Quantitative imaging of gold nanoparticle distribution in a tumor-bearing mouse using benchtop x-ray fluorescence computed tomography

X-ray fluorescence (XRF) analysis, which has a long history of use for biological samples1,2,3, operates on the premise of stimulating the production of XRF photons (i.e., characteristic x-rays) from a sample, typically using an excitation source of monochromatic x-rays, such as a synchrotron source. The resulting XRF and scattered photons are detected and analyzed in order to identify and quantify the elements contained within. Detection of XRF photons also allows a tomographic imaging technique known as x-ray fluorescence computed tomography (XFCT). With XFCT, the identity, quantity, and spatial distribution of elements within imaging objects can be simultaneously determined4,5,6,7,8,9,10,11. Owing to the spectroscopic nature of XRF-based analysis, XFCT offers fundamentally distinct advantages over attenuation/contrast-based imaging modalities (e.g., x-ray CT and its variants), such as inherent specificity due to the element-specific energies of XRF photons. Therefore, XFCT can be a powerful modality for molecular imaging of high-atomic-number (high-Z) probes such as metallic nanoparticles. Among various metallic nanoparticles, gold (Z = 79) nanoparticles (GNPs) have attained much popularity in recent years for biomedical applications, including cancer imaging and therapy12,13,14,15,16,17,18. For the purpose of x-ray imaging, in particular, GNPs offer noteworthy advantages over conventional contrast agents (e.g., iodine), such as higher photoelectric interaction probability and more favorable biochemical properties (e.g., slower clearance and more specific tumor targeting). GNPs are also biologically non-reactive and chemically inert. Moreover, gold is an ideal element for XFCT of small-animal size objects (e.g., <10 cm in diameter) due to its high fluorescence yield and relatively energetic (i.e., penetrating) K-shell XRF photons (e.g., Kα1 and Kα2 at 68.8 and 67.0 keV, respectively). In order to produce K-shell XRF photons from gold, the excitation x-ray energy must be above the K-edge of gold (80.7 keV). The implementation of XFCT with a synchrotron as a monochromatic source of x-rays above the K-edge of gold, while ideal, is generally regarded as impractical for routine biomedical imaging tasks, mainly because of the limited accessibility of synchrotron facilities and the high dose rate of synchrotron x-rays. We therefore explored the possibility of implementing XFCT using ordinary polychromatic x-ray sources in a benchtop setting (benchtop XFCT). While earlier investigations of benchtop XFCT produced disappointing results, our initial experimental study19 in which we used a pencil beam of polychromatic x-rays resulted in the first successful demonstration of the use of benchtop XFCT to image small-animal-size objects containing biologically relevant concentrations of GNPs (≤ ~1–2% by weight, or wt%). Subsequently, adopting similar pencil beam approaches, other studies20,21,22 explored multiplexed imaging with other metal probes and the many nuances of a pencil beam-based benchtop XFCT system. Meanwhile, we have developed a cone beam implementation of XFCT23,24 that, while technically more complex25, offers distinct advantages over the pencil beam approach, such as parallel XRF signal acquisition, and is also absolutely crucial for making XFCT suitable for in vivo imaging under the practical constraints of x-ray dose and scan time. In a more recent study, we investigated optimization of the incident polychromatic x-ray spectrum (e.g., filtration and quasi-monochromatization)26 as it pertains to the interplay among XRF signal production/detection, dose, and overall scanning time, in order to expedite the development of a benchtop XFCT system capable of in vivo imaging. The basic concepts and an overall workflow for benchtop XFCT with GNPs are illustrated in Fig. 1. Figure 1 Schematic representation of benchtop XFCT imaging of a mouse injected with GNPs. Here, we report the latest advancement in the course of benchtop XFCT development: a postmortem animal imaging study in which we have successfully applied benchtop XFCT to image a tumor-bearing mouse injected with GNPs. In contrast to previous phantom studies, this investigation was performed with an actual animal exhibiting a realistic biodistribution of GNPs in various organs/tissues and a tumor. This study clearly demonstrates the unique capabilities of benchtop XFCT under the conditions most relevant to in vivo imaging, charting a clear path toward in vivo imaging using benchtop XFCT.