Comparison of methods for rapid evaluation of lifetimes of exponential decays.

For evaluating exponential luminescence decays, there are a variety of computational rapid integral methods based on the areas of the decay under different binned intervals. Using both Monte Carlo methods and experimental photon counting data, we compare the standard rapid lifetime determination method (SRLD), optimized rapid lifetime determination methods (ORLD), maximum likelihood estimator method (MLE), and the phase plane method (PPM). The different techniques are compared with respect to precision, accuracy, sensitivity to binning range, and the effect of baseline interference. The MLE provides the best overall precision, but requires 10 bins and is sensitive to very small uncorrected baselines. The ORLD provides nearly as good precision using only two bins and is much more immune to uncompensated baselines. The PPM requires more bins than the MLE and has systematic errors, but is largely resistant to baseline issues. Therefore, depending on the data acquisition method and the number of bins that can be readily employed, the ORLD and MLE are the preferred methods for reasonable signal-to-noise ratios.

Self-referencing intensity measurements based on square-wave gated phase-modulation fluorimetry.

An adaptation of square-wave gated phase-modulation (GPM) fluorimetry allows for self-referenced intensity measurements without the complexity of dual excitation or dual emission wavelengths. This AC technique utilizes square-wave excitation, gated detection, a reference emitter, and a sensor molecule. The theory and experimental data demonstrating the effectiveness and advantages of the adapted GPM scheme are presented. One component must have an extremely short lifetime relative to the other. Both components are affected identically by changes in intensity of the excitation source, but the sensor intensity also depends on the concentration of the analyte. The fluctuations of the excitation source and any optical transmission changes are eliminated by ratioing the sensor emission to the reference emission. As the concentration of the analyte changes, the corresponding sensor intensity changes can be quantified through several schemes including digitization of the signal and digital integration or AC methods. To measure pH, digital methods are used with Na3[Tb(dpa)3] (dpa = 2,6-pyridinedicarboxylic acid) as the long-lived reference molecule and fluorescein as the short-lived sensor molecule. Measurements from the adapted GPM scheme are directly compared to conventional ratiometric measurements. Good agreement between the data collection methods is demonstrated through the apparent pKa. For the adapted GPM measurements, conventional measurements, and a global fit the apparent pKa values agree within less than 2%. A key element of the adapted GPM method is its insensitivity to fluctuations in the source intensity. For a roughly 8-fold change in the excitation intensity, the signal ratio changes by less than 3%.

Elimination of fluorescence and scattering backgrounds in luminescence lifetime measurements using gated-phase fluorometry.

A new gated form of phase fluorometry for measuring lifetimes is presented. The technique uses a square-wave excitation and gates the detector on only during the off period of the excitation. Using a long-lived sample, this eliminates or reduces errors from scattered light and short-lived fluorescences. Using a square-wave modulated excitation source with a 50% duty cycle, traditional data treatment can be used after, at most, a simple pi/2 phase adjustment. A combination of theory and experimental results demonstrates the validity of this new gated method and its utility for eliminating or reducing background. The results are precise, accurate, eliminate scattering errors, and greatly reduce errors due to short-lived fluorescence impurities. Errors from fluorescence bleed-through into the detection period or a slow excitation source turn off can be mitigated by using an offset time prior to gating the detector on.