Figure 4
Optimisation of scanning-LIEI injury size (ROI diameter). Injuries were generated using different sized scanning ROIs. (a) Quantification of platelet volume 5 min after injury using different ROI diameter (25 μm, 50 μm, 75 μm, or 100 μm). (b) Quantification of fibrin volume 5 min after injury using different scanning ROI diameter (25 μm, 50 μm, 75 μm, or 100 μm) (n = 3–11 injuries from 3 to 5 mice). (c) Representative XY surface 3D reconstructions of thrombi (platelets blue, fibrin red), 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: Thrombus side view (YZ 3D surface reconstruction) 5 min after injury. The ROI diameter was (1) 25 μm (note the autofluorescence of damaged tissue in the 488 nm (green) and 546 nm (red) channels (seen as yellow in the fibrin image) causing an artificially high signal in the fibrin (564 nm) channel (#) (see also Supplementary Fig. 1C) (2) 50 μm (3) 75 μm or (4) 100 μm. Note the height of the thrombus exceeding our 100 um piezo stage Z-range limit when using the 100 um ROI. Scale bar and grid size 50 μm.
