4D imaging model of hemostatic plug formation. (A-C) Experimental setup. (A) Exposed saphenous vein. (B) Laser injury to saphenous vein was induced with Ablate! photoablation system with a 532-nm pulse laser. Accumulation and localization of various hemostatic plug components were recorded using a ZEISS Axio Examiner Z1 microscope with a 20×/1 numerical aperture water immersion objective lens and Slidebook software. (C) Sequential 150-μm Z-stacks of the forming hemostatic plug were acquired with 7.5-μm step size and 4 × 4 binning. Image analysis was performed with ImageTank Software (Visual Data Tools). Image shows surface view of platelets (gray) and ECs (yellow). Icons indicate intravascular, extravascular, and side perspective. (D-E) Visualization of platelets, ECs, fibrin, and PS at sites of vascular injury. Mice with green fluorescent protein (GFP)–expressing platelets (gray) and tdTomato-expressing endothelium (yellow) were infused with AF647-labeled antibody to fibrin (blue) (D) or AF647-labeled aV (red) (E). Images were taken at the indicated time points after laser injury. (F) Overlay of fluorescence intensity histograms for platelets (gray), ECs (yellow), and fibrin (blue) or PS (red) measured along 1 cross-sectional slice of a Z-max projection. Scale bar indicates 50 μm.