Abstract Advanced Imaging Core C is essential to accurately define spatial and temporal organization of processes governing macrophage plasticity. Surface markers determined by flow cytometry have been primarily used to identify and sort diverse macrophage subpopulations. However, each Project makes clear that macrophage diversity is fundamentally a consequence of specific spatiotemporal intracellular signaling and coordinated transcriptional programming dictated by the niche environment. These processes control macrophage plasticity during both inflammatory lung injury and its resolution. All Projects will address how inter-cellular and paracrine signals interact with intracellular cues to alter macrophage fate in response to lung injury and resolution. Thus, the function of Core C is to provide advanced reagents and imaging methodologies required for both in vitro and in vivo studies and enable testing of hypotheses in each Project. We will provide the tools to address the fundamental question that a given signaling pathway or transcriptional program is essential for the active regulation of macrophage differentiation, specialization, and function. To gain the mechanistic signaling insights needed for the success of each Project, Core C will provide methods at both conventional and super-resolution levels to interrogate the signaling pathways in live macrophages. Spatiotemporal metabolic, transcriptional, and signaling landscape changes in response to macrophage activation by ATP, β-catenin, CREB, STAT family transcription factors and other key signals will be studied by multi-color super-resolution microscopy and super- resolution activity imaging. These analyses of macrophages will permit a fine-grained assessment of the time- sequence and spatial organization of the programs employed by macrophage variants. Although intravital imaging is the most direct way to interrogate lung injury and recovery outcomes, respiratory motion in the live animal represents a major obstacle for obtaining meaningful results by in vivo lung imaging. Thus, a major Core C function will also be to provide advanced two-photon intravital lung imaging using a novel approach to reliably identify myeloid cells in living mouse lung (in vessels and tissue) and track single cell motion, allowing each Project to directly examine the functional effects of their pathway of interest during lung injury and repair phases. By providing a seamless visual examination of signaling activity in macrophages as they differentiate, migrate, and resolve immune challenges, Core C is essential for the Program’s overall success.