Activation of arteriolar endothelium in vivo by laser-induced injury. The endothelium of the cremaster microcirculation was loaded with either Fluo-4 AM or DIOC₆ by systemic infusion of Fluo-4 AM/Cremophore or DIOC₆ via the femoral artery. A period of 20 minutes was allowed after infusion for uptake and de-esterification of Fluo-4 AM or uptake of DIOC₆ within the endothelium. Concurrently platelet accumulation was inhibited by infusion of eptifibatide. After laser-induced vessel wall injury, changes in endothelial Ca²⁺ levels were observed by excitation at 488 nm. (A) Representative composite fluorescence and brightfield images after vessel injury show Ca²⁺ elevation in the endothelium in the absence of platelet accumulation. The fluorescence signal is presented as a pseudocolor intensity map where blue represents the least intense and red represents most intense fluorescence signal. (B) Calcium elevation after vessel injury as determined by the median integrated fluorescence intensity (y-axis) from Fluo-4–loaded endothelium (top black curve, 27 thrombi from 3 mice) in comparison to the median integrated fluorescence of the inert dye DiOC₆ (bottom gray curve, 15 thrombi from 3 mice). (C) Propagation of Ca²⁺ elevation and endothelial activation along the vessel after injury is presented as a pseudocolor intensity map as in panel A. Image is representative of peak endothelial activation, the site of injury is marked (X) and one line demarking the vessel wall on the same side as the injury (purple) and a second demarking the opposing side (gray). (D) Representative trace from a single experiment showing quantitation of the Fluo-4 fluorescence signal longitudinally along each vessel wall. A line was drawn along each wall and the intensity of the pixels at each step along this line was determined and plotted. The start of the line (0 pixels) is bottom right and the end (∼ 300 pixels) is top left.