Stabilized imaging of immune surveillance in the mouse lung

Real-time imaging of cellular and subcellular dynamics in vascularized organs requires image resolution and image registration to be simultaneously optimized without perturbing normal physiology. This problem is particularly pronounced in the lung, in which cells may transit at speeds >1 mm s(-1) and in which normal respiration results in large-scale tissue movements that prevent image registration. Here we report video-rate, two-photon imaging of a physiologically intact preparation of the mouse lung that is stabilizing and nondisruptive. Using our method, we obtained evidence for differential trapping of T cells and neutrophils in mouse pulmonary capillaries, and observed neutrophil mobilization and dynamic vascular leak in response to stretch and inflammatory models of lung injury in mice. The system permits physiological measurement of motility rates of >1 mm s(-1), observation of detailed cellular morphology and could be applied in the future to other organs and tissues while maintaining intact physiology.