The application of fluorescence decay measurements in studies of biological systems

Synchronously pumped, cavity dumped dye lasers provide moderately intense stable pulses of circa 10 ps duration, at a high repetition rate. Such a laser system is an ideal excitation source for the measurement of energy-resolved fluorescence decay profiles. When used in conjunction with photon counting electronics and well tested deconvolution procedures the system is capable of measuring complex (multi-component) fluorescence decays in the time range of < 100 ps- >100 ns. Different fluorophores, when incorporated into membranes or proteins (i.e., extrinsic probing), exhibit different types of interactions with each system. Many simple aromatics are relatively insensitive to the medium, thus the fluorescence decay contains little information of interest. However, in these cases the decay of the anisotropy, which may be extracted from the fluorescence decays, will yield information about the environment. Many heteroaromatic systems are more polar in the first excited singlet state than in the ground state. Consequently there is a strong interaction between the excited state and the surrounding field, this results in a relaxation of the environment about the excited state, resulting in a shift in the fluorescence spectrum to lower energy. When the relaxation is slow, the spectral shift can be time resolved, which can give further information about the structure of the probed system, usually a biological membrane. These types of measurements are discussed, as are some of the problems involved in extrinsic probing, such as multiple siting of the probe.