Purpose: To develop a high sensitivity L-shell x-ray fluorescence imaging system that locates and quantifies sparse concentrations of gold nanoparticles (GNPs) using a polychromatic benchtop x-ray source. Methods: An L-shell x-ray fluorescence imaging system was built with a benchtop polychromatic x-ray source and a Si-PIN detector. Water-filled cylindrical tubes (12 mm in diameter) loaded with GNPs at 2%, 1%, 0.5%, 0.05%, and 0.005% GNP by weight were served as calibration phantoms. An imaging phantom was created using the same cylindrical tube but filled with water-equivalent gel containing structures mimicking GNP-loaded blood vessel and 1 cc tumor. The phantoms were irradiated by a 3-mm diameter 65 kVp x-rays filtered by 1 mm aluminum. Fluorescence/scatter photons from the phantoms were detected at 90° with respect to the beam direction using a Si-PIN detector with a pin-hole collimation. For the imaging phantom, the detector was translated horizontally and vertically in 0.3-mm steps to create a 2-D fluorescence image of the phantom. The net L-shell fluorescence signal from GNPs was extracted from background, and then corrected for detector efficiency and in-phantom attenuation using a fluorescence-to-scatter normalization algorithm. The corrected signal from the calibration phantoms was used to create a calibration curve showing a linear relationship between corrected fluorescence signal and GNP mass per image voxel. Results: The results suggest that the current setup can detect a GNP mass of 500 ng (or 0.5 ppm) contained within each image voxel (0.0173 cc). The 2-D fluorescence image properly correlated the known spatial distribution and amount of GNPs within the imaging phantom. Conclusion: L-shell fluorescence imaging can be a highly sensitive tool that has the capability of simultaneously imaging the spatial distribution and determining the local concentration of GNPs presented within ex-vivo samples and superficial tumors during pre-clinical small animal studies. Supported by NIH/NCI grant R01CA155446; NIH/NCI grant R01CA155446
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