We describe a method to correct for background fluorescence and to measure the intensity of long-lifetime probes using phase-modulation fluorometry. The theoretically predicted and simulated data were supported by two experiments. The fractional contribution of background fluorescence in the sample was determined by measurement of phase angle and/or modulation at single modulation frequency. In certain experimental situations, where the mean decay times of the background and the long lifetime probe are widely separated, determination of background signals in the sample does not require a blank sample or information about the nature of intensity decay of the background. Hence, phase-modulation fluorometry can be used to directly determine the intensity of the long-lifetime probe in the presence of an unknown short-lifetime background. We also discuss the effects of ambient light (indefinitely long lifetime) and scattered excitation light (zero lifetime) on phase-modulation measurements.
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