Enabling Coarse X-ray Fluorescence Imaging Scans with Enlarged Synchrotron Beam by Means of Mosaic Crystal Defocusing Optics.

Trace elements, functionalized nanoparticles and labeled entities can be localized with sub-mm spatial resolution by X-ray fluorescence imaging (XFI). Here, small animals are raster scanned with a pencil-like synchrotron beam of high energy and low divergence and the X-ray fluorescence is recorded with an energy-dispersive detector. The ability to first perform coarse scans to identify regions of interest, followed by a close-up with a sub-mm X-ray beam is desirable, because overall measurement time and X-ray dose absorbed by the (biological) specimen can thus be minimized. However, the size of X-ray beams at synchrotron beamlines is usually strongly dependent on the actual beamline setup and can only be adapted within specific pre-defined limits. Especially, large synchrotron beams are non-trivial to generate. Here, we present the concept of graphite-based, convex reflection optics for the one-dimensional enlargement of a 1 mm wide synchrotron beam by a factor of 5 to 10 within a 1 m distance. Four different optics are tested and characterized and their reflection properties compared to ray tracing simulations. The general shape and size of the measured reflection profiles agree with expectations. Enhancements with respect to homogeneity and efficiency can be expected with improved optics manufacturing. A mouse phantom is used for a proof-of-principle XFI experiment demonstrating the applicability of coarse and fine scans with the suggested optics design.

Feasibility of Monitoring Tumor Response by Tracking Nanoparticle-Labelled T Cells Using X-ray Fluorescence Imaging-A Numerical Study.

Immunotherapy has been a breakthrough in cancer treatment, yet only a subgroup of patients responds to these novel drugs. Parameters such as cytotoxic T-cell infiltration into the tumor have been proposed for the early evaluation and prediction of therapeutic response, demanded for non-invasive, sensitive and longitudinal imaging. We have evaluated the feasibility of X-ray fluorescence imaging (XFI) to track immune cells and thus monitor the immune response. For that, we have performed Monte Carlo simulations using a mouse voxel model. Spherical targets, enriched with gold or palladium fluorescence agents, were positioned within the model and imaged using a monochromatic photon beam of 53 or 85 keV. Based on our simulation results, XFI may detect as few as 730 to 2400 T cells labelled with 195 pg gold each when imaging subcutaneous tumors in mice, with a spatial resolution of 1 mm. However, the detection threshold is influenced by the depth of the tumor as surrounding tissue increases scattering and absorption, especially when utilizing palladium imaging agents with low-energy characteristic fluorescence photons. Further evaluation and conduction of in vivo animal experiments will be required to validate and advance these promising results.

X-ray Fluorescence Uptake Measurement of Functionalized Gold Nanoparticles in Tumor Cell Microsamples.

Quantitative cellular in vitro nanoparticle uptake measurements are possible with a large number of different techniques, however, all have their respective restrictions. Here, we demonstrate the application of synchrotron-based X-ray fluorescence imaging (XFI) on prostate tumor cells, which have internalized differently functionalized gold nanoparticles. Total nanoparticle uptake on the order of a few hundred picograms could be conveniently observed with microsamples consisting of only a few hundreds of cells. A comparison with mass spectroscopy quantification is provided, experimental results are both supported and sensitivity limits of this XFI approach extrapolated by Monte-Carlo simulations, yielding a minimum detectable nanoparticle mass of just 5 pg. This study demonstrates the high sensitivity level of XFI, allowing non-destructive uptake measurements with very small microsamples within just seconds of irradiation time.

Enabling X-ray fluorescence imaging for in vivo immune cell tracking.

The infiltration of immune cells into sites of inflammation is one key feature of immune mediated inflammatory diseases. A detailed assessment of the in vivo dynamics of relevant cell subtypes could booster the understanding of this disease and the development of novel therapies. We show in detail how advanced X-ray fluorescence imaging enables such quantitative in vivo cell tracking, offering solutions that could pave the way beyond what other imaging modalities provide today. The key for this achievement is a detailed study of the spectral background contribution from multiple Compton scattering in a mouse-scaled object when this is scanned with a monochromatic pencil X-ray beam from a synchrotron. Under optimal conditions, the detection sensitivity is sufficient for detecting local accumulations of the labelled immune cells, hence providing experimental demonstration of in vivo immune cell tracking in mice.

Assessing Cellular Uptake of Exogenous Coenzyme Q10 into Human Skin Cells by X-ray Fluorescence Imaging.

X-ray fluorescence (XRF) imaging is a highly sensitive non-invasive imaging method for detection of small element quantities in objects, from human-sized scales down to single-cell organelles, using various X-ray beam sizes. Our aim was to investigate the cellular uptake and distribution of Q10, a highly conserved coenzyme with antioxidant and bioenergetic properties. Q10 was labeled with iodine (I2-Q10) and individual primary human skin cells were scanned with nano-focused beams. Distribution of I2-Q10 molecules taken up inside the screened individual skin cells was measured, with a clear correlation between individual Q10 uptake and cell size. Experiments revealed that labeling Q10 with iodine causes no artificial side effects as a result of the labeling procedure itself, and thus is a perfect means of investigating bioavailability and distribution of Q10 in cells. In summary, individual cellular Q10 uptake was demonstrated by XRF, opening the path towards Q10 multi-scale tracking for biodistribution studies.

Size- and Ligand-Dependent Transport of Nanoparticles in Matricaria chamomilla as Demonstrated by Mass Spectroscopy and X-ray Fluorescence Imaging.

Matricaria chamomilla flowers were incubated with gold nanoparticles of different sizes ranging from 1.4 to 94 nm. After different incubation times of 6, 12, 24, and 48 h, the gold distribution in the flowers was destructively measured by inductively coupled plasma mass spectrometry (ICP-MS) and non-destructively measured by X-ray fluorescence imaging (XFI) with high lateral resolution. As a control, the biodistribution of iodine ions or iodine-containing organic molecules (iohexol) was determined, in order to demonstrate the feasibility of mapping the distribution of several elements in parallel. The results show a clear size-dependent transport of the nanoparticles. In addition, the surface chemistry also plays a decisive role in disposition. Only the 1.6 nm nanoparticles coated with acetylcysteine could be efficiently transported through the stem of the flowers into the petals. In this case, almost 80% of the nanoparticles which were found within each flower were located in the petals. The study also highlights the potential of XFI for in situ recording of in vivo analyte biodistribution.

In-situ x-ray fluorescence imaging of the endogenous iodine distribution in murine thyroids.

X-ray fluorescence imaging (XFI) is a non-invasive detection method of small quantities of elements, which can be excited to emit fluorescence x-ray photons upon irradiation with an incident x-ray beam. In particular, it can be used to measure nanoparticle uptake in cells and tissue, thus making it a versatile medical imaging modality. However, due to substantially increased multiple Compton scattering background in the measured x-ray spectra, its sensitivity severely decreases for thicker objects, so far limiting its applicability for tracking very small quantities under in-vivo conditions. Reducing the detection limit would enable the ability to track labeled cells, promising new insights into immune response and pharmacokinetics. We present a synchrotron-based approach for reducing the minimal detectable marker concentration by demonstrating the feasibility of XFI for measuring the yet inaccessible distribution of the endogenous iodine in murine thyroids under in-vivo conform conditions. This result can be used as a reference case for the design of future preclinical XFI applications as mentioned above.

X-ray-Fluorescence Imaging for In Vivo Detection of Gold-Nanoparticle-Labeled Immune Cells: A GEANT4 Based Feasibility Study.

The growing field of cellular therapies in regenerative medicine and oncology calls for more refined diagnostic tools that are able to investigate and monitor the function and success of said therapies. X-ray Fluorescence Imaging (XFI) can be applied for molecular imaging with nanoparticles, such as gold nanoparticles (GNPs), which can be used in immune cell tracking. We present a Monte Carlo simulation study on the sensitivity of detection and associated radiation dose estimations in an idealized setup of XFI in human-sized objects. Our findings demonstrate the practicability of XFI in human-sized objects, as immune cell tracking with a minimum detection limit of 4.4 × 105 cells or 0.86 µg gold in a cubic volume of 1.78 mm3 can be achieved. Therefore, our results show that the current technological developments form a good basis for high sensitivity XFI.

X-ray-Based Techniques to Study the Nano-Bio Interface.

X-ray-based analytics are routinely applied in many fields, including physics, chemistry, materials science, and engineering. The full potential of such techniques in the life sciences and medicine, however, has not yet been fully exploited. We highlight current and upcoming advances in this direction. We describe different X-ray-based methodologies (including those performed at synchrotron light sources and X-ray free-electron lasers) and their potentials for application to investigate the nano-bio interface. The discussion is predominantly guided by asking how such methods could better help to understand and to improve nanoparticle-based drug delivery, though the concepts also apply to nano-bio interactions in general. We discuss current limitations and how they might be overcome, particularly for future use in vivo.