Nanoscopic X-ray Research Articles

Synchrotron XRF and Histological Analyses Identify Damage to Digestive Tract of Uranium NP-Exposed Daphnia magna.

Micro- and nanoscopic X-ray techniques were used to investigate the relationship between uranium (U) tissue distributions and adverse effects to the digestive tract of aquatic model organism Daphnia magna following uranium nanoparticle (UNP) exposure. X-ray absorption computed tomography measurements of intact daphnids exposed to sublethal concentrations of UNPs or a U reference solution (URef) showed adverse morphological changes to the midgut and the hepatic ceca. Histological analyses of exposed organisms revealed a high proportion of abnormal and irregularly shaped intestinal epithelial cells. Disruption of the hepatic ceca and midgut epithelial tissues implied digestive functions and intestinal barriers were compromised. Synchrotron-based micro X-ray fluorescence (XRF) elemental mapping identified U co-localized with morphological changes, with substantial accumulation of U in the lumen as well as in the epithelial tissues. Utilizing high-resolution nano-XRF, 400-1000 nm sized U particulates could be identified throughout the midgut and within hepatic ceca cells, coinciding with tissue damages. The results highlight disruption of intestinal function as an important mode of action of acute U toxicity in D. magna and that midgut epithelial cells as well as the hepatic ceca are key target organs.

Synchrotron-based characterization of arthroprosthetic CoCrMo particles in human bone marrow

Particles released from cobalt-chromium-molybdenum (CoCrMo) alloys are considered common elicitors of chronic inflammatory adverse effects. There is a lack of data demonstrating particle numbers, size distribution and elemental composition of bone marrow resident particles which would allow for implementation of clinically relevant test strategies in bone marrow models at different degrees of exposure. The aim of this study was to investigate metal particle exposure in human periprosthetic bone marrow of three types of arthroplasty implants. Periprosthetic bone marrow sections from eight patients exposed to CoCrMo particles were analyzed via spatially resolved and synchrotron-based nanoscopic X-ray fluorescence imaging. These analyses revealed lognormal particle size distribution patterns predominantly towards the nanoscale. Analyses of particle numbers and normalization to bone marrow volume and bone marrow cell number indicated particle concentrations of up to 1 × 1011 particles/ml bone marrow or 2 × 104 particles/bone marrow cell, respectively. Analyses of elemental ratios of CoCrMo particles showed that particularly the particles’ Co content depends on particle size. The obtained data point towards Co release from arthroprosthetic particles in the course of dealloying and degradation processes of larger particles within periprosthetic bone marrow. This is the first study providing data based on metal particle analyses to be used for future in vitro and in vivo studies of possible toxic effects in human bone marrow following exposure to arthroprosthetic CoCrMo particles of different concentration, size, and elemental composition.Graphical abstract

Single-exposure X-ray phase imaging microscopy with a grating interferometer.

The advent of hard X-ray free-electron lasers enables nanoscopic X-ray imaging with sub-picosecond temporal resolution. X-ray grating interferometry offers a phase-sensitive full-field imaging technique where the phase retrieval can be carried out from a single exposure alone. Thus, the method is attractive for imaging applications at X-ray free-electron lasers where intrinsic pulse-to-pulse fluctuations pose a major challenge. In this work, the single-exposure phase imaging capabilities of grating interferometry are characterized by an implementation at the I13-1 beamline of Diamond Light Source (Oxfordshire, UK). For comparison purposes, propagation-based phase contrast imaging was also performed at the same instrument. The characterization is carried out in terms of the quantitativeness and the contrast-to-noise ratio of the phase reconstructions as well as via the achievable spatial resolution. By using a statistical image reconstruction scheme, previous limitations of grating interferometry regarding the spatial resolution can be mitigated as well as the experimental applicability of the technique.

Nanoscopic X-ray tomography for correlative microscopy of a small meiofaunal sea-cucumber

In the field of correlative microscopy, light and electron microscopy form a powerful combination for morphological analyses in zoology. Due to sample thickness limitations, these imaging techniques often require sectioning to investigate small animals and thereby suffer from various artefacts. A recently introduced nanoscopic X-ray computed tomography (NanoCT) setup has been used to image several biological objects, none that were, however, embedded into resin, which is prerequisite for a multitude of correlative applications. In this study, we assess the value of this NanoCT for correlative microscopy. For this purpose, we imaged a resin-embedded, meiofaunal sea cucumber with an approximate length of 1 mm, where microCT would yield only little information about the internal anatomy. The resulting NanoCT data exhibits isotropic 3D resolution, offers deeper insights into the 3D microstructure, and thereby allows for a complete morphological characterization. For comparative purposes, the specimen was sectioned subsequently to evaluate the NanoCT data versus serial sectioning light microscopy (ss-LM). To correct for mechanical instabilities and drift artefacts, we applied an alternative alignment procedure for CT reconstruction. We thereby achieve a level of detail on the subcellular scale comparable to ss-LM images in the sectioning plane.

Effect of sample preparation techniques upon single cell chemical imaging: A practical comparison between synchrotron radiation based X-ray fluorescence (SR-XRF) and Nanoscopic Secondary Ion Mass Spectrometry (nano-SIMS)

Analytical capabilities of Nanoscopic Secondary Ion Mass Spectrometry (nano-SIMS) and Synchrotron Radiation based X-ray Fluorescence (SR nano-XRF) techniques were compared for nanochemical imaging of polymorphonuclear human neutrophils (PMNs). PMNs were high pressure frozen (HPF), cryo-substituted, embedded in Spurr’s resin and cut in thin sections (500 nm and 2 μm for both techniques resp.) Nano-SIMS enabled nanoscale mapping of isotopes of C, N, O, P and S, while SR based nano-XRF enabled trace level imaging of metals like Ca, Mn, Fe, Ni, Cu and Zn at a resolution of approx. 50 nm. The obtained elemental distributions were compared with those of whole, cryofrozen PMNs measured at the newly developed ID16A nano-imaging beamline at the European Synchrotron Radiation Facility (ESRF) in Grenoble, France. Similarities were observed for elements more tightly bound to the cell structure such as phosphorus and sulphur, while differences for mobile ions such as chlorine and potassium were more pronounced. Due to the observed elemental redistribution of mobile ions such as potassium and chlorine, elemental analysis of high pressure frozen (HPF), cryo-substituted and imbedded cells should be interpreted critically. Although decreasing analytical sensitivity occurs due to the presence of ice, analysis of cryofrozen cells - close to their native state - remains the golden standard. In general, we found nanoscale secondary ion mass spectrometry (nano-SIMS) and synchrotron radiation based nanoscopic X-ray fluorescence (SR nano-XRF) to be two supplementary alternatives for nanochemical imaging of single cells at the nanoscale.

Nanoscopic X-ray imaging and quantification of the iron cellular architecture within single fibroblasts of Friedreich's ataxia patients.

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.

Revealing the Microscopic Structure of Human Renal Cell Carcinoma in Three Dimensions.

For fully characterizing renal cell carcinoma (RCC), information about the 3D tissue microstructure is essential. Histopathology, which represents the current diagnostic gold standard, is destructive and only provides 2D information. 3D X-ray histology endeavors to overcome these limitations by generating 3D data. In a laboratory environment, most techniques struggle with limited resolution and the weak X-ray attenuation contrast of soft tissue. We recently developed a laboratory-based method combining nanoscopic X-ray CT with a cytoplasm-specific X-ray stain. Here, we present the application of this method to human RCC biopsies. The NanoCT slices enable pathological characterization of crucial structures by reproducing tissue morphology with a similar detail level as corresponding histological light microscopy images. Beyond that, our data offer deeper insights into the 3D configuration of the tumor. By demonstrating the compatibility of the X-ray stain with standard pathological stains, we highlight the feasibility of integrating staining based NanoCT into the pathological routine.

Cellular Fractionation and Nanoscopic X-Ray Fluorescence Imaging Analyses Reveal Changes of Zinc Distribution in Leaf Cells of Iron-Deficient Plants.

Multilevel interactions among nutrients occur in the soil-plant system. Among them, Fe and Zn homeostasis in plants are of great relevance because of their importance for plant and human nutrition. However, the mechanisms underlying the interplay between Fe and Zn in plants are still poorly understood. In order to elucidate how Zn interacts with Fe homeostasis, it is crucial to assess Zn distribution either in the plant tissues or within the cells. In this study, we investigated the subcellular Zn distribution in Fe-deficient leaf cells of cucumber plants by using two different approaches: cellular fractionation coupled with inductively coupled plasma mass spectrometry (ICP/MS) and nanoscopic synchrotron X-ray fluorescence imaging. Fe-deficient leaves showed a strong accumulation of Zn as well as a strong alteration of the organelles’ ultrastructure at the cellular level. The cellular fractionation-ICP/MS approach revealed that Zn accumulates in both chloroplasts and mitochondria of Fe deficient leaves. Nano-XRF imaging revealed Zn accumulation in chloroplast and mitochondrial compartments, with a higher concentration in chloroplasts. Such results show that (i) both approaches are suitable to investigate Zn distribution at the subcellular level and (ii) cellular Fe and Zn interactions take place mainly in the organelles, especially in the chloroplasts.

Nonequilibrium Phase Precursors during a Photoexcited Insulator-to-Metal Transition in V_{2}O_{3}.

Here, we photoinduce and directly observe with x-ray scattering an ultrafast enhancement of the structural long-range order in the archetypal Mott system V_{2}O_{3}. Despite the ultrafast increase in crystal symmetry, the change of unit cell volume occurs an order of magnitude slower and coincides with the insulator-to-metal transition. The decoupling between the two structural responses in the time domain highlights the existence of a transient photoinduced precursor phase, which is distinct from the two structural phases present in equilibrium. X-ray nanoscopy reveals that acoustic phonons trapped in nanoscale twin domains govern the dynamics of the ultrafast transition into the precursor phase, while nucleation and growth of metallic domains dictate the duration of the slower transition into the metallic phase. The enhancement of the long-range order before completion of the electronic transition demonstrates the critical role the nonequilibrium structural phases play during electronic phase transitions in correlated electrons systems.

Correction: Nanoscopic X-ray fluorescence imaging and quantification of intracellular key-elements in cryofrozen Friedreich's ataxia fibroblasts.

[This corrects the article DOI: 10.1371/journal.pone.0190495.].

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.

Combined Computed Nanotomography and Nanoscopic X-ray Fluorescence Imaging of Cobalt Nanoparticles in Caenorhabditis elegans.

Synchrotron radiation phase-contrast computed nanotomography (nano-CT) and two- and three-dimensional (2D and 3D) nanoscopic X-ray fluorescence (nano-XRF) were used to investigate the internal distribution of engineered cobalt nanoparticles (Co NPs) in exposed individuals of the nematode Caenorhabditis elegans. Whole nematodes and selected tissues and organs were 3D-rendered: anatomical 3D renderings with 50 nm voxel size enabled the visualization of spherical nanoparticle aggregates with size up to 200 nm within intact C. elegans. A 20 × 37 nm2 high-brilliance beam was employed to obtain XRF elemental distribution maps of entire nematodes or anatomical details such as embryos, which could be compared with the CT data. These maps showed Co NPs to be predominantly present within the intestine and the epithelium, and they were not colocalized with Zn granules found in the lysosome-containing vesicles or Fe agglomerates in the intestine. Iterated XRF scanning of a specimen at 0° and 90° angles suggested that NP aggregates were translocated into tissues outside of the intestinal lumen. Virtual slicing by means of 2D XRF tomography, combined with holotomography, indicated presumable presence of individual NP aggregates inside the uterus and within embryos.

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.

X-ray nanoscopy study on metal nano-structure formation at a metal-organic interface

Scanning transmission X-ray microscopy (STXM) with ~30-nm spatial resolution revealed at annealing effect on the metal-organic interface of an Al layer with a poly (3-hexylthiophene) (P3HT) organic semiconducting layer with and without phenyl-C61-butyric acid methyl ester (PCBM). In the case of a P3HT/Al layer with a high-tensile-strain organic over-layer, the overall interface changed to a zagged morphology with Al nano pillars after thermal annealing. On the contrary, a P3HT:PCBM/Al layer with a less-tensile-strain organic over-layer showed a smooth interface except for a few nano pillars. These interfacial morphology changes were related to the initial strain status and to the relaxation process and the phase separation of P3HT crystals relating to the PCBM. Grazing-incident wide-angle X-ray scattering (GI-WAXS) measurements were also conducted to examine the residual strain and the crystalline properties of P3HT in the presence of PCBM.

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.

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.

Hard-X-ray magnetic microscopy and local magnetization analysis using synchrotron radiation.

X-ray measurement offers several useful features that are unavailable from other microscopic means including electron-based techniques. By using X-rays, one can observe the internal parts of a thick sample. This technique basically requires no high vacuum environment such that measurements are feasible for wet specimens as well as under strong electric and magnetic fields and even at a high pressure. X-ray spectroscopy using core excitation provides element-selectivity with significant sensitivities to the chemical states and atomic magnetic moments in the matter. Synchrotron radiation sources produce a small and low-divergent X-ray beam, which can be converged to a spot with the size of a micrometer or less using X-ray focusing optics. The recent development in the focusing optics has been driving X-ray microscopy, which has already gone into the era of X-ray nanoscopy. With the use of the most sophisticated focusing devices, an X-ray beam of 7-nm size has successfully been achieved [1]. X-ray microscopy maintains above-mentioned unique features of X-ray technique, being a perfect complement to electron microscopy.In this paper, we present recent studies on magnetic microscopy and local magnetic analysis using hard X-rays. The relevant instrumentation developments are also described. The X-ray nanospectroscopy station of BL39XU at SPring-8 is equipped with a focusing optics consisting of two elliptic mirrors, and a focused X-ray beam with the size of 100 × 100 nm(2) is available [2]. Researchers can perform X-ray absorption spectroscopy: nano-XAFS (X-ray absorption fine structure) using the X-ray beam as small as 100 nm. The available X-ray energy is from 5 to 16 keV, which allows nano-XAFS study at the K edges of 3d transition metals, L edges of rare-earth elements and 5d noble metals. Another useful capability of the nanoprobe is X-ray polarization tunability, enabling magnetic circular dichroism (XMCD) spectroscopy with a sub-micrometer resolution. Scanning XMCD imaging, XMCD measurement in local areas, and element-specific magnetometry for magnetic particles/magnetic devices as small as 100 nm can be performed. Nano-XAFS application includes visualization of the chemical state in a particle catalyst [3] and phase-change memory devices [4]. For magnetic microscopic study, magnetization reversal processes of an individual magnetic CoPt dot in bit-patterned media have directly been observed [2]. Imaging of the chemical distribution and magnetic domain evolution in a Nd-Fe-B sintered magnet in demagnetization processes is presented.

Hierarchical nanoparticle clusters induced by block copolymer self-assembly.

The hierarchical relation of microscale structures with those of zoomed-in nanoscale in a composite material is one of the challenging issues. In terms of multiscale regional analogy of fractal, identical motifs are repeated on different size scales. However, idealized packing schemes are typically known to break down beyond a length scale of a few clusters. Here, we propose a new packing scheme - the structural analogy investigated by gold nanoparticle (AuNP) clusters describes a deformed self-similarity in multiscales. The clusters are directly imaged in microscale and in submicroscale using X-ray microscopy (XM) and X-ray nanoscopy (XN) without sample distortion in the hierarchical field-of-view up to 600 μm. We show that the broad-range ordering has the characteristics of a fractal with a dimension (D) between 1 and 2. This D value is smaller in XN scale than in XM scale, indicating dense cluster formation in large scale. The D values are independent from the pathways of cluster growth and compatibility of AuNPs with the polymer template. The nanoparticle cluster growth induced by block copolymer self-assembly is characteristically different from those occurring in free-space where the conventional diffusion- or reaction-limited aggregations are dominant. Our findings provide a new perspective in organic-inorganic hybrid nanomaterials and has broad implications in understanding their structure-property relationship for various applications.

Controlled cellular uptake and drug efficacy of nanotherapeutics

Cellular uptake pathway of nanoparticle (NP) is different from that of free drugs. Therefore, NP-mediated nanotherapeutics can be designed to overcome the adverse effects of free drugs. However, synthetic NPs are typically trapped in the endosome and have difficulty to reach the cytosol because of the characteristic endocytosis, where the endosomal membranes wrap-up the introduced NPs. In this study, the Spacer molecules linking the apoptotic anticancer drug and the gold NP (AuNP) are designed and cellular uptake procedure and drug deployment in the cancer cells are controlled. X-ray nanoscopy and two-photon microscopy are employed to observe the AuNPs in a cell in-situ without additional dye molecule or imaging agent introduction on an AuNP. We confirm that the effective design of the Spacer molecules importantly control the cellular interaction of the AuNPs. This technology can be generalized to broad biomedical applications utilizing nanotherapeutics-mediated diagnosis and new-concepted disease treatment technologies.

X-ray nanoscopy of cobalt Fischer–Tropsch catalysts at work

Transmission X-ray microscopy has been used to investigate individual Co/TiO2 Fischer-Tropsch (FT) catalyst particles in 2-D and 3-D with 30 nm spatial resolution. Tomographic elemental mapping showed that Co is heterogeneously concentrated in the centre of the catalyst particles. In addition, it was found that Co is mostly metallic during FT at 250 °C and 10 bar. No evidence for Co oxidation was found.