Correction of systematic phase errors in frequency-domain fluorometry

In frequency-domain fluorometry, the phase shift and relative modulation are measured with respect to a reference solution. Systematic timing errors can be avoided if a fluorophore of known lifetime is used as a reference. However, this reference fluorophore must fulfil several stringent requirements which are not always met by common reference fluorophores. The authors suggest an alternative method in which the measurements of phase shift and relative modulation are performed by using a scattering suspension as a reference, and the systematic phase errors are corrected afterwards thanks to a separate calibration of the instrument. Such a calibration is possible because it is demonstrated that the timing errors result in a frequency-independent time shift. The value of this time shift can be readily determined by analysis of the phase and modulation values obtained with a fluorophore which is excitable and observable at wavelengths comparable with those used in the experiment to be performed. No previous knowledge of the lifetime is required. The validity of this method is demonstrated by the resolution of closely spaced two-component decays and by the determination of very short lifetimes.