Correction for contaminant fluorescence in frequency-domain fluorometry.

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.