biostudies-literatureHendriks TFEInterreg Vlaanderen-NederlandDutch Research Council (NWO)Fonds Wetenschappelijk OnderzoekNWO-STEM42660https://www.ebi.ac.uk/biostudies/studies/S-EPMC12663239biostudies-literatureUnknown15(1)Understanding tissue complexity requires spatially resolved multi-omics data at single-cell resolution. Here, we present a workflow integrating high-resolution matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) with Xenium spatial transcriptomics (SPT) on a single tissue section. This strategy ensures pixel-scale spatial correspondence between metabolic and transcriptomic features, avoiding misalignment issues of serial sections, where even minor offsets result in sampling different cells. We investigated MALDI-MSI compatibility with downstream SPT revealing that the number of transcripts per cell decreased by ~ 30% after MSI, whilst cell recovery and cell-type assignments are preserved. Validated using mouse brain and demonstrated using human glioblastoma tiScientific reportsOne section, two worlds: single-cell integration of MALDI-MSI and spatial transcriptomics on the same single tissue section.PMC12663239TBM T001919N19013Molecular Brain Tumor DetectorVisvikis THeeren RMAEijkel GBCuypers EBalluff BHendriks TFEfalseOne section, two worlds: single-cell integration of MALDI-MSI and spatial transcriptomics on the same single tissue section.Understanding tissue complexity requires spatially resolved multi-omics data at single-cell resolution. Here, we present a workflow integrating high-resolution matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) with Xenium spatial transcriptomics (SPT) on a single tissue section. This strategy ensures pixel-scale spatial correspondence between metabolic and transcriptomic features, avoiding misalignment issues of serial sections, where even minor offsets result in sampling different cells. We investigated MALDI-MSI compatibility with downstream SPT revealing that the number of transcripts per cell decreased by ~ 30% after MSI, whilst cell recovery and cell-type assignments are preserved. Validated using mouse brain and demonstrated using human glioblastoma ti2025-01-01T00:00:00Z2025 Nov2026-06-05T21:35:14.514Z2026-05-22T03:12:24.23ZS-EPMC126632394131539610.1038/s41598-025-26735-1