The analog electronics commonly found in frequency domain fluorometers are limited to collecting only a single frequency at a time, and can be a source of systematic errors. We have developed an instrument in which most of the analog electronics are replaced with a computercontrolled digital-acquisition system. The computer is used for the direct collection of multifrequency data; filtering and calculation of the phase and modulation ratio are done by the software. From these values, fluorescence lifetimes can be determined. This new approach reduces most oft he systematic errors due to the analog electronics' hardware and allows for reconfiguration of the instrument with only minor changes of the software. This digitalacquisition system is not a simple substitution of an analog element, but it provides a new function and new capabilities for frequency domain fiuorometers. We have used this digital approach to build a "parallel" phase fl.uorometer which simultaneously collects and processes several modulation frequencies. INTRODUCTIONFluorescence decay measurements can be performed in the time domain using the correlated single photon counting technique or in the frequency domain by measuring the phase shift. and demodulation of the emission with respect to a sinusoidal excitation as a function of frequency. Among the advantages of frequency domain fiuorimct ry are high accuracy and rapid determination of fluorescence lifetimes. 1 -3 Lifetime accuracy is ultimately limited by spurious signals which are not properly processed by the analog electronics, and thereby result in systematic errors. 4 The rapid determination of single-or double-exponential fluorescence decays, which can be obtained by measurements at only one or two frequencies is not possible for systems where complex fluorescence decays must be resolved. In this case, a large number of modulation frequencies is needed to obtain the full decay information. 5 A block diagram of the analog electronics of a crosscorrelation frequency domain fluorometer 4 is shown in Fig. 1. The analog electronics begin with current-to-voltage converters that change the current output from the photomultiplier tubes (PMT) to voltages. The amplified output waveform is split, with one part passing through a low-pass filter, which gives the de value, and the other pa.rt going into an active analog band pass filter. The analog filter selects the low-frequency cross··COtTelated signal from the rest of the PMT output. The output of the filter is amplified, and fed into the zero-crossing detector to determine the phase and into another low-pass filter-rectifier circuit combination to determine the average ac value. Unwanted effocts, not properly accounted for by the analog electronics, are caused mainly by the relatively large bandwidth and nonlinearity of the analog filters. Systematic errors can also be introduced by the peculiar noise characteristics of the zero-crossing detector used to determine the phase. The analog filters must reject harmonic frequencies by a factor of at...

No abstract

The duty cycle problem referred to by Alcala has been discussed in our original manuscript, and we calculated the reduction of the signal quite explicitly. His comments on the harmonic content are in error, and the correct values were given in the original paper.

No abstract

No abstract