Quantitative Pulmonary Neutrophil Dynamics using Computer-Vision Stabilized Intravital Imaging.

In vivo intravital imaging in animals models in the lung remains challenging due to respiratory motion artifacts. Here we describe a novel intravital imaging approach based on the computer vision stabilization algorithm, Computer-Vision STabilized intravital imaging (CoVSTii). This method corrects lung movements and deformations at sub-micron precision in respiring mouse lungs. The precision enables high-throughput quantitative analysis of intravital pulmonary polymorphonuclear neutrophil (PMN) dynamics in lungs. We quantified real-time PMN patrolling dynamics of microvessels in the basal state and PMN recruitment resulting from sequestration in a model of endotoxemia in mice. We focused on determining the marginated pool of PMNs in the lung. Direct visualization of marginated PMNs revealed that they are not static but highly dynamic and undergo repeated cycles of "catch and release". PMNs briefly arrest in larger diameter capillary junction (~10 μm) and then squeeze into narrower ~5 μm diameter vessels through PMN deformation. We also observed that the sequestered PMNs in lung microvessels lost their migratory capabilities in association with cell morphological change following prolonged endotoxemia. These observations underscore the value of direct visualization and quantitative analysis of PMN dynamics in lungs to study PMN physiology and pathophysiology and role in inflammatory lung injury.