ABSTRACT The overall goal of this Program Project is to test the novel hypothesis that vascular endothelial cells (EC) serve as an immune active niche in the lungs during injury and repair. The Cellular Imaging Core (Core C) is essential for accurate identification and tracking of the spatiotemporal organization and expression of specific molecules in the EC regulating the immune niche. The function of the Core is to provide advanced reagents and imaging methodologies required for both in vitro and in vivo analysis of immune function and to enable testing of the hypotheses across the Program. We will provide the tools to address how the active regulation of endothelial cell-surface receptor signaling and mitophagy-mediated recognition of phagocytes by EC promote innate immune signaling in the lung. To gain the mechanistic signaling insights needed for the success of each Project, the Core will provide methods at both the conventional and super-resolution levels to identify and interrogate the signaling pathways. Using multi-channel microscopy and super-resolution activity imaging, we will study spatiotemporal signaling induced through internalization of tyrosine phosphorylated sphingosine-1-phosphate receptor1 (S1PR1) in EC and the role of the EC-expressed ubiquitin ligase CFHR in regulating VE-cadherin expression and function. These analyses will permit a fine-grained assessment of the time-sequence and spatial organization of the immune signaling in lung EC and how it impacts lung function. By providing a seamless visual examination of a wide range of signaling activities in individual EC downstream of above listed EC-receptors during inflammatory injury and repair, the Core is essential for supporting and testing the molecular and functional hypotheses advanced by the Projects, and crucial to the Program’s overall success.