Brecht Laforce

Nanoscopic X-ray fluorescence imaging of meteoritic particles and diamond inclusions

The new ESRF ID16B-NA Nanoanalysis beamline has been applied for the first time for XRF imaging with a resolution level down to a few tens of nanometers on rare geological materials: meteoritic fragments from achondrite NWA 6693 and diamond inclusions. The instrument proved to be an extremely valuable tool for mapping samples containing submicrometer heterogeneities. It was discovered that the track of bubblelike inclusions in NWA 6693 consists mainly of Cr-rich phases. Some inclusions containing Ni and Ca were also detected. In diamond SL05, originating from the Juina region in Brazil, multiple inclusions were analyzed with dimensions smaller than 1 μm. Raman spectrometry measurements indicated the presence of a ringwoodite inclusion in this diamond; the detection of several iron-rich inclusions justifies further investigation of this material.

Full-field fluorescence mode micro-XANES imaging using a unique energy dispersive CCD detector.

X-ray absorption near-edge structure (XANES) spectroscopy is a well-known nondestructive technique that allows for chemical state and local structure determination. Spatially resolved oxidation state imaging is possible performing full-field transmission mode XANES experiments, providing chemical state information on the illuminated sample area, but these experiments are limited to thin, concentrated samples. Here we present the use of a unique energy dispersive (ED) pnCCD detector, the SLcam, for full-field fluorescence mode XANES experiments, thereby significantly relaxing the constraints on sample thickness. Using this new detection methodology, spatially resolved chemical state information on millimeter-sized sample areas can be obtained with microscopic resolution in moderate measuring times (less than 15 h), obtaining a XANES profile for each of nearly 70,000 points in a single measurement without the need of scanning the sample through the beam. Besides a description of the use of this detector for micro-XANES applications, we also present the proof of concept for fluorescence mode micro-XANES using a Fe(0)/Fe2O3 model sample and a Nitisol soil sample, which was measured to obtain iron chemical state distribution information.

Nanoscopic tumor tissue distribution of platinum after intraperitoneal administration in a xenograft model of ovarian cancer

There is increasing interest in the treatment of advanced stage ovarian cancer (OC) using intraperitoneal (IP) delivery of platinum (Pt)-based chemotherapy. The antitumor efficacy of IP chemotherapy is determined by efficient tumor tissue penetration. Although it is assumed that Pt penetration is limited to a few millimeters after IP delivery, little is known on the distribution of Pt in different tumor compartments at the ultrastructural level following IP administration. Here, using synchrotron radiation X-ray fluorescence spectrometry (SR-XRF) and laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS), Pt distribution and penetration in OC peritoneal xenografts were determined at nanometer scale after IP chemoperfusion of cisplatin at 37-38°C or 40-41°C (hyperthermic). Using principal component analysis (PCA) the presence of phosphorus, manganese, calcium, zinc, iron, bromine, and sulfur was correlated with the distribution of Pt, while k-means analysis was used to quantify the amount of Pt in weight% in tumor stroma and in tumor cells. The results showed a heterogeneous distribution of Pt throughout the tumor, with an accumulation in the extracellular matrix. LA-ICP-MS mappings indicated significantly higher concentrations of Pt (P=0.0062) after hyperthermic chemoperfusion of cisplatin, while SR-XRF demonstrated a deeper tissue Pt penetration after hyperthermic treatment. Using PCA, it was showed that Pt co-localizes with bromine and sulfur. No differences were observed in Pt distribution regarding tumor cells and stroma, when comparing normo- vs. hyperthermic treatment. In conclusion, SR-XRF and LA-ICP-MS are suitable and highly sensitive techniques to analyze the penetration depth and distribution of Pt-based drugs after IP administration. To the best of our knowledge, this is the first experiment in which the distribution of Pt is analyzed at the cellular level after IP administration of cisplatin.

Laboratory Scale X-ray Fluorescence Tomography: Instrument Characterization and Application in Earth and Environmental Science

A new laboratory scale X-ray fluorescence (XRF) imaging instrument, based on an X-ray microfocus tube equipped with a monocapillary optic, has been developed to perform XRF computed tomography experiments with both higher spatial resolution (20 μm) and a better energy resolution (130 eV @Mn-K(α)) than has been achieved up-to-now. This instrument opens a new range of possible applications for XRF-CT. Next to the analytical characterization of the setup by using well-defined model/reference samples, demonstrating its capabilities for tomographic imaging, the XRF-CT microprobe has been used to image the interior of an ecotoxicological model organism, Americamysis bahia. This had been exposed to elevated metal (Cu and Ni) concentrations. The technique allowed the visualization of the accumulation sites of copper, clearly indicating the affected organs, i.e. either the gastric system or the hepatopancreas. As another illustrative application, the scanner has been employed to investigate goethite spherules from the Cretaceous-Paleogene boundary, revealing the internal elemental distribution of these valuable distal ejecta layer particles.

Integrated Three-Dimensional Microanalysis Combining X-Ray Microtomography and X-Ray Fluorescence Methodologies

A novel 3D elemental and morphological analysis approach is presented combining X-ray computed tomography (μCT), X-ray fluorescence (XRF) tomography, and confocal XRF analysis in a single laboratory instrument (Herakles). Each end station of Herakles (μCT, XRF-CT, and confocal XRF) represents the state-of-the-art of currently available laboratory techniques. The integration of these techniques enables linking the (quantitative) spatial distribution of chemical elements within the investigated materials to their three-dimensional (3D) internal morphology/structure down to 1-10 μm resolution level, which has not been achieved so-far using laboratory X-ray techniques. The concept of Herakles relies strongly on its high precision (around 100 nm) air-bearing motor system that connects the different end-stations, allowing combined measurements based on the above X-ray techniques while retaining the coordinate system. In-house developed control and analysis software further ensures a smooth integration of the techniques. Case studies on a Cu test pattern, a Daphnia magna model organism and a perlite biocatalyst support material demonstrate the attainable resolution, elemental sensitivity of the instrument, and the strength of combining these three complementary methodologies.

Assessment of Ovarian Cancer Tumors Treated with Intraperitoneal Cisplatin Therapy by Nanoscopic X-ray Fluorescence Imaging.

Ovarian cancer is amongst the most common types of cancer in women, with a relatively low overall cure rate of approximately 30%. This is therefore an important incentive to urge for further research in order to maximize the chances of survival for these patients. Intraperitoneal chemotherapy with Cisplatin is an effective treatement for ovarian cancer; however, many questions still remain concerning the ideal treatment protocol and tumor resistance towards the drug, which should be resolved for optimal application of this therapy. For the first time in-vivo grown tumors treated with both hyper- and normothermic intraperitoneal chemotherapy have been studied using nano-XRF spectroscopy to examine the platinum (Pt) distribution within the analyzed tissues. These measurements prove Pt resides predominantly outsides the cancer cells in the stroma of the tissue. These findings indicate the resistance mechanism of the cancer cells prevents Cisplatin from diffusing through their cell membranes. This is an important addition to the existing knowledge on the resistance mechanism providing insights which might help to overcome this effect. In our aim to find the optimal treatment protocol, no significant differences were found between the two examined procedures. A more extensive data set will be needed to draw definite conclusions.

Imaging the 3D trace metal and metalloid distribution in mature wheat and rye grains via laser ablation-ICP-mass spectrometry and micro-X-ray fluorescence spectrometry

Toxic trace metals and metalloids in human nutrient sources pose a severe health risk, and the processes governing metal accumulation should hence be well understood. In this work, the spatial distribution of toxic trace metals/metalloids and micronutrients (Cr, Mn, Ni, Cu, Zn, As, Cd, Hg and Pb) in mature wheat (Triticum aestivum L.) and rye (Secale cereale L.) grains at typical exposure levels was visualized and quantified via laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) by serial sectioning. The bulk concentrations of these elements were also determined by pneumatic nebulisation-ICP-MS. Furthermore, longitudinal sections were scanned using m-X-ray fluorescence spectrometry to confirm the major element distribution. Serial sectioning in this study was realized via a polishing strategy. Although the methodology is time-consuming and laborious, it enables to access 3D information for samples which cannot be sectioned using a microtome on a depth scale that would otherwise be inaccessible by a laser probe. In the elemental images, strong local enrichment patterns for Mn and Zn are apparent in the aleurone layer/seed coat, vascular tissue of the crease, and embryonic tissue, whereas Cr, As, Cd and Pb have been mainly accumulated in the grain endosperm as a result of different transport and storage dynamics.

Three-Dimensional Reconstruction of the Tissue-Specific Multielemental Distribution within Ceriodaphnia dubia via Multimodal Registration Using Laser Ablation ICP-Mass Spectrometry and X-ray Spectroscopic Techniques

In this work, the three-dimensional elemental distribution profile within the freshwater crustacean Ceriodaphnia dubia was constructed at a spatial resolution down to 5 μm via a data fusion approach employing state-of-the-art laser ablation-inductively coupled plasma-time-of-flight mass spectrometry (LA-ICP-TOFMS) and laboratory-based absorption microcomputed tomography (μ-CT). C. dubia was exposed to elevated Cu, Ni, and Zn concentrations, chemically fixed, dehydrated, stained, and embedded, prior to μ-CT analysis. Subsequently, the sample was cut into 5 μm thin sections that were subjected to LA-ICP-TOFMS imaging. Multimodal image registration was performed to spatially align the 2D LA-ICP-TOFMS images relative to the corresponding slices of the 3D μ-CT reconstruction. Mass channels corresponding to the isotopes of a single element were merged to improve the signal-to-noise ratios within the elemental images. In order to aid the visual interpretation of the data, LA-ICP-TOFMS data were projected onto the μ-CT voxels representing tissue. Additionally, the image resolution and elemental sensitivity were compared to those obtained with synchrotron radiation based 3D confocal μ-X-ray fluorescence imaging upon a chemically fixed and air-dried C. dubia specimen.

Probing Intracellular Element Concentration Changes during Neutrophil Extracellular Trap Formation Using Synchrotron Radiation Based X-Ray Fluorescence

High pressure frozen (HPF), cryo-substituted microtome sections of 2 μm thickness containing human neutrophils (white blood cells) were analyzed using synchrotron radiation based X-ray fluorescence (SR nano-XRF) at a spatial resolution of 50 nm. Besides neutrophils from a control culture, we also analyzed neutrophils stimulated for 1–2 h with phorbol myristate acetate (PMA), a substance inducing the formation of so-called Neutrophil Extracellular Traps (or NETs), a defense system again pathogens possibly involving proteins with metal chelating properties. In order to gain insight in metal transport during this process, precise local evaluation of elemental content was performed reaching limits of detection (LODs) of 1 ppb. Mean weight fractions within entire neutrophils, their nuclei and cytoplasms were determined for the three main elements P, S and Cl, but also for the 12 following trace elements: K, Ca, Mn, Fe, Co, Ni, Cu, Zn, Se, Br, Sr and Pb. Statistical analysis, including linear regression provided objective analysis and a measure for concentration changes. The nearly linear Ca and Cl concentration changes in neutrophils could be explained by already known phenomena such as the induction of Ca channels and the uptake of Cl under activation of NET forming neutrophils. Linear concentration changes were also found for P, S, K, Mn, Fe, Co and Se. The observed linear concentration increase for Mn could be related to scavenging of this metal from the pathogen by means of the neutrophil protein calprotectin, whereas the concentration increase of Se may be related to its antioxidant function protecting neutrophils from the reactive oxygen species they produce against pathogens. We emphasize synchrotron radiation based nanoscopic X-ray fluorescence as an enabling analytical technique to study changing (trace) element concentrations throughout cellular processes, provided accurate sample preparation and data-analysis.

Nanoscopic X-ray fluorescence imaging and quantification of intracellular key-elements in cryofrozen Friedreich’s ataxia fibroblasts

Synchrotron radiation based nanoscopic X-ray fluorescence (SR nano-XRF) analysis can visualize trace level elemental distribution in a fully quantitative manner within single cells. However, in-air XRF analysis requires chemical fixation modifying the cell’s chemical composition. Here, we describe first nanoscopic XRF analysis upon cryogenically frozen (-150°C) fibroblasts at the ID16A-NI ‘Nano-imaging’ end-station located at the European Synchrotron Radiation Facility (ESRF) in Grenoble (France). Fibroblast cells were obtained from skin biopsies from control and Friedreich’s ataxia (FRDA) patients. FRDA is an autosomal recessive disorder with dysregulation of iron metabolism as a key feature. By means of the X-ray Fundamental Parameter (FP) method, including absorption correction of the ice layer deposited onto the fibroblasts, background-corrected mass fraction elemental maps of P, S, Cl, K, Ca, Fe and Zn of entire cryofrozen human fibroblasts were obtained. Despite the presence of diffracting microcrystals in the vitreous ice matrix and minor sample radiation damage effects, clusters of iron-rich hot-spots with similar mass fractions were found in the cytoplasm of both control and FRDA fibroblasts. Interestingly, no significant difference in the mean iron concentration was found in the cytoplasm of FRDA fibroblasts, but a significant decrease in zinc concentration. This finding might underscore metal dysregulation, beyond iron, in cells derived from FRDA patients. In conclusion, although currently having slightly increased limits of detection (LODs) compared to non-cryogenic mode, SR based nanoscopic XRF under cryogenic sample conditions largely obliterates the debate on chemical sample preservation and provides a unique tool for trace level elemental imaging in single cells close to their native state with a superior spatial resolution of 20 nm.