Project Summary The brain is composed of unique cell types, such as neurons and astrocytes, that actively coordinate to enable higher order functions including learning, memory, and cognition. Even slight deviances in the molecular or cellular states of the brain can result in debilitating neurological symptoms whose severity, treatment course, and overall treatment outcome vary widely from patient to patient. This level of complexity likely contributes to promising therapeutics failing within clinical trials and, thus, requires further exploration. To date, most of our foundational knowledge of neuroscience stems from a neuron-centric focus. Recent literature has demonstrated that other cell types, such as astrocytes, may participate in signaling and communication beyond their known passive, supporting roles. As such, exploring the molecular and cellular diversity of astrocytes in their native tissue context will help us better understand neurological function and dysfunction, particularly in Alzheimer’s disease (AD). Because of the inherent spatial and chemical complexity in physiological processes and interconnectedness of cells, we will develop workflows for integrating multiresolution and multimodal imaging methods for the characterization of spatial relationships among cell phenotypes in AD compared to healthy controls. To investigate the molecular diversity within the hippocampus, we are employing a combination of mass spectrometry imaging (MSI) and multiplexed immunofluorescence (MxIF) to gain rich metabolomic information that is associated with cell type and state. MSI can detect hundreds to thousands of endogenous molecules while maintaining their spatial distributions. While chemically informative, these datasets are often difficult to correlate directly to cell type or functional state without an orthogonal technique, such as immunohistochemistry. Because the number of cell types exceed what can be probed by traditional IF approaches within a single experiment, we have chosen to incorporate MxIF, using Cell DIVE, to increase the number of imageable targets compared to traditional fluorescence microscopy. By staining for traditional cell-specific markers, we can use MxIF to connect metabolomic profiles uncovered using MSI to functionally important cellular neighborhoods in AD. Ultimately, we will establish small molecule, lipid, and cellular spatial differences between hippocampal regions of AD and control subjects using high spatial resolution MALDI mass spectrometry imaging (MSI) and highly multiplexed immunofluorescence (MxIF) (aim 1) as well as integrate multimodal data sets to examine the molecular neighborhoods associated with typical and atypical cellular neighborhoods surrounding astrocytes within AD (aim 2). By conclusion of these two aims, we will have developed workflows for integrating MSI and MxIF on the cortex from human AD and control subjects to identify molecular and cellular phenotypes and characterize their spatial r...