| Abstract: | ABSTRACT We describe the construction and operation of a cross-correlation phase and modulation fluorometer which uses the harmonic content of a high repetition rate mode-locked laser as the excitation source. A mode-locked argon ion laser is used to synchronously pump a dye laser. The pulse train output from the dye laser is amplitude modulated by an acousto-optic modulator and then frequency doubled with an angle tuned frequency doubler. With the particular dye utilized in these studies, the ultraviolet light obtained was continuously tunable over the range 280-310 nm. In the frequency domain the high repetition rate pulsed source gives a large series of equally spaced harmonic frequencies. The frequency spacing of the harmonics is determined by the repetition frequency of the laser. Amplitude modulation of the pulse train permits variation of the frequency quasi-continuously from a few hertz to gigahertz. Use of cross-correlation techniques permits precise isolation of individual frequencies. The cross-correlation frequency required for the analysis of the phase delay and modulation ratio is obtained using coupled frequency synthesizers. In the present instrument three synthesizers are used. One drives the pump mode-locker head, a second drives the acousto-optic modulator and the third is used to modulate the response of the photomultiplier tubes which detect the signal. The accuracy, reproducibility and sensitivity of the instrumentation have been determined. Experimental data are provided to show use of this high frequency cross-correlation phase-modulation fluorometer for the determination of fluorescence lifetimes and rotational motions of tryptophan in solution and in proteins. |
