Frequency-Domain Lifetime Measurements

Abstract

In the preceding chapter we described the theory and instrumentation for measuring fluorescence intensity decays using time-domain measurements. In the present chapter we continue this discussion, but describe frequency-domain fluorometry. In this method the sample is excited with light that is intensity modulated at a high frequency comparable to the reciprocal of the lifetime. When this is done, the emission is intensity modulated at the same frequency. However, the emission does not precisely follow the excitation, but rather shows time delays and amplitude changes that are determined by the intensity decay law of the sample. The time delay is measured as a phase angle shift between the excitation and emission, as was shown in Figure 4.2. The peak-to-peak height of the modulated emission is decreased relative to the modulated excitation, and provides another independent measure of the lifetime. Time-resolved measurements, whether performed in the time domain or in the frequency domain, provides information about intensity decay of the sample. Samples with multiple fluorophores typically display multi-exponential decays. Even samples with a single fluorophore can display complex intensity decays due to conformational heterogeneity, resonance energy transfer, and transient effects in diffusive quenching or fluorophore—solvent interactions, to name just the most common origins. The goal of the time-resolved measurement is to determine the form of the intensity decay law, and to interpret the decay in terms of molecular features of the sample.