Resolvability of fluorescence lifetime distributions using phase fluorometry.

J. R. Alcala, E. Gratton, F. G. Prendergast

Research output: Contribution to journalArticle

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Abstract

The analysis of the fluorescence decay using discrete exponential components assumes that a small number of species is present. In the absence of a definite kinetic model or when a large number of species is present, the exponential analysis underestimates the uncertainty of the recovered lifetime values. A different approach to determine the lifetime of a population of molecules is the use of probability density functions and lifetime distributions. Fluorescence decay data from continuous distributions of exponentially decaying components were generated. Different magnitudes of error were added to the data to simulate experimental conditions. The resolvability of the distributional model was studied by fitting the simulated data to one and two exponentials. The maximum width of symmetric distributions (uniform, gaussian, and lorentzian), which cannot be distinguished from single and double exponential fits for statistical errors of 1 and 0.1%, were determined. The width limits are determined by the statistical error of the data. It is also shown that, in the frequency domain, the discrete exponential analysis does not uniformly weights all the components of a distribution. This systematic error is less important when probability and distribution functions are used to recover the decay. Finally, it is shown that real lifetime distributions can be proved using multimodal probability density functions. In the companion paper that follows we propose a physical approach, which provides lifetime distribution functions for the tryptophan decay in proteins. In the third companion paper (Alcala, J.R., E. Gratton, and F.J. Prendergast, 1987, Biophys. J., in press) we use the distribution functions obtained to fit data from the fluorescence decay of single tryptophan proteins.

Original languageEnglish (US)
Pages (from-to)587-596
Number of pages10
JournalBiophysical Journal
Volume51
Issue number4
StatePublished - Apr 1987
Externally publishedYes

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Fluorometry
Fluorescence
Tryptophan
Normal Distribution
Uncertainty
Proteins
Weights and Measures
Population

ASJC Scopus subject areas

  • Biophysics

Cite this

Alcala, J. R., Gratton, E., & Prendergast, F. G. (1987). Resolvability of fluorescence lifetime distributions using phase fluorometry. Biophysical Journal, 51(4), 587-596.

Resolvability of fluorescence lifetime distributions using phase fluorometry. / Alcala, J. R.; Gratton, E.; Prendergast, F. G.

In: Biophysical Journal, Vol. 51, No. 4, 04.1987, p. 587-596.

Research output: Contribution to journalArticle

Alcala, JR, Gratton, E & Prendergast, FG 1987, 'Resolvability of fluorescence lifetime distributions using phase fluorometry.', Biophysical Journal, vol. 51, no. 4, pp. 587-596.
Alcala JR, Gratton E, Prendergast FG. Resolvability of fluorescence lifetime distributions using phase fluorometry. Biophysical Journal. 1987 Apr;51(4):587-596.
Alcala, J. R. ; Gratton, E. ; Prendergast, F. G. / Resolvability of fluorescence lifetime distributions using phase fluorometry. In: Biophysical Journal. 1987 ; Vol. 51, No. 4. pp. 587-596.
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