Time-domain measurement of fluorescence lifetime variation with pH

Advances in the design and miniaturization of the lasers and electronics required for Time Correlated Single Photon Counting (TCSPC) measurement of fluorescence lifetime have simplified the use of the time domain method. We have assembled a compact portable system that is capable of measuring lifetimes down to ∼200 ps (with deconvolution) and that can operate at a range of excitation and emission wavelengths. The excitation sources are pulsed LEDs and laser diodes with a maximum pulse rate of 40 MHz and are easily interchanged. Furthermore, the development of violet and blue GaN LEDs and laser diodes is expanding the range of fluorophores available for fluorescence lifetime measurement of ion concentrations. pH sensitive fluorophores have a wide range of biological and clinical applications. The use of fluorescence lifetime rather than intensity to measure pH has a number of advantages including the reduction of effects due to photobleaching, scattering, and intensity variations in the excitation source. Using our compact TCSPC instrumentation we have measured the dependence of fluorescence lifetimes on pH for a range of dyes in phosphate buffer over the physiologically important range of 6.0 to 8.0. Most dyes exhibit only a small variation in lifetime (<1.0 ns) over the 6.0 to 8.0 pH range; however, acridine exhibits a large variation in lifetime and hence shows promise as a pH indicator.