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Mass Spectrometry Imaging (MSI) has evolved as an exciting technology that can be used to map lipids on tissue surfaces, with many research, industrial and clinical applications. A wide variety of lipids can be spatially localised in both positive and negative modes (e.g. highlighted in cancer, skin and brain) and ionisation approaches such as Matrix-Assisted Laser Desorption/Ionisation (MALDI), Secondary Ion Mass Spectrometry (SIMS) and Desorption Electrospray Ionisation (DESI) bring different strengths and weaknesses, including mass ranges and resolution, fragmentation profiles and spatial resolution. This chapter explores the technical differences of the ionisation types applied to lipid MSI, and offers insight into appropriate choices for spatially profiling specific lipid classes. Novel approaches to enhance ionisation through derivatisation, matrix chemistry and nanomaterials offer the potential to reveal a greater proportion of the lipidome. MSI offers opportunities to profile lipid classes, but in the absence of chromatography cannot yet confidently assign stereochemistry, a weakness that may be addressed using ion mobility. Advances in integrating ion mobility and improvements in handling the large datasets generated, in combination with current methodologies, offer exciting future opportunities to decipher the complex lipid patterns in health and disease.

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