Background suppression in frequency-domain fluorometry

Gated detection is often used in time-domain measurements of long-lived flu orophores for suppression of interfering short-lived autofluorescence. Howe ver, no direct method has been available for gated detection and background suppression when using frequency-domain fluorometry, We describe a direct method for real-time suppression of autofluorescence in frequency-domain fl uorometry, The method uses a gated detector and the sample is excited by a pulsed train. The detector is gated on following each excitation pulse afte r a suitable time delay for decay of the prompt autofluorescence. Under the same experimental conditions a detectable reference signal is obtained by using a long lifetime standard with a known decay time. Because the sample and reference signals are measured under identical excitation, gating and i nstrumental conditions, the data can be analyzed as usual for frequency-dom ain data without further processing. We show by simulations that this metho d can be used to resolve single and multiexponential decays in the presence of short lifetime autofluorescence. (C) 2000 Academic Press.