Abstract
Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) is a rapidly advancing technology for biomedical research. As spatial resolution increases, however, so do acquisition time, file size, and experimental cost, which increases the need to perform precise sampling of targeted tissue regions to optimize the biological information gleaned from an experiment and minimize wasted resources. The ability to define instrument measurement regions based on key tissue features and automatically sample these specific regions of interest (ROIs) addresses this challenge. Herein, we demonstrate a workflow using standard software that allows for direct sampling of microscopy-defined regions by MALDI IMS. Three case studies are included, highlighting different methods for defining features from common sample types─manual annotation of vasculature in human brain tissue, automated segmentation of renal functional tissue units across whole slide images using custom segmentation algorithms, and automated segmentation of dispersed HeLa cells using open-source software. Each case minimizes data acquisition from unnecessary sample regions and dramatically increases throughput while uncovering molecular heterogeneity within targeted ROIs. This workflow provides an approachable method for spatially targeted MALDI IMS driven by microscopy as part of multimodal molecular imaging studies.