With this high-end instrumentation grant (HEI), we apply for funds to purchase a Bruker timsTOF fleX MALDI-2 instrument for advanced matrix-assisted laser desorption/ionization (MALDI) imaging. This mass spectrometry imaging (MSI) instrument is capable of fast, high-spatial-resolution, high-spectral separation MALDI imaging, which is enabled by trapped ion mobility spectrometry (TIMS) combined with mass spectrometry in imaging mode. This instrument comes with the cutting-edge addition of a second post-ionization laser for MALDI-2 to boost signal intensity for increased sensitivity and spatial resolution. These technological improvements make the timsTOF fleX MALDI-2 MSI instrument an attractive molecular discovery tool with unprecedented molecular depth and coverage for visualizing the spatial distributions of drugs, metabolites, lipids, tryptic peptides, proteins, and N-glycans from tissue sections. The timsTOF fleX MALDI-2 will be placed in the Johns Hopkins Medical Institutions (JHMI) Applied Imaging Mass Spectrometry (AIMS) Core facility, established and directed by Dr. Glunde, the principal investigator of this HEI S10 application, who has 15 years of experience in MALDI imaging. A group of 10 NIH-funded investigators, along with Dr. Glunde, will comprise a dedicated group of major users, whose projects will greatly benefit from the availability of the Bruker timsTOF fleX MALDI-2 MSI instrument. Each major user is requesting 7-10% of instrument time. These major users are supported by a total of 14 NIH-funded R01/R21/R35 research grants on whose research outcomes the timsTOF fleX MALDI-2 instrument would have a transformative impact. They are spread across 8 Departments, which include the Departments of Radiology, Oncology, Pharmacology, Pediatrics, Infectious Disease, Biological Chemistry, Neurology, and Plastic Surgery. Several minor users will be requesting instrument time as well. Major and minor users have diverse research projects on cancer, infectious disease, antiretroviral drugs, immunotherapy, neuroscience, pediatrics, biological chemistry. Three of the users are from institutions on the East Coast, including the University of Maryland, New York University, and the University of Scranton, demonstrating that the JHMI AIMS core serves as a regional resource. The Radiology Department and JHMI have committed significant institutional funds towards the operation of this instrument, which includes the superb infrastructure of the JHMI AIMS Core facility, as well as covering running costs for the instrument, and providing dedicated technical personnel to help users with efficient sample preparation, instrument set-up and data analysis for their respective MALDI imaging applications. Online booking and billing for this instrument is available through the iLAB software in the AIMS Core.