Two-photon fluorescence lifetime imaging (FLIM) of molecules can reveal important information on the local microenvironment. NADH, an intrinsic fluorescent molecule and ubiquitous metabolic co-enzyme, has a lifetime that depends strongly on enzymatic binding. We present a custom image-processing algorithm for raw fluorescence lifetime and amplitude data that produces an image showing spatially distinct NADH fluorescence lifetimes in slices of rat and human brain. NADH FLIM images were collected in control and epileptic rat tissue. Differences in spatial patterns of NADH lifetimes support the hypothesis that NADH binding, and thus metabolic capacity, is significantly different between groups. This type of analysis can provide information on metabolic states in pathological material.