Quantitative MSI Evaluation of Pathogen Inactivation Strategies for Spatial Lipidomics

Abstract

Pathogen inactivation methods are essential for safe downstream molecular analyses of tissues containing infectious organisms, yet their impact on lipid integrity and spatial resolution in mass spectrometry imaging (MSI) remains incompletely understood. Here, we systematically evaluate the effects of three common pathogen inactivation methods: gamma irradiation, heat inactivation, and neutral buffered formalin (NBF) fixation on phospholipid profiles in mouse kidney tissue using matrix-assisted laser desorption/ionization MSI (MALDI-MSI). Tissue sections were analyzed in positive and negative ion modes after inactivation, with normalization to lipid class-specific deuterated internal standards enabling in-class quantitative MSI (Q-MSI) analysis of the inactivation effects. All inactivation methods generally preserved renal morphology and spatial lipid organization across cortical, medullary, and pelvic regions. Gamma irradiation and heat inactivation induced moderate, broadly distributed reductions in phospholipid signals, with limited class-specific effects and minimal spatial disruption. In contrast, NBF fixation produced pronounced and selective suppression of phosphatidylethanolamine (PE) and phosphatidylserine (PS), consistent with formalin-mediated cross-linking of amine-containing headgroups. Despite these chemical effects, lipid localization patterns remained intact across all conditions. While the general tissue distribution patterns remained intact for all inactivation methods, fine-level spatial defects were noted across all and were unique by ion. Normalization to deuterated standards significantly reduced apparent variability, demonstrating that observed differences are primarily driven by localized ionization conditions and efficiency rather than spatial redistribution. Overall, our results demonstrate that pathogen inactivation strategies differentially affect lipid abundances while largely preserving spatial organization─although with loss of detail in fine tissue structures─emphasizing the importance of method selection and appropriate normalization in MALDI-MSI lipidomics of inactivated tissues.