In vivo imaging of platelet accumulation concomitantly with endothelial calcium elevation after laser-induced injury. Platelets were labeled with anti–mouse CD41 Fab fragments conjugated to Alexa 647 infused via the jugular vein, and the endothelium of the cremaster microcirculation was loaded with Fluo-4 AM. After laser-induced vessel wall injury activation of the endothelium and thrombus formation were observed and recorded over time. (A) Composite fluorescence and brightfield images after vessel injury show Ca2+ elevation (green) in the endothelium in conjunction with and preceding platelet accumulation (red) or presence of both signals (yellow). The fluorescence signal is shown binarized for ease of visual interpretation. (B) Kinetic curves displaying the median integrated Fluo-4 fluorescence (gray curve on left y-axis) and median integrated platelet fluorescence (black curve on right y-axis) for 34 thrombi in 3 wild-type mice. Fluo-4 fluorescence originating from platelets and not the endothelium was eliminated by subtracting any Fluo-4 fluorescence in pixels where Alexa 647 fluorescence was observed.
