1) Project Summary/Abstract The estimated 20,000 genes in the human genome are variably expressed in the body’s organs and tissue, each made of heterogeneous cell types characterized by shared and cell-type-specific gene expression profiles. The biological function of a gene frequently depends on the spatial context within a healthy organ and tissue. Many human disorders, including developmental diseases, cancer, and neurodegenerative diseases, result from the deregulation of the spatial organization of cells within a tissue as well as their impaired gene expression. Systematic annotation of gene and protein expression is a crucial step in understanding the biological complexity, elucidating cellular identity, deciphering disease mechanisms, etc. However, most routine transcriptomics and proteomics technologies overlook the relationship between the disease state and spatially delineated alteration in gene and protein expression that exists in many human diseases. Preserving the spatial context of gene and protein expression is essential to gain deeper insights into tissue biology and the manifestation of disease pathology. Digital spatial profiling based on in situ RNA imaging and in situ sequencing has emerged as promising tools that could allow an analysis of cellular transcriptomes within their spatial context in tissue sections. The GeoMx Digital Spatial Profiling platform, introduced in 2019, represents a recent advancement that provides morphological context to high-plex protein or gene expression profiling from just one tissue section on a slide. In this system, spatial profiling of RNA and protein is performed on the GeoMx DSP platform, which includes imaging and fluidic components to capture spatial context as micropipette aspirates into 96-well plates. The samples are read on the nCounter, which provides a multiplexed measurement of transcripts and protein with a high level of precision while retaining spatial resolution using a direct, digital counting technology. Remarkably, the implementation of the technology allows one to profile up to 96 protein targets, ~800 RNA targets, or even multiplexing RNA and protein quantification with spatial resolution on the same formalin-fixed, paraffin-embedded tissue section on a microscope slide. Even within a year of its introduction, the GeoMx platform has been successfully used in a range of biological investigations, elucidating the versatility of the system for biomedical discovery and translational research. Implementation of this technology in a shared microscopy core at the University of South Florida Morsani College of Medicine will significantly benefit a large number of NIH-funded investigators engaged in biomedical research in fields as broad as Alzheimer’s disease, aging, stress and trauma, neuroinflammation, addiction, oncogenesis, innate immunity, infectious diseases, parasite-host interaction, computational and integrative biology, and gene therapy. The seamlessly integrated digita...