Measurement of the intensity of long-lifetime luminophores in the presenceof background signals using phase-modulation fluorometry

We describe a method to correct for background fluorescence and to measure the intensity of long-lifetime probes using phase-modulation fluorometry. T he theoretically predicted and simulated data were supported by two experim ents. The fractional contribution of background fluorescence in the sample was determined by measurement of phase angle and/or modulation at single mo dulation frequency. In certain experimental situations, where the mean deca y 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 backgroun d. Hence, phase-modulation fluorometry can be used to directly determine th e intensity of the long-lifetime probe in the presence of an unknown short- lifetime background. We also discuss the effects of ambient light (indefini tely long lifetime) and scattered excitation light (zero lifetime) on phase -modulation measurements.