Abstract
I. SUMMARY Phase-modulation fluorometry is the frequency-domain analogue of time-resolved fluorescence spectroscopy. During the past three years we witnessed the development of variable-frequency phase-modulation fluorometers with modulation frequencies from 1 to 220 MHz. These instruments provide impressive resolution of multi or non-exponential fluorescence decays. To introduce these instruments we describe their design and operational principles. To illustrate the obtainable resolution we present results for the resolution of two and three-component mixtures of fluorophores, the resolution of complex anisotropy decays from non-spherical molecules and the determination of time-resolved emission spectra in the presence of time-dependent spectral relaxation. At present, the resolution obtainable with the frequency-domain fluorometers appears to be at least equivalent to that obtained with pulsed mode-locked laser sources with single photon counting. These mode-locked sources can also be used for phase flu...
Highlights
During the past three years we witnessed the development of variable-frequency phasemodulation fluoraneters with modulation frequencies from 1 to 220 MHz
These instruments provide impressive resolution of multi or non-exponential fluorescence decays. To introduce these instruments we describe their design and operational principles
To illustrate the obtainable resolution we present results for the resolution of two and three-component mixtures of fluorophores, the resolution of complex anisotropy decays from non-spherical
Summary
Phase-modulation fluorometry is the frequency-domain analogue of time-resolved fluorescence spectroscopy. Phase-modulation fluorometry is less widely utilized for the resolution of complex fluorescence decays This is somewhat surprising in that phase-modulation fluorometers were constructed prior to the development of time-domain instruments [10,11,12,13,14]. Phase sensitive detection has been found useful [17,19], and recently it has become possible to resolve three lifetime components using phase sensitive spectra at a single modulation frequency [20], the resolution of multiple lifetimes using phase sensitive spectra relies upon knowing, at least approximately, the steady state spectra of the individual components Because of these limitations phase-modulation fluorometers have not provided, in a general way, for the resolution of complex decay laws. Fitting is performed directly in the frequency domain [32]
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