Parallel acquisition of fluorescence decay using array detectors

Frequency domain fluorometry provides an alternative method for the recording of fluorescence decay kinetics (1), The major advantages of this method are the time resolution and the fast measurement of lifetime values. There are several systems, where a change in lifetime occurs across the emission band. These systems include heterogeneous ground state systems, where species emit at different wavelengths; excited state reactions, with products emitting at different parts of the spectrum; and dipolar relaxation processes, where the emission spectra change with time. To characterize the decay of the systems, it is necessary to collect the emission decay at several wavelengths. Generally, this process is obtained by successive measurements using a monochromator or a series of bandpass filters. For steady-state spectra, optical multichannel analyzers (OMA) are available, which can collect the entire emission spectra at once. By gating the image intensifier, used with some of these analyzers, it is possible to obtain time windows of the order of a few nanoseconds (2), This time resolution is inadequate for most of the fluorescence substances. We have developed a new method based of frequency domain fluorometry which extends the time resolution of an OMA to the picosecond region. The entire emission decay is collected in a few seconds and the lifetime information across the entire emission band is analyzed. The same method can be applied to other array detectors such as TV and CCD cameras used in fluorescence microscopy (3).