Cryogenic ToF-SIMS 3D imaging of SKOV3 ovarian cancer spheroids: Preserving native lipid distributions with Ar cluster sputtering

Mass spectrometry imaging (MSI) enables spatial mapping of molecular distributions in biological samples, but the reliability of results strongly depends on sample preparation. In this study, we investigated how air-dried and cryogenic conditions affect three-dimensional molecular imaging of multicellular spheroids using time-of-flight secondary ion mass spectrometry. A conceptual scheme illustrated the rationale: ambient drying leads to water loss and artificial redistribution of lipids, whereas cryogenic sampling preserves near-native molecular states. MSI in both positive and negative ion modes revealed pronounced differences between the two conditions. Under room-temperature drying, water-derived ions such as H3O+ were depleted, and lipids including cholesterol, sphingomyelins, and fatty acids migrated toward the spheroid surface. These patterns could be misinterpreted as compartmentalized lipid remodeling in two-dimensional images, but three-dimensional analysis demonstrated that they were artifacts of drying-induced separation and aggregation. By contrast, cryogenic sampling preserved water and maintained uniform molecular distributions throughout the spheroid volume. Key lipid species were further validated by liquid chromatography–tandem mass spectrometry, confirming characteristic fragment patterns of cholesterol, sphingomyelins, and phosphatidylinositols 38:4. Together, these results establish cryogenic preparation as essential for accurate three-dimensional MSI of soft biological systems. Although certain limitations remain—such as reduced sputtering efficiency and blurred boundaries due to water retention—cryogenic MSI provides a more faithful representation of native molecular organization, advancing its potential for biomedical research and molecular pathology.