Enabling High-Throughput Tissue Sectioning for Robust Lipid Mass Spectrometry Imaging

Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) has been established as a powerful tool for the spatial characterization of molecules. Recent advances have been driven by improvements in the spatial resolution, acquisition time, and sensitivity. Although MALDI-MSI excels in the label-free, multiplexed detection of biomolecules, variations in sample preparation remain a significant source of experimental variability. Here, we present a workflow demonstrating the integration of a robust, completely automated tissue section processing system into a MALDI workflow. Importantly, PFA fixation and agarose embedding are demonstrated to preserve lipid compositional distributions. Combined, it enables robust and stable lipid imaging across a completely sectioned mouse brain. The integration of the Marinus MP system advances mass-spectrometry imaging toward a fully automated workflow, significantly reduces sample-preparation variability, and enables more consistent, reliable data acquisition and improved biological interpretation. Parallel with sectioning, serial two-photon (STP) tomography is used to acquire high-resolution digital images of every single section, providing a structural reference for downstream analyses. We demonstrate how the Allen Mouse Brain Atlas can be seamlessly integrated with these multimodal imaging data sets. This workflow enables precise region-specific comparisons and enriches the anatomical context with robust molecular information.