ABSTRACT The focus of this Program Project is to investigate the molecular and signaling mechanisms underlying the immune regulatory role of the pulmonary endothelial niche during inflammatory lung injury and resolution of injury. The Intravital Imaging and Physiology Core (Core D) is essential to precisely define how dynamic changes in the pulmonary endothelium during injury and repair programs the immune cells to either promote or resolve inflammatory lung injury. Each Project makes clear that spatio-temporal signaling regulated by endothelial cell-surface receptors and mitochondria is fundamentally important in regulating the endothelial immune niche and defining the outcome of lung injury and resolution. Thus, all Projects, using endothelial- specific genetically altered living mouse models, will address how signals emanating from basal and activated endothelial cells alter neutrophil/monocyte/macrophage fate in response to lung injury and contribute to resolution. Respiratory motion in the live animal represents a major obstacle for obtaining meaningful results from in vivo imaging studies of the normally respiring lung. We have developed a novel imaging approach to monitor pulmonary microvessels and immune cell trafficking during injury and repair. Thus, a major Core D function will be to provide advanced two-photon intravital lung imaging expertise to reliably quantify the activation of endothelium, to measure lung vascular leak, and to identify the role of activated pulmonary vascular niche in regulating transendothelial migration of myeloid cells in the living mouse lung. Each Project in conjunction with Core D will examine the functional effects of their pathways of interest during inflammatory lung injury and repair phases. Imaging analyses will include the assessment of transmigration of phagocytic cells and in situ phagocytic and efferocytotic activities of immune cells as programmed by endoplasmic reticulum-localized S1PR1 (sphingosine-1-phosphate receptor-1) in endothelial cells in Project 1; CHFR (an E3 ligase, checkpoint with Forkhead and ring finger domains)-induced degradation of VE-cadherin in endothelial cells in Project 2; and activation of endothelial cell mitophagy and mitochondrial biogenesis in Project 3. By providing visual in vivo examination of endothelial activity and its impact on myeloid cell functions and physiological assessments during inflammatory injury and repair, Core D will be essential for the Program’s success.