For patients presenting with acute myocardial infarction (AMI) or stroke, the primary goal of therapy is to promptly re-open blocked arteries (recanalization) to salvage the dying ischemic tissue. Despite successful recanalization of major arteries, blood perfusion in surrounding microvasculature supplying the tissue can remain poor, a common complication of ischemia reperfusion (IR) injury, known as microvascular obstruction (MVO). MVO occurs in 60% of AMI patients, and persistent MVO can lead to progressive worsening of heart function and infarction. Several pathogenic processes have been implicated in MVO, however targeted therapies have not been effective in improving microvascular perfusion. This is due in part to the lack of suitable animal models and technical difficulties associated with performing real-time imaging on the microvasculature. Using an in vivo model of gut IR injury which allows us access to microvasculature in living animals during IR injury, in combination with precision imaging systems (confocal and scanning electron microscopy), we have observed previously unappreciated in vivo changes within the microvasculature during IR. Our studies demonstrate that ischemia and reperfusion phases trigger phenotypically distinct endothelial dysfunction and death, associated with the onset of distinct vessel occlusion mechanisms involving red blood cells, platelets and neutrophils – ultimately cooperating to lead to MVO in local gut and remote organs. These findings not only demonstrate an intimate spatiotemporal relationship between endothelial injury and vaso-occlusion mechanisms, they also help explain why existing therapies remain ineffective.