Quantitative Assessment on Optical Properties as a Basis for Bioluminescence Imaging: An Experimental and Numerical Approach to the Transport of Optical Photons in Phantom Materials.

Bioluminescence imaging (BLI) is a widely used technique in preclinical scientific research, particularly in the development of mRNA-based medications and non-invasive tumor diagnostics. It has become an essential tool in current science. However, the current state of bioluminescence imaging is primarily qualitative, making it challenging to obtain quantitative measurements and to draw accurate conclusions. This fact is caused by the unique properties of optical photons and tissue interactions. In this paper, we propose an experimental setup and Geant4-simulations to gain a better understanding of the optical properties and processes involved in bioluminescence imaging. Our goal is to advance the field towards more quantitative measurements. We will discuss the details of our experimental setup, the data we collected, the outcomes of the Geant4-simulations, and additional information on the underlying physical processes.

Review of Development and Recent Advances in Biomedical X-ray Fluorescence Imaging.

The use of X-rays for non-invasive imaging has a long history, which has resulted in several well-established methods in preclinical as well as clinical applications, such as tomographic imaging or computed tomography. While projection radiography provides anatomical information, X-ray fluorescence analysis allows quantitative mapping of different elements in samples of interest. Typical applications so far comprise the identification and quantification of different elements and are mostly located in material sciences, archeology and environmental sciences, whereas the use of the technique in life sciences has been strongly limited by intrinsic spectral background issues arising in larger objects, so far. This background arises from multiple Compton-scattering events in the objects of interest and strongly limits the achievable minimum detectable marker concentrations. Here, we review the history and report on the recent promising developments of X-ray fluorescence imaging (XFI) in preclinical applications, and provide an outlook on the clinical translation of the technique, which can be realized by reducing the above-mentioned intrinsic background with dedicated algorithms and by novel X-ray sources.

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.

Observation of High Transformer Ratio Plasma Wakefield Acceleration.

Particle-beam-driven plasma wakefield acceleration (PWFA) enables various novel high-gradient techniques for powering future compact light-source and high-energy physics applications. Here, a driving particle bunch excites a wakefield response in a plasma medium, which may rapidly accelerate a trailing witness beam. In this Letter, we present the measurement of ratios of acceleration of the witness bunch to deceleration of the driver bunch, the so-called transformer ratio, significantly exceeding the fundamental theoretical and thus far experimental limit of 2 in a PWFA. An electron bunch with ramped current profile was utilized to accelerate a witness bunch with a transformer ratio of 4.6_{-0.7}^{+2.2} in a plasma with length ∼10 cm, also demonstrating stable transport of driver bunches with lengths on the order of the plasma wavelength.

Numerical Study towards In Vivo Tracking of Micro-/Nanoplastic Based on X-ray Fluorescence Imaging.

There is a rising awareness of the toxicity of micro- and nanoplastics (MNPs); however, fundamental precise information on MNP-biodistribution in organisms is currently not available. X-ray fluorescence imaging (XFI) is introduced as a promising imaging modality to elucidate the effective MNP bioavailability and is expected to enable exact measurements on the uptake over the physical barriers of the organism and bioaccumulation in different organs. This is possible because of the ability of XFI to perform quantitative studies with a high spatial resolution and the possibility to conduct longitudinal studies. The focus of this work is a numerical study on the detection limits for a selected XFI-marker, here, palladium, to facilitate the design of future preclinical in vivo studies. Based on Monte Carlo simulations using a 3D voxel mouse model, the palladium detection thresholds in different organs under in vivo conditions in a mouse are estimated. The minimal Pd-mass in the scanning position at a reasonable significance level is determined to be <20 ng/mm2 for abdominal organs and <16 µg/mm2 for the brain. MNPs labelled with Pd and homogeneously distributed in the organ would be detectable down to a concentration of <1 µg/mL to <2.5 mg/mL in vivo. Long-term studies with a chronic MNP exposure in low concentrations are therefore possible such that XFI measurements could, in the future, contribute to MNP health risk assessment in small animals and humans.

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.

FLASHlab@PITZ: New R&D platform with unique capabilities for electron FLASH and VHEE radiation therapy and radiation biology under preparation at PITZ.

At the Photo Injector Test facility at DESY in Zeuthen (PITZ), an R&D platform for electron FLASH and very high energy electron radiation therapy and radiation biology is being prepared (FLASHlab@PITZ). The beam parameters available at PITZ are worldwide unique. They are based on experiences from 20 + years of developing high brightness beam sources and an ultra-intensive THz light source demonstrator for ps scale electron bunches with up to 5 nC bunch charge at MHz repetition rate in bunch trains of up to 1 ms length, currently 22 MeV (upgrade to 250 MeV planned). Individual bunches can provide peak dose rates up to 1014 Gy/s, and 10 Gy can be delivered within picoseconds. Upon demand, each bunch of the bunch train can be guided to a different transverse location, so that either a "painting" with micro beams (comparable to pencil beam scanning in proton therapy) or a cumulative increase of absorbed dose, using a wide beam distribution, can be realized at the tumor. Full tumor treatment can hence be completed within 1 ms, mitigating organ movement issues. With extremely flexible beam manipulation capabilities, FLASHlab@PITZ will cover the current parameter range of successfully demonstrated FLASH effects and extend the parameter range towards yet unexploited short treatment times and high dose rates. A summary of the plans for FLASHlab@PITZ and the status of its realization will be presented.

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.

Localising functionalised gold-nanoparticles in murine spinal cords by X-ray fluorescence imaging and background-reduction through spatial filtering for human-sized objects.

Accurate in vivo localisation of minimal amounts of functionalised gold-nanoparticles, enabling e.g. early-tumour diagnostics and pharmacokinetic tracking studies, requires a precision imaging system offering very high sensitivity, temporal and spatial resolution, large depth penetration, and arbitrarily long serial measurements. X-ray fluorescence imaging could offer such capabilities; however, its utilisation for human-sized scales is hampered by a high intrinsic background level. Here we measure and model this anisotropic background and present a spatial filtering scheme for background reduction enabling the localisation of nanoparticle-amounts as reported from small-animal tumour models. As a basic application study towards precision pharmacokinetics, we demonstrate specific localisation to sites of disease by adapting gold-nanoparticles with small targeting ligands in murine spinal cord injury models, at record sensitivity levels using sub-mm resolution. Both studies contribute to the future use of molecularly-targeted gold-nanoparticles as next-generation clinical diagnostic and pharmacokinetic tools.

Molecular imaging based on x-ray fluorescent high-Z tracers.

We propose a novel x-ray fluorescence imaging setup for the in vivo detection of high-Z tracer distributions. The main novel aspect is the use of an analyzer-based, energy-resolved detection method together with a radial, scatter reducing collimator. The aim of this work is to show the feasibility of this method by measuring the Bragg reflected K-fluorescence signal of an iodine solution sample in a proof of principle experiment and to estimate the potential of the complete imaging setup using a Monte Carlo simulation, including a quantification of the minimal detectable tracer concentration for in vivo imaging. The proof of principle experiment shows that even for a small detector area of approximately 7 mm(2), the collimated and Bragg reflected K-fluorescence signal of a sample containing an iodine solution with a concentration of 50 µg ml(-1) can be detected. The Monte Carlo simulation also shows that the proposed x-ray fluorescence imaging setup has the potential to image distributions of high-Z tracers in vivo at a radiation dose of a few mGy and at tracer concentrations down to 1 µg ml(-1) for iodine in small animals.

Gold nanoparticles functionalized with a fragment of the neural cell adhesion molecule L1 stimulate L1-mediated functions.

The neural cell adhesion molecule L1 is involved in nervous system development and promotes regeneration in animal models of acute and chronic injury of the adult nervous system. To translate these conducive functions into therapeutic approaches, a 22-mer peptide that encompasses a minimal and functional L1 sequence of the third fibronectin type III domain of murine L1 was identified and conjugated to gold nanoparticles (AuNPs) to obtain constructs that interact homophilically with the extracellular domain of L1 and trigger the cognate beneficial L1-mediated functions. Covalent conjugation was achieved by reacting mixtures of two cysteine-terminated forms of this L1 peptide and thiolated poly(ethylene) glycol (PEG) ligands (~2.1 kDa) with citrate stabilized AuNPs of two different sizes (~14 and 40 nm in diameter). By varying the ratio of the L1 peptide-PEG mixtures, an optimized layer composition was achieved that resulted in the expected homophilic interaction of the AuNPs. These AuNPs were stable as tested over a time period of 30 days in artificial cerebrospinal fluid and interacted with the extracellular domain of L1 on neurons and Schwann cells, as could be shown by using cells from wild-type and L1-deficient mice. In vitro, the L1-derivatized particles promoted neurite outgrowth and survival of neurons from the central and peripheral nervous system and stimulated Schwann cell process formation and proliferation. These observations raise the hope that, in combination with other therapeutic approaches, L1 peptide-functionalized AuNPs may become a useful tool to ameliorate the deficits resulting from acute and chronic injuries of the mammalian nervous system.