Correction for contaminant fluorescence in frequency-domain fluorometry

Joseph R. Lakowicz, Ranjith Jayaweera, Nanda Joshi, Iganzy Gryczynski

Research output: Contribution to journalArticle

25 Scopus citations

Abstract

We describe a general method to correct for contaminant fluorescence when using the technique of frequency-domain fluorometry. The method can be applied regardless of the origin of the background signal, from scattered light, impurity fluorescence, or both. The procedure requires measurement of the frequency-dependent phase and modulation of the background at enough frequencies to approximate the decay law of the background. We also describe a general method to propagate the uncertainties in the measured phase and modulation values into the corrected values. This propagation is necessary to ensure proper weighting of the frequency-dependent data in the least-squares fitting algorithms. The practical usefulness of this correction method is demonstrated using frequency-domain data for one and two component mixtures which were deliberately contaminated with scattered light and/or other fluorophores.

Original languageEnglish
Pages (from-to)471-479
Number of pages9
JournalAnalytical Biochemistry
Volume160
Issue number2
DOIs
StatePublished - 1 Feb 1987

Keywords

  • background correction
  • fluorescence spectroscopy
  • frequency-domain fluorometry
  • scattered light
  • time-resolved fluorescence

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