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Due to movement of the specimen, vasodilation, and intense clutter, the intravital location of a vessel boundary from video microscopy is a difficult but necessary task in analyzing the mechanics of inflammation and the structure of the microvasculature. This paper details an active contour model for vessel boundary detection and tracking. In developing the method, two innovations are introduced. First, the B-spline model is combined with the gradient vector flow (GVF) external force. Second, a multiscale gradient vector flow (MSGVF) is employed to elude clutter and to reliably localize the vessel boundaries. Using synthetic experiments and video microscopy obtained via transillumination of the mouse cremaster muscle, we demonstrate that the MSGVF approach is superior to the fixed-scale GVF approach in terms of boundary localization. In each experiment, the fixed scale approach yielded at least a 50% increase in root mean squared error over the multiscale approach. In addition to delineating the vessel boundary so that cells can be detected and tracked, we demonstrate the boundary location technique enables automatic blood flow velocity computation in vivo.