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.
Highlights
In recent years there has been a marked interest in the use of fluorescence spectroscopy for the study of dynamics of macromolecules
Even proteins containing a single tryptophan or tyrosine emitter, may demonstrate complex decay schemes, i.e., heterogeneity may exist in the lifetime as well as rotational modes of the fluorophore
Our particular interest has been the development of multifrequency phase fluorometry and especially its application to the study of subnanosecond raacromolecular dynamics using intrinsic fluorescence probes such as tryptophan and tyrosine residues
Summary
In recent years there has been a marked interest in the use of fluorescence spectroscopy for the study of dynamics of macromolecules. The fluorescence response can be measured in the frequency domain, using a light source with an Intensity which is modulated sinusoidally, by determining the phase delay and the relative modulation of the fluorescence signal with respect to the exciting light. In the time domain the response to the delta function excitation of an emitting system comprising N exponentially decaying components is given by the following equation,. The measurement of phase and modulation as a function of the frequency is equivalent to determining the time evolution of the emitting system to delta pulse excitation. The function r(t) can be expressed as a sum of exponentials.''" In the frequency domain the phase delay A and modulation ratio, A, between the parallel and perpendicular components of the fluorescence are measured. Notice again that relations 15 to 18 imply delta function excitation
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