • global analysis