Sample preparation of Drosophila melanogaster larvae for MALDI imaging

Abstract Mass spectrometry-based lipidomics has emerged as a crucial field for unraveling the complexity of biological systems through comprehensive profiling of lipid species with unparalleled sensitivity and specificity. Among the available techniques, matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI imaging, or MALDI-IMS) stands out as a powerful spatial lipidomics tool, enabling the visualization of distribution and localization of individual lipid classes and species directly within biological tissues. The spatial context of lipids provided by MALDI imaging is especially valuable for studying nutritional and developmental processes in heterogenic tissues. The reliability and quality of MALDI imaging data critically depend on sample preparation, which must preserve both tissue morphology and analyte integrity. Here we introduce a sample preparation protocol for Drosophila melanogaster larval tissue, addressing key limitations of standard protocols such as thin-sectioning. Owing to the small size of Drosophila tissues and the flat morphology of organs such as the fat body, our approach enables direct imaging of the inner organs of the larvae without sectioning. This not only streamlines the workflow and minimizes tissue disruption but also allows simultaneous analysis of all major internal organs in a single measurement. Our approach not only facilitates high-quality MALDI-IMS data acquisition from complex larval tissues but also enables the localization of a broad spectrum of lipid species complementary to data gained by established techniques like liquid chromatography mass spectrometry (LC-MS). These advances position MALDI imaging, when coupled with our optimized preparation workflow, as an indispensable technique for spatially resolved lipidomics studies in small model organisms such as Drosophila larvae. Graphical Abstract