Comprehensive MSI-based protocols for the spatial lipidomics characterization of microscale organisms

Spatial omics technologies are essential for characterizing molecular disruptions induced by pathological conditions or environmental stressors while preserving the native spatial context of biomolecules. Among these, mass spectrometry imaging (MSI) has become a central platform for spatial lipidomics. However, applying MSI to small and fragile organisms such as zebrafish embryos (ZFEs) presents significant challenges. In this study, we introduce an integrative MSI-based workflow for spatial lipidomics, optimized for ZFEs but broadly adaptable to other delicate biological microsystems. The workflow integrates a refined sample preparation protocol that enabled high-quality consecutive tissue sections with a systematic comparison of ionization strategies, including matrix-assisted laser desorption/ionization (MALDI), surface-assisted laser desorption/ionization (SALDI), and advanced laser post-ionization (MALDI-2), achieving reproducible multimodal MSI analysis. Beyond comparative assessment, our results provide new insights into the ionization yields for each MSI platform. MALDI-2 stands out as a technique for spatial lipidomics, demonstrating superior ionization efficiency and enabling high-resolution imaging down to a pixel size of 5 μm. This pixel size enhancement improves visualization of anatomical structures and detailed lipid mapping, which are critical for understanding lipid biology in these small models. While MALDI proved highly effective for glycerophospholipids and sphingolipids, SALDI showed better performance for low-molecular-weight and neutral lipids. These results establish a conceptual model linking experimental conditions and analyte properties to optimize ionization across lipid classes, providing a robust framework for MSI-based spatial lipidomics in ZFEs. Therefore, this advancement contributes to the broader application of spatial omics using novel biological systems on biomedical, pharmacological and toxicological research. • Spatial analysis of small organisms like ZFEs requires tailored approaches. • Embedding and orientation are key for successful spatial analysis of ZFEs. • MALDI-2 enabled significant improvement on the spatial characterization of ZFEs. • Significant ionization differences were observed across MSI modalities. • Research hypotheses should guide the selection of MSI modality used.