Gold nanoparticles (GNPs) as promising radiation sensitizers have been increasingly studied in a wide range of radiotherapy applications. By detecting the characteristic x-ray fluorescence (XRF) photons, x-ray fluorescence computed tomography (XFCT) can simultaneously determine both the spatial distribution and concentration of GNPs in vivo, affording for cancer diagnosis and irradiation guidance. However, the long scanning time of current single-pixel detectorbased configuration hinders the translation of XFCT to preclinical and clinical applications. This study presents a conebeam XFCT system using pixelated photon-counting detector with pinhole collimator to acquire XRF projection image in one motion, eliminating the previously step-by-step translation of objects, which allows fast whole-body GNP imaging. We have 3D printed a heat-resistance mold kit to cast a cone-beam x-ray source collimator using Cerrobend alloy. We selected HECITEC (High Energy X-ray Imaging Technology) as the XRF detector, in view of its high spatial resolution (0.25 mm of pitch) and energy resolution (800 eV FWHM at 60 keV). We have customized a 2-mm pinhole collimator to provide spatial information of XRF signals. We have also evaluated the roles of pixel binning and spectrum denoising in aspects of XRF peal extraction. Phantom experiments with GNP of different concentrations (0.078~2.5wt.%) were used to evaluate the sensitivity of GNP detection. In vivo experiments on mouse intravenously administered GNPs were used to validate the feasibility of the proposed system in terms of GNP biodistribution imaging. The results of this study will be helpful to guide XFCT development for routine in vivo GNP imaging
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