In conventional fluorescence microscopy only the information conferred by the fluorescence intensity is used. However, a fluorophore is not only characterized by its absorption and emission spectra, but also by its lifetime in the excited state, which is, in the case of most fluorophores, in the nanosecond time range.
The fluorescence lifetime does not only provide another dimension of contrast, but can be exploited for functional imaging. As compared to the fluorescence intensity the fluorescence lifetime has the additional advantage from being independent of the fluorophore concentration, the excitation intensity and the geometry of the light path. Thus, for measurements in biomedical samples, animals or patients, where these factors cannot be controlled, the fluorescence lifetime is an ideal parameter to measure and monitor the concentration of physiologically important ions, biomolecules and metabolites. Moreover, the fluorescence lifetime is often sensitive to the physical and chemical environment of the fluorophore. As such it is an excellent reporter of its surroundings facilitating in some way molecular imaging on the stage of a fluorescence microscope despite its limited resolution.