This work has provided the first study investigating the advantages of MALDI-2 for the MSI of metabolites in tissue sections. Compared to conventional MALDI, MALDI-2 enabled the detection of a broader range of metabolites and increased overall sensitivity for many others that are also detected using MALDI. The advantages enable MSI to provide a deeper understanding of altered metabolism throughout heterogeneous tissues and will likely enable improvements in spatial resolution of metabolite MSI that leverage the gains in sensitivity.