Summary/Abstract A central goal of the NIH Brain Initiative is to develop new imaging tools sufficiently powerful to spatially map at high resolution the neuronal circuitry and underlying molecular composition of the brain. While cutting edge imaging tools and related labeling techniques have been developed, it is still a major challenge to map the spatial distribution at different length scales of the thousands of biomolecules, including expressed proteins, which play key roles in brain function. The goal of this Phase II project is to evaluate the ability of a new tissue imaging technology developed by AmberGen, termed MALDI-IHC, to rapidly create a highly multiplexed, multiomic and multimodal 3D molecular map of the mouse brain. The development of MALDI-IHC for whole brain imaging will provide neuroscientists with an important new tool for exploring the underlying molecular basis of brain function and neurodegenerative disorders. MALDI-IHC is based on the use of novel photocleavable mass-tags (PC-MTs) developed by AmberGen which when linked to antibody or lectin probes enable targeted biomolecules to be identified in the mass spectrometric image. This approach significantly exceeds the multiplex capability of fluorescence immunohistochemistry (IHC) and previous cleavable mass-tag based methods which are generally limited to 5 biomarkers or require extensive cycling procedures. It also exceeds the capability of metal-tagged antibody techniques such as IMC and MIBI which can probe small mm2 regions at subcellular resolution but are limited to approximately 40 antibody probes and require several days to scan a whole tissue section. In contrast, MALDI-IHC can image an entire mouse brain FFPE section for over 100 targeted proteins at 40 µm resolution in less than one hour. The ability of MALDI-IHC to perform label-free, untargeted small molecule mass spectrometric imaging (MSI), fluorescence imaging using unique dual-labeled fluorescent-PC-MT probes and high-plex imaging of intact expressed proteins including glycosylation patterns on the same tissue section greatly extends the power of this approach. During Phase I, we demonstrated the feasibility of this combined approach on mouse brain FF and FFPE tissue specimens. During Phase II, we will develop methods using MALDI-IHC to reconstruct whole mouse brain protein expression maps at 40 µm voxel resolution. FFPE sagittal and coronal mouse brain tissue slices from mouse brain will be probed by MALDI-IHC using a panel of 50 NeuroMab PC-MT antibodies and 25 PC-MT lectins. Validation of individual PC-MT probes will be performed by comparing MALDI-IHC and fluorescence IHC images. A 3D tri-modal map of the mouse brain merging both metabolites and expressed proteins will also be reconstructed based on a demonstrated workflow that involves MSI of unlabeled small molecules from successive FF specimens, IHC staining with a 75-plex panel of PC-MT probes including some dual-labeled PC-MT antibodies, and fluores...