Color rendition of the shape and height of thrombi formed on type I collagen fibrils at different wall shear rates. Washed blood cells and adhesive proteins were used as described in the legend to Fig3. Note that both at 300 s⁻¹ and 1,500 s⁻¹, larger thrombi were present at later perfusion times in the presence of vWF and fibrinogen (Fb) combined than either of the two ligands alone. To obtain these pseudocolor images, stacks of confocal sections taken throughout the height of thrombi at 1-μm intervals in thez-axis were processed after fixing brightness and contrast. Noise was reduced by applying a 3 × 3 median filter, and images were binarized so that the gray scale value of all the pixels above a set threshold was 255. The value of each pixel composing individual images in a shear rate group was then multiplied by a factor, p/N, where p is the plane number (distance from the surface in μm) corresponding to the image, and N is the number of images in the stack including the thrombus of maximum height in that group. After this operation, the value of each pixel in a stack of confocal sections was proportional to the position in the z axis, ie, to height, and could be used to generate single pseudocolor images using for each pixel the maximum calculated intensity value in the stack. Thrombi were larger in the experiments performed at 300 s⁻¹ than at 1,500 s⁻¹ owing to longer perfusion times; thus, distinct height calibration color scales were calculated for the two groups, as shown.