FLUORESCENCE LIFETIME IMAGING - AN EMERGING TECHNIQUE IN FLUORESCENCEMICROSCOPY

Fluorescence microscopy is an important tool for biological research, in part because of the extremely high detection sensitivity that can b e achieved, but also because fluorescent molecules can be used as prob es on account of their environmental responsiveness, for example to me asure intracellular pH or metal ion concentration. Unfortunately, the environmental sensitivity can sometimes be a source of problems becaus e of enhancement or 'quenching', which can make it very difficult to r elate emission intensity to the amount of fluorophore present. The mea sured intensity is essentially proportional to the product of the amou nt of fluorophore present in the sample and the local quantum yield of the fluorophore (the quantum yield can be thought of as the probabili ty that an excited molecule decays by fluorescence emission rather tha n by other non-radiative processes). This is a particular difficulty i n an environment such as a cell or tissue slice in which quantum yield and fluorophore concentration can both vary within the sample. Ideall y we would wish to be able to measure the quantum yield of fluorescenc e as well as the fluorescence intensity, as this would allow environme ntal effects to be compensated for. Unfortunately, this is not at all easy, and indirect means to achieve the same goal are more appropriate . A recently introduced technique, fluorescence lifetime imaging (Morg an et al. 1992, Wang et al. 1992), offers one such means to improve qu antification of fluorescence microscopy. In addition, as will be expla ined, the technique offers the prospect of significantly improving det ection sensitivity in appropriate circumstances.