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An optical landmark-based pose measurement and tracking system has been developed to provide 3D animal position data for a single photon emission computed tomography (SPECT) imaging system for non-anesthetized, unrestrained laboratory animals. The animal position and orientation data provides the opportunity for motion correction of the SPECT data. The tracking system employs infrared (IR) markers placed on the animal's head along with strobed IR LEDs to illuminate the reflectors. A stereo CMOS camera system acquires images of the markers through a transparent enclosure. Software routines segment the markers, reject unwanted reflections, determine marker correspondence, and calculate the 3D pose of the animal's head. Recent improvements have been made in this tracking system including enhanced pose measurement speed and accuracy, improved animal burrow design, and more effective camera positioning for enhanced animal viewing. Furthermore, new routines have been developed to calibrate the SPECT detector head positions relative to one another and to align the coordinate systems of the optical tracking cameras with the SPECT detectors. This alignment enables motion-corrected SPECT image reconstruction. Phantom experiments validate the accuracy of the tracking system to better than 0.1 mm accuracy, and live mouse tracking results demonstrate that reliable, accurate tracking measurements can be consistently achieved during the entire 360-degree SPECT image acquisition.