Fluorescence lifetime resolution with phase fluorometry

A phase fluorometer for the measurement of fluorescence lifetimes was constructed from commercially available components. The instrument was tested by using optical delays of 4 and 2 cm, showing an accuracy of 10 ps. Lifetimes, as short as 0.3 ns, obtained by the quenching of fluorescein by KI, were analyzed with a standard deviation of 3 ps. The lifetime resolving power was checked using mixtures of acridine and quinine–sulphate and least-squares fitting procedures. Accurate amplitude ratios were obtained with the technique of phase-sensitive detection [J. R. Lakowicz and H. Cherek, J. Biochem. Biophys. Methods 5, 19 (1981)].