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Besides conventional scanning X-ray fluorescence imaging at synchrotron sources, full-field X-ray fluorescence (FF-XRF) imaging techniques that do not implicitly require spatial scanning of the sample have become available. FF-XRF has become achievable thanks to the development of a new type of energy dispersive CCD-based 2D detector, also referred to as a ‘color X-ray camera (CXC)’ or ‘SLcam’. We report on different imaging schemes for biological samples using FF-XRF imaging: (a) 2D ‘zoom’ imaging with pinhole optics using the ‘camera obscura’ principle; (b) 2D ‘fixed magnification’ imaging using magnifying polycapillary optics; and (c) 3D-FF-XRF imaging using an X-ray sheet beam or computed tomography (CT). The different FF-XRF imaging modes are illustrated using the crustacean Daphnia magna, a model organism for investigating the effects of metals on organism/ecosystem health, and foraminifera, a class of amoeboid protist. Detailed analytical characterization of the set-up is performed through analyzing various reference materials in order to determine limits of detection (LODs) and sensitivities. Experiments were performed using the BAMline at the BESSY synchrotron (Berlin, Germany) and using the P06 Hard X-ray Microprobe at the PETRAIII synchrotron (Hamburg, Germany).

Beam hardening and scattering effects can seriously degrade image quality in polychromatic X-ray CT imaging. In recent years, polychromatic image reconstruction techniques and scatter estimation using Monte Carlo simulation have been developed to compensate for beam hardening and scattering CT artifacts, respectively. Both techniques require knowledge of the X-ray tube energy spectrum. In this work, Monte Carlo simulations were used to calculate the X-ray energy spectrum of FleXCT, a novel prototype industrial micro-CT scanner, enabling beam hardening and scatter reduction for CT experiments. Both source and detector were completely modeled by Monte Carlo simulation. In order to validate the energy spectra obtained via Monte Carlo simulation, they were compared with energy spectra obtained via a second method. Here, energy spectra were calculated from empirical measurements using a step wedge sample, in combination with the Maximum Likelihood Expectation Maximization (MLEM) method. Good correlation was achieved between both approaches, confirming the correct modeling of the FleXCT system by Monte Carlo simulation. After validation of the modeled FleXCT system through comparing the X-ray spectra for different tube voltages inside the detector, we calculated the X-ray spectrum of the FleXCT X-ray tube, independent of the flat panel detector response, which is a prerequisite for beam hardening and scattering CT artifacts.

Laboratory based X-ray micro-CT is a non-destructive testing method that enables three dimensional visualization and analysis of the internal and external morphology of samples. Although a wide variety of commercial scanners exist, most of them are limited in the number of degrees of freedom to position the source and detector with respect to the object to be scanned. Hence, they are less suited for industrial X-ray imaging settings that require advanced scanning modes, such as laminography, conveyor belt scanning, or time-resolved imaging (4DCT). We introduce a new X-ray scanner FleXCT that consists of a total of ten motorized axes, which allow a wide range of non-standard XCT scans such as tiled and off-centre scans, laminography, helical tomography, conveyor belt, dynamic zooming, and X-ray phase contrast imaging. Additionally, a new software tool ‘FlexRayTools’ was created that enables reconstruction of non-standard XCT projection data of the FleXCT instrument using the ASTRA Toolbox, a highly efficient and open source set of tools for tomographic projection and reconstruction.

Biointerphases 16(1), 011004 (1-8) (2021). doi:10.1116/6.0000593, X-ray fluorescence analysis enables the study of trace element distributions in biological specimens. When this analysis is done under cryogenic conditions, cells are cryofixed as closely as possible to their natural physiological state, and the corresponding intracellular elemental densities can be analyzed. Details about the experimental setup used for analysis at the P06 beamline at Petra III, DESY and the used cryo-transfer system are described in this work. The system was applied to analyze the elemental distribution in single HeLa cells, a cell line frequently used in a wide range of biological applications. Cells adhered to silicon nitride substrates were cryoprotected within an amorphous ice matrix. Using a continuous scanning scheme and a KB x-ray focus, the distribution of elements in the cells was studied. We were able to image the intracellular potassium and zinc levels in HeLa cells as two key elements relevant for the physiology of cells., Published by AVS, New York, NY

Journal of analytical atomic spectrometry 35, 10.1039.D0JA00067A (2020). doi:10.1039/D0JA00067A, Published by ChemSoc, Cambridge

International audience; Friedreich's ataxia (FRDA) is a neurodegenerative disease characterized by an increase in intracytoplasmic iron concentration. Here the nanoscale iron distribution within single fibroblasts from FRDA patients was investigated using synchrotron-radiation-based nanoscopic X-ray fluorescence and X-ray in-line holography at the ID16A nano-imaging beamline of the ESRF. This unique probe was deployed to uncover the iron cellular two-dimensional architecture of freeze-dried FRDA fibroblasts. An unsurpassed absolute detection capability of 180 iron atoms within a 30 nm  50 nm nanoscopic X-ray beam footprint was obtained using state-of-the-art X-ray focusing optics and a large-solid-angle detection system. Various micrometre-sized iron-rich organelles could be revealed for the first time, tentatively identified as endoplasmic reticulum, mitochondria and lysosomes. Also a multitude of nanoscopic iron hot-spots were observed in the cytosol, interpreted as chaperoned iron within the fibroblast's labile iron pool. These observations enable new hypotheses on the storage and trafficking of iron in the cell and ultimately to a better understanding of iron-storage diseases such as Friedreich's ataxia.

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

International audience; 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 degrees 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.

A new three-dimensional (3D) micro X-ray fluorescence (μXRF) methodology based on a novel 2D energy dispersive CCD detector has been developed and evaluated at the P06 beamline of the Petra-III storage ring (DESY) in Hamburg, Germany. This method is based on the illumination of the investigated sample cross-section by a horizontally focused beam (vertical sheet beam) while fluorescent X-rays are detected perpendicularly to the sheet beam by a 2D energy dispersive (ED) CCD detector allowing the collection of 2D cross-sectional elemental images of a certain depth within the sample, limited only by signal self-absorption effects. 3D elemental information is obtained by a linear scan of the sample in the horizontal direction across the vertically oriented sheet beam and combining the detected cross-sectional images into a 3D elemental distribution data set. Results of the 3D μXRF analysis of mineral inclusions in natural deep Earth diamonds are presented to illustrate this new methodology.

Larvae of Mytilus spp. are among the most Cu sensitive marine species. In this study we assessed the combined effect of salinity and dissolved organic carbon (DOC) on Cu accumulation on mussel larvae. Larvae were exposed for 48 h to three Cu concentrations in each of nine salinity/DOC treatments. Synchrotron radiation X-ray fluorescence was used to determine the Cu concentration in 36 individual larvae with a spatial resolution of 10 × 10 μm. Cu body burden concentrations varied between 1.1 and 27.6 μg/g DW larvae across all treatments and Cu was homogeneously distributed at this spatial resolution level. Our results indicate decreasing Cu accumulation with increasing DOC concentrations which can be explained by an increase in Cu complexation. In contrast, salinity had a nonlinear effect on Cu. This cannot be explained by copper speciation or competition processes and suggests a salinity-induced alteration in physiology.

Extraction of As, Cd, Cu, Mn, Ni and Ti from two white as well as from one brown rice samples was studied by inductively coupled plasma sector field mass spectrometry (ICP-SF-MS). Samples were subjected to cold and hot water extraction (rice mass to deionised water volume ratio = 1:6, and 1:3, respectively). The discarded liquids were freeze-dried and digested by microwave-assisted digestion. About 50% of the content of the investigated elements could be extracted from the white rice. In case of the brown rice, the boiling water contained As, Cd, Ni and Ti in significant percentages. Elemental distributions in the rice grains determined by synchrotron radiation confocal micro-X-ray fluorescence analysis revealed that a surface layer having a thickness of about 80 μm is the richest region in elements. Ti was detectable only in this so-called skin region. Good correlation was observed for the extraction of As, Mn, Ni and Ti by the above-mentioned two techniques. Thus, in regions affected by heavy metal and other toxic element contamination, those rice dishes would be preferred whose preparation should need abundant amounts of water for washing and cooking, which later should be discarded.

Visualization of elemental distributions in thin sections of biological tissue is gaining importance in many disciplines of biological and medical research. Laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) and scanning micro-X-ray fluorescence spectrometry (micro-XRF) are two widely used microanalytical techniques for elemental mapping. This article compares the capabilities of the two techniques for imaging the distribution of selected elements in the model organism Daphnia magna in terms of detection power and spatial resolution. Sections with a thickness of 10 and 20 microm of the fresh water crustacean Daphnia magna were subjected to LA-ICP-MS and micro-XRF analysis. The elemental distributions obtained for Ca, P, S and Zn allow element-to-tissue correlation. LA-ICP-MS and micro-XRF offer similar limits of detection for the elements Ca and P and thus, allow a cross-validation of the imaging results. LA-ICP-MS was particularly sensitive for determining Zn (LOD 20 microg g(-1), 15 microm spot size) in Daphnia magna, while the detection power of micro-XRF was insufficient in this context. However, LA-ICP-MS was inadequate for the measurement of the S distributions, which could be better visualized with micro-XRF (LOD 160 microg g(-1), 5 s live time). Both techniques are thus complementary in providing an exhaustive chemical profiling of tissue samples.

Synchrotron radiation based confocal micro-XRF was employed to unravel the tissue-specific three-dimensional (3D) distribution of metals down to trace concentration levels in a non-destructive manner within the crustacean Daphnia magna, an ecotoxicological model organism. Next to the analytical characterization of the employed confocal micro-XRF set-up, specific areas of metal accumulation in different cross-sections of interest within the organism were investigated. The use of a fast dynamic (continuous) scanning approach delivered 3D information on major, minor and trace element distributions in specific sub-regions within Daphnia magna. Coupling the obtained elemental information with microscopic morphological data obtained by laboratory absorption microtomography, full 3D element-to-tissue correlation could be derived, allowing a more detailed interpretation of the obtained results with respect to metal accumulation within this model organism.