Mass spectrometry imaging (MSI) is a powerful tool that has advanced our understanding of complex biological processes by enabling unprecedented details of metabolic biology to be uncovered. Through the use of high-spatial resolution MSI, metabolite localizations can be obtained with high precision. Here we describe our recent progress to enhance the spatial resolution of matrix-assisted laser desorption/ionization (MALDI) MSI from ∼50 μm with the commercial configuration to ∼5 μm. Additionally, we describe our efforts to develop a 'multiplex MSI' data acquisition method to allow more chemical information to be obtained on a single tissue in a single instrument run, and the development of new matrices to improve the ionization efficiency for a variety of small molecule metabolites. In combination, these contributions, along with the efforts of others, will bring MSI experiments closer to achieving metabolomic scale.
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