Frequency domain instrument for measuring phosphorescence lifetime distributions in heterogeneous samples

The luminescence lifetime distribution can be used to determine the distribution of quencher concentrations in a heterogeneous sample. We describe a frequency domain instrument for real-time measurements of phosphorescence lifetime distributions in microheterogeneous objects. In this system (1) an array of harmonics (typically 100–200 frequencies) is used to modulate the excitation source, a light emitting diode. Due to the relatively long triplet state lifetimes, the frequencies required for the modulation are typically below 40 000 kHz, which allows direct digitization of both excitation and emission signals. (2) The dependence of the phase/amplitude factor on the modulation frequency is determined by linear least-squares analysis of the emission signal, which is sampled and summed over the multiple excitation cycles. (3) The phase/amplitude relationship obtained is analyzed in real time using a “light” version of the maximum entropy algorithm, which provides a complete phosphorescence lifetime distribution. (4) The lifetime distribution is converted into the distribution of quencher concentrations using an appropriate model of quenching. The instrument is also capable of measuring phosphorescence in “single-frequency” mode, which is useful for rapid evaluation of apparent luminescence lifetimes. In this mode, a correction for an in-phase signal, which is due to backscattering and fluorescence, is applied to improve the accuracy of lifetime measurements. The instruments were tested in Stern–Volmer calibrations of Pd-porphyrin based phosphors for oxygen measurements and used for preliminary evaluation of oxygen distributions in rat tumor tissues. The instruments were found to be capable of accurate determination of lifetimes in the range of 10–3000 μs. The average duration of a single lifetime distribution measurement was about 15 s, depending on the sample and on the density of the lifetime grid in the maximum entropy method analysis. In the single-frequency mode, the measurement time was reduced to about 0.2–0.5 s. The instruments provide complete correction for the in-phase signal of up to 40% of the total emission intensity.