New developments in molecular imaging techniques like small animal SPECT systems are important tools to analyze mouse models of human diseases. The main subjects of this thesis are simulation, construction and image reconstruction algorithms needed for the development of a small-animal SPECT system called U-SPECT. With the U-SPECT it is possible to study the function of organs and tissue in vivo at sub-half millimeter scale using radioactively labeled tracers. The first prototype (U-SPECT-I) is based on a clinical SPECT system with three gamma cameras. A custom build collimator contains 75 gold pinholes, focused to a small field of view to obtain a high photon count yield from the organ under investigation. Since the pinholes simultaneously observe the animal from a large number of angles, there is no need to rotate the animal or the gamma cameras. The performance in terms of achievable resolution and sensitivity of this system are characterized. To scan a volume larger than the field of view, the animal is stepwise moved through the focus of the pinholes. A specially adapted iterative reconstruction method is presented, which exploits all projections simultaneously to reconstruct the entire volume sampled. This method is validated using simulation studies, physical phantom experiments as well as total-body in vivo mouse imaging. Accurate modelling of the gamma photons and the detector response in the system matrix is important to obtain good reconstructed images. The system matrix for the U-SPECT is obtained using point source measurements. The U-SPECT-II is based on the same fundamentals of the U-SPECT-I but is a stand alone system, specially designed for small animal imaging. Several collimators are available for both rats and mice with a variety of pinhole diameters. Optical images of the animal are used to mark the boundaries of the volume to be scanned. A user friendly interface allows easy selection of this volume from single organ to total body scanning. In vivo cardiac, kidney, tumor, and bone images show that U-SPECT-II can be used for novel applications in the study of dynamic biological systems with a resolution of 0.35 mm for mice and 0.8 mm for rats. The static design, high resolution and high sensitivity of the U-SPECT systems are well suited for dynamic imaging. This is demonstrated by imaging dopamine transporters in the mouse brain during a range of points in time. Applied to the dopamine system of different models of disease, we expect that this will aid the understanding of dynamic processes of this neurotransmitter. The U-SPECT-I and U-SPECT-II systems are equipped with large scintillation cameras which have a spatial resolution of a few millimeters. We expect that significant improvements are possible by using high resolution detectors. The design of U-SPECT-III is presented, which uses a set of high resolution detectors based on CCD’s. The U-SPECT-III is compared with a dual pinhole SPECT system through simulation. Reconstructed images show much more detail with U-SPECT-III than with dual pinhole SPECT
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