Sample Preparation and Data Acquisition Strategies for High-Resolution MS Imaging of Organoids

Abstract Organoids are in vitro-generated three-dimensional multicell aggregates that are designed to mimic the structural and functional features of their corresponding organ. The field of organoids has attracted significant attention in recent years, particularly in the context of disease research, drug screening, and most notably personalized medicine. Mass spectrometry imaging (MSI), a technique yet to be fully incorporated within organoid research, facilitates the potential for nontargeted broadband molecular analysis at the single-cell level. Due to the small size and fragility of organoids, it is imperative that meticulous sample preparation be undertaken for high-resolution MSI experiments. In this study, we propose a series of methodologies for the preparation of samples, encompassing the processes of embedding and cutting, the selection of a suitable matrix, and the deposition of matrices. Additionally, we explore the acquisition of data through a range of methods, with the objective of generating sufficient MSI data at the single-cell level. These experiments, which employ high-mass-resolution, high-spatial-resolution AP-SMALDI MSI, demonstrate its potential in advancing research into cell-type-specific metabolites in organoids.