The intrinsic fluorescence of the metabolic cofactors NADH and NADPH has been used as an indicator of the metabolic state of living tissues for more than 50 years. Combining this approach with modern developments in laser -scanning microscopy, pulsed laser sources, and time-resolved fluorescence detection allows measurement of the fluorescence lifetime of NAD(P)H within cells and tissues using fluorescence lifetime imaging microscopy (FLIM). As the fluorescence lifetime of a molecule is extremely sensitive to its local environment at the nanoscale, NAD(P)H FLIM can provide information on the metabolic state of complex biological preparations at a molecular level. Here, we describe experimental and theoretical considerations to the application of the technique in the study of neuroscience.