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Our group has recently developed a cone-beam micro-CT imaging system that is capable of imaging a variety of ex vivo and in vivo structures with varying levels of magnification and spatial resolution. The micro-CT imaging system consists of a microfocal X-ray source, a custom designed 7-axis micro-positioning system and a high-resolution X-ray image intensifier coupled to a 2048×2048 CCD camera. A tent-FDK filtered backprojection method based on a circular data acquisition is used for 3D reconstruction of the micro-CT data sets. An open-source software application, developed at the Ohio Supercomputer Center (OSC), is used for real-time visualization and graphics display of the 3D micro-CT data. This imaging system has been used in a variety of novel bone imaging applications, i.e., to monitor bone resorption longitudinally in vivo in a rat fibula osteotomy model, to evaluate the trabecular structure in tibias from 2-week old transgenic 'knock-out' mice, and to obtain high-resolution images of the human temporal bone for use in a surgical training simulator.