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Mass spectrometry imaging (MSI) is a powerful technology in biomedical research, made available to the Johns Hopkins research community in the Applied Imaging Mass Spectrometry (AIMS) Core. The most widely used ionization technique used in overall MSI is matrix-assisted laser desorption/ionization (MALDI) imaging because of its high imaging speed and high spatial resolution. Spatially resolved MALDI imaging measurements are directly taken from frozen, formalin-fixed, or formalin-fixed paraffin-embedded (FFPE) tissue sections or cells deposited or grown on slides without destroying them. MALDI imaging combines mass spectrometric analyses of biomolecules with simultaneous histological evaluation to analyze intact proteins, tryptic peptides (on-tissue tryptic digest), N-glycans (on-tissue PNGase digest), peptides, lipids, metabolites, drug molecules, and drug metabolites in a spatially resolved manner. This course will cover the basics of MSI and then focus on MALDI imaging in including sample preparation, on-tissue digests and derivatizations, and data analysis and consists of online modules, quizzes, journal club, and a hands-on MALDI imaging in the AIMS Core including data analysis.