Information regarding the energetic state of tissue is important in a wide range of experimental fields, particularly in the study of metabolic stress, such as hypoxia or ischaemia. Metabolic stress leads to the degradation of adenine nucleotides in brain tissue, but little is known about any changes in the relative spatial distribution of these molecules. Ion mobility enabled MALDI imaging mass spectrometry has been employed to investigate the localisation of the adenine nucleotides ATP, ADP and AMP in mouse brain. The aim of the work is to develop a reproducible method for detecting changes in localisation and relative intensity of these nucleotides after metabolic stress. We demonstrate improved selectivity when ion mobility separation for the identification and localisation of the adenine nucleotides in tissue is employed. Tissue fixation methods have been evaluated to overcome rapid post-mortem degradation of adenine nucleotides such that biologically relevant localisation images can be obtained. These studies highlight the crucial importance of appropriate biological sample presentation in MALDI imaging experiments.