FLIM stands for Fluorescence Lifetime Imaging. As the name suggests, it is an imaging technique based on the detection of the lifetime of fluorescent molecules present in the sample, rather than their fluorescence emission. Any fluorophore, once excited by a photon of the proper energy, is driven from its ground state to an excited short-living state. Fluorescence emission occurs when the molecule decays to the ground state while spontaneously emitting another photon (with an energy lower than the exciting one).

The average time that the molecule spends in the excited state before coming back to the ground state is called fluorescence lifetime, whose value is different for diverse fluorophores. Therefore, the image of the sample is based on the different decay rates and not on the intensity of the fluorescence emission. FLIM is a strong technique when it comes to study the cell environment, since fluorescent lifetime is sensitive to physiological changes of the sample.