Development and quantitative applications of a digitizer-based time-resolved fluorometer

Recent improvements in the capabilities of commercially available digitization electronics provided an opportunity for the improvement of time-resolved fluorometric instrumentation using real time data acquisition and software processing modules. These modules emulate and often improve the performance of their hardware counterparts. The functions of boxcar averager, discriminator, photon counter, pulse height analyzer and time array detection were implemented. The flexibility inherent in this instrumentation approach is augmented by the ability to construct instruments that are not realizable with commercially available hardware components. Anode current processing software, capable of either time slice or time interval detection, provides both display of the fluorescence decay and integration of its area in the same operation; offers a multiplex advantage to improve the data collection rate; and allows the definition of a gating function with sharp 1 ns edges and ideal rectangular shape. The 1-ns time resolution and single shot capabilities of the waveform digitizer, along with the definition of a software gate, allow the development of coincident pulse height analysis software. With the reduced duty cycle instrument, a well-resolved pulse height spectrum was observed for the inexpensive circular-cage geometry photomultipliers. Of particular interest, the dark pulse height spectrum showed no dark counts having peak voltages higher than the local minimum of the light spectrum. The results of pulse height analysis led to the application of the photon counting method to a low excitation rate fluorometer. A multistop, multilevel discrimination processing algorithm increased the effective usable detection rate. The use of photon counting permits a more rigorous statistical treatment of the data and provides nearly transit time-spread limited resolution