Visualization of single-cell lipidomes with MALDI-MSI

Lipids are central to cellular structure, metabolism, and signaling, and yet their distribution within and across cells is highly heterogeneous. Traditional bulk mass spectrometry masks this diversity, whereas single-cell lipidomics can uncover distinct lipid configurations that define cellular states and organize tissues. Matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) has emerged as a powerful tool to probe lipid heterogeneity beyond bulk analyses. Here, we describe a workflow for single-cell lipidomics that integrates optimized matrix deposition, high-resolution acquisition, and optical co-registration. This approach enables label-free, spatially resolved detection of endogenous lipid species with cellular precision and minimal sample perturbation. Applied to cultured cells, the method uncovers pronounced cell-to-cell variability and reveals coherent lipid domains across neighboring cells in tissues. By providing a robust and scalable strategy for visualizing lipidomes at single-cell resolution, MALDI-MSI bridges the gap between lipid localization and metabolic diversity, advancing lipidomics toward the study of cellular identity, tissue organization, and disease mechanisms.