The ability to visualize the presence, abundance, location and functional state of many proteins at once in cells and tissues with preserved morphology has long been a goal. This proposal seeks to optimize recently demonstrated mass-spec-based imaging methods that can, when fully developed, detect as many as 40 to 100+ molecular targets in cells and tissues using simultaneously applied mass-tag-labeled antibodies, with outstanding morphology. The technique is analogous to laser-scanning confocal microscopy, except that the scanning is performed using a tightly focused ion beam, rather than a similarly sized point of laser light. And instead of fluorophores, the labels detected are clustes of metal atoms attached to any given probe. Preliminary data using a highly focused, scanning oxygen ion beam and 10 antibodies applied simultaneously to clinical breast cancer tissues demonstrate histology-like images that reveal bound antibody location with subcellular resolution. Going forward, we would like to optimize sensitivity and reliability, with the goal of making this a standard, high-performance, imaging approach for basic and translational science discovery, drug development, and possible clinical deployment. The present project will address: reliable pre-analytical sample preparation methods and labeling techniques for protein analytes; ion-beam imaging method improvements based on current and projected instrumentation; powerful, easy-to-use software for equipment control, display and analysis; and validation in several important biological test cases.