Figure 2
Optimisation of scanning-LIEI laser targeting depth. The scanning laser was targeted at the level of the endothelium (0 μm), 7 μm luminally of the endothelium (7 μm), 10 μm luminally of the endothelium (10 μm), or 14 μm luminally of the endothelium (14 μm). (a) Quantification of platelet volume 5 min after injury. (b) Quantification of fibrin volume 5 min after injury (n = 3–11 injuries from 3–5 mice). (c) Representative XY surface 3D reconstructions of thrombi (platelets blue, fibrin red), annexin V (white), and autofluorescent elastin layer (green). Blood flow is from right to left. Top row: platelet accumulation 5 min after injury. Middle row: Fibrin formation 5 min after injury. Bottom row: Annexin V binding > 30 min after injury. The laser was targeted (1) at the endothelium, (2) 7 μm luminally of the endothelium, (3) 10 μm luminally of the endothelium, or (4) 14 μm luminally of the endothelium. Note the strong annexin V binding to transverse smooth muscle cells and minimum fibrin formation after endothelium + 0 μm targeting. Also note the annexin V-positive endothelium without stable thrombus formation in the + 14 μm injury indicating that endothelial damage/activation alone is not sufficient for stable thrombus formation in this model. Scale bar and grid size 50 μm.
