Fluorescence lifetime imaging microscopy (FLIM) is a powerful tool for producing an image based on the differences in the exponential decay rate of the fluorescence from a fluorescent sample. This technique can provide information, not only concerning the localization of specific fluorophores, but also about the local fluorophore environment. It can be used in scanning confocal, multi-photon microscopes, or in wide-field microscopes and endoscopes. FLIM systems can be implemented both in the frequency domain, using sinusoidally modulated excitation light and in the time domain, using pulsed excitation sources. The power of this technique lies in the fact that the measured fluorescent lifetime of a fluorophore is sensitive to the molecular environment of that fluorophore. Due to this phenomenon FLIM has recently found use in several applications: in the analysis of protein-protein interactions with high spatial and temporal specificity, in ion concentration imaging as well as in measuring of oxygen concentration and in medical applications.