A method for on-line background subtraction in frequency domain fluorometry.

Time-resolved fluorescence determinations on biochemical samples are often complicated by contributions from background. In the time-correlated single-photon counting method, background subtraction is a routine procedure. A limitation of frequency domain fluorometry, however, arises from the difficulty of performing this operation. This limitation has become increasingly significant as frequency domain methods are being applied to evermore complex biological systems using the higher-frequency capabilities of modern instrumentation. We have devised a method for such a correction in the frequency domain, regardless of the complexity of the background decay, based on measurement of the background phasor and subsequent subtraction from the sample phasor. This method is applicable to both lifetime and dynamic polarization measurements, and it can be readily implemented on commercially available frequency domain fluorometers. Decay curves may be accurately recovered from samples containing background contributions ranging from less than 5% to greater than 90% of the total signal intensity.