Enhancing Lipid Detection and Spatial Accuracy in Carotid Plaques Using Mass Spectrometry Imaging Techniques

Abstract Matrix-assisted laser desorption/ionisation mass spectrometry imaging (MALDI-MSI) is a powerful technique for studying lipid distribution in carotid plaques, key to understanding atherosclerosis. This study aimed to improve sample preparation for MALDI-MSI-based spatial lipidomics of carotid plaques by improving both matrix application and tissue handling. Human carotid plaques were collected from endarterectomy patients with ethical approval and sectioned at 10 µm thickness for MALDI-MSI. We compared eight sample preparation methods, including hydroxypropyl methylcellulose-polyvinylpyrrolidone (HPMC-PVP) embedding media and Cryofilm-type IMS® to provide support and maintain tissue structural integrity during sectioning. Methods were assessed for signal intensity, lipid diffusion, lipid coverage, tissue morphology, and image co-registration which each criterion scored from 1-3. Cryofilm-based methods scored highest for preserving tissue morphology and minimising folding artifacts (2.9-3.0) but were limited in co-registration (2.0) due to reliance on adjacent sections. Sublimation methods generally produced greater lipid coverage with reduced lateral diffusion, while automated sprayer methods scored higher in signal intensity/sensitivity (3.0) but had increased lipid delocalisation, particularly for highly hydrophobic species such as triacylglycerols and sterols. The results highlight clear trade-offs between tissue structural preservation, lipid detection sensitivity, and spatial integrity in MALDI-MSI. Because spatial integrity cannot be compromised for imaging lipids in carotid atherosclerotic plaques, Cryofilm combined with sublimation offers a clear advantage. This work strengthens MALDI-MSI workflows enabling more precise spatial mapping and deeper biological interpretation of atherosclerotic lipid distributions.