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 Na-3[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 pK(a). For the adapted GPM measurements, conventional measurements, and a global fit the apparent pK(a) 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%.