Experimental investigation of fluorescence quenching

Care must be taken with the preparation of solutions. The materials must be freed from fluorescent or absorbing impurities, especially as the concentration of the fluorophore species may be less than 10 M. Oxygen, which often acts as a strong quencher, may 

This technique is the most accurate and sensitive available, and has been increasingly adopted [15], superseding a pulse-sampling method previously used before dye lasers became available [16]. Its great advantage is that the measurements are almost free from electronic noise. Each photon is independent of its predecessors. The detector is used merely to time the arrival of individual photons and to count them, not to measure the 

The mean fluorescence lifetime may also be determined by continuous intensity measurements, if the exciting light intensity is modulated at a high frequency. Fluorescence is excited by light modulated sinusoidally at a known frequency (ajln Hz). The emission is a forced response to the excitation, and is therefore modulated at the same frequency, but with a phase shift, due to the time-lag between absorption and emission. The intensities of the two beams are monitored by photomultipliers. The difference in phase (0) between the two intensities is determined electronically. The lifetime r is given by cox = tan . The modulation frequency must be made comparable to the decay rate, e.g., around 30 MHz for a mean lifetime of 30 ns. Such frequencies can be achieved by using a hydrogen lamp actuated by a suitably modulated current source. Commercial equipment is available. The method has been applied to quinine sulphate, fluorescein, and acridine, for example, with a precision of 1-2%. It is especially useful for very short (sub-nanosecond) lifetimes. 

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