First, we optimised ion source parameters for the detection of low molecular weight (LMW) metabolites. We observed that reducing the high-pressure funnel RF amplitudes and reducing the ion source pressure from the conventional setting of 7.5 Torr to 4 Torr increased ion signals observed between m/z 100-1000 (Fig. ). Next, we optimised the time delay between the MALDI and PI laser pulses. The optimal delay time depends on the distance of the PI laser above the sample surface and the velocity of the MALDI plume. The optimal delay time was found to be 6 µs (Fig. , ESI †) for the detection of low mass (<m/z 400) metabolites. These optimised parameters were used for all subsequent experiments. Next, we investigated the metabolite coverage obtained using conventional MALDI and MALDI-2 using mouse kidney tissue (n=3 for MALDI and MALDI-2) at a pixel size of 60 µm. Fig. shows the averaged MALDI (black) and MALDI-2 (red) spectra from one kidney section following the removal of background peaks (see ESI † for details). MALDI-2 increased both signal intensity, e.g. the base peak at m/z 124.0078 assigned to the [M-H] -of taurine increased 4-fold (Fig. ) and the number of tissue-related signals. To further evaluate the added metabolite coverage using MALDI-2 we utilised the METASPACE annotation platform 7 and compared MALDI-2 metabolite annotations obtained at 10% false discovery rate and detected in at least 2/3 technical replicates. Compared to MALDI datasets using NEDC matrix from 140 human and mouse kidney datasets, and only considering annotations found in ≥10% of these datasets, MALDI-2 resulted in 63 unique annotations (Figure ). These results further highlight the added metabolite coverage achieved using MALDI-2.