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We recently developed a dedicated focusing multi-pinhole collimator for a stationary SPECT system that offers down to 120  µ m (or 1.7 nL) spatial resolution SPECT images of cryo-cooled tissue samples (EXIRAD-3D). This collimator is suitable for imaging isotopes that are often used in small animal and diagnostic SPECT such as  125 I (27 keV),  201 Tl (71 keV),  99m Tc (140 keV), and  111 In (171 and 245 keV). The goal of the present work is to develop high-resolution pinhole imaging of tissue samples containing isotopes with high-energy photon emissions, for example, therapeutic alpha and beta emitters that co-emit high energy gammas (e.g.  213 Bi (440 keV) and  131 I (364 keV)) or 511 keV annihilation photons from PET isotopes. To this end, we optimise and evaluate a new high energy small-bore multi-pinhole collimator through simulations. The collimator-geometry was first optimised by simulating a Derenzo phantom scan with a biologically realistic activity concentration of  18 F at two system sensitivities (0.30% and 0.60%) by varying pinhole placements. Subsequently, the wall thickness was selected based on reconstructions of a Derenzo phantom and a uniform phantom. The obtained collimators were then evaluated for  131 I (364 keV),  213 Bi (440 keV),  64 Cu (511 keV), and  124 I (511 + 603 keV) with biologically realistic activity concentrations, and also for some high activity concentrations of  18 F, using digital resolution, mouse knee joint, and xenograft phantoms. Our results show that placing pinhole centres at a distance of 8 mm from the collimator inner wall yields good image quality, while a wall thickness of 43 mm resulted in sufficient shielding. The collimators offer resolutions down to 0.35 mm, 0.6 mm, 0.5 mm, 0.6 mm, and 0.5 mm when imaging  131 I,  213 Bi,  18 F,  64 Cu, and  124 I, respectively, contained in tissue samples at biologically achievable activity concentrations.

EXIRAD-HE: multi-pinhole high-resolution ex vivo imaging of high-energy isotopes