To help overcome this limitation and broaden the overall analyte coverage and sensitivity of MSI, post-ionisation (PI) techniques that enhance ionisation efficiencies by up to 2-orders of magnitude have seen rapid development in recent years. These PI methods add an additional ionisation event that helps ionise analytes not ionised by the initial ionisation event (e.g., MALDI). A variety of PI methods have been reported including those reliant on the interaction of neutral analytes with reactive species generated by a cold plasma , dopants ionised by VUV photons and laser-post-ionisation (MALDI-2) . MALDI-2 is the most widely adopted PI method. It involves the irradiation of the MALDI plume 10-20 µs after generation by the MALDI laser with a second laser pulse below the two-photon threshold of the matrix (often <280 nm). When performed under elevated pressure conditions to maintain plume density (3-10 mbar), this induces a second MALDI-like ionisation event spatially and temporally separated from the MALDI process. MALDI-2 has shown significant benefits for lipids , pharmaceuticals , N-glycans and tryptic peptides . For example, applied to liver tissue at 20 µm pixel size, MALDI-2 was capable of detecting ~7-times more lipid species than conventional MALDI in positive-ion mode . The sensitivity gain offered by MALDI-2 has also facilitated spatial resolution improvement in MALDI-MSI of lipids to 1 µm 31 . However, the benefits of MALDI-2 for imaging low molecular weight metabolites have not been investigated. Herein, we have investigated MALDI-2 for metabolite imaging of kidney and tumour tissue and show it allows detection and imaging of a broader range of metabolites than conventional MALDI.