Measurements of fluorescence lifetimes by use of a hybrid time-correlated and multifrequency phase fluorometer.

Measurements of homogeneous and heterogeneous fluorescence intensity decays using a hybrid time-correlated single photon counting/multifrequency phase fluorometer are reported. A trio of fluorophores exhibiting a range of decay profiles was selected. p-Terphenyl, 1,4-bis[2-(4-methyl-5-phenyloxazolyl)]benzene [(Me)2POPOP], and p-bis[2-(5-phenyloxazolyl)]benzene (POPOP), commonly used reference fluorophores, were analyzed initially; their emissions were characterized by monoexponential decay functions. Additionally, emissions from two single tryptophan proteins with different decay profiles were measured. Scorpion neurotoxin variant 3 required three exponentials to fit the emission decay properly (average lifetime approximately 500 ps). At pH 5.5, the fluorescence emission of ribonuclease T1 showed a monoexponential decay with a measured lifetime of approximately 4.0 ns. Thus, in each case, the results from both measurements were consistent between the two detection systems, confirming the view that the two approaches for measuring fluorescence lifetimes are equivalent.