Imaging energy metabolites as markers of the energy shuttle between glia and neurons following ischemia is an ongoing challenge. Traditional microscopies in combination with histochemistry reveal glycogen accumulation within glia following ischemia, indicating an altered metabolic profile. Although semiquantitative histochemical glycogen analysis is possible, the method suffers from typical confounding factors common to histochemistry, such as variation in reagent penetration and binding. In addition, histochemical detection of glycogen does not reveal information on the metabolic fate of glycogen (i.e., lactate production). Therefore, validation of a direct semiquantitative method to simultaneously image both brain glycogen and lactate in the same tissue section would benefit this research field. In this study, we demonstrate the first application of Fourier transform infrared (FTIR) spectroscopy for simultaneous direct spectroscopic imaging of brain glycogen and lactate, in situ within ex vivo tissue sections. Serial tissue sections were analyzed with anti-glial fibrillary acidic protein (GFAP) immunohistochemistry to provide a comparison between the glycogen and lactate distribution revealed by FTIR and the glial distribution revealed by GFAP immunohistochemistry. The distribution of glycogen revealed by FTIR spectroscopic imaging has been further compared with histochemical detection of glycogen on the adjacent tissue sections. This approach was then applied to study spatiotemporal disturbances in metabolism, relative to glia and neuronal populations, following cerebral ischemia in a murine model of stroke.