X-ray Fluorescence Imaging Research Articles

Recent Development of Gold Nanoparticles as Contrast Agents for Cancer Diagnosis.

Simple SummaryThe development of nanotechnology has brought revolution to the diagnosis and therapy of diseases, with a high precision and efficacy. Because nanoparticles can integrate multifunctions together including imaging, targeting, and therapeutics, they are more efficient than the standalone diagnostic or therapeutic entities. Among which, gold nanoparticles are most extensively investigated due to their excellent biocompatibility, versatility and ease of functionalization. Excepting the using of gold nanoparticles as vehicles for therapeutics delivery, they are also good candidates as contrast agents for imaging diagnosis, from magnetic resonance imaging, CT and nuclear imaging, fluorescence imaging, photoacoustic imaging to X-ray fluorescence imaging. We summarize their recent applications in these imaging modalities and challenges for their clinical translation.The last decade has witnessed the booming of preclinical studies of gold nanoparticles (AuNPs) in biomedical applications, from therapeutics delivery, imaging diagnostics, to cancer therapies. The synthetic versatility, unique optical and electronic properties, and ease of functionalization make AuNPs an excellent platform for cancer theranostics. This review summarizes the development of AuNPs as contrast agents to image cancers. First, we briefly describe the AuNP synthesis, their physical characteristics, surface functionalization and related biomedical uses. Then we focus on the performances of AuNPs as contrast agents to diagnose cancers, from magnetic resonance imaging, CT and nuclear imaging, fluorescence imaging, photoacoustic imaging to X-ray fluorescence imaging. We compare these imaging modalities and highlight the roles of AuNPs as contrast agents in cancer diagnosis accordingly, and address the challenges for their clinical translation.

Elemental mapping of Portuguese ceramic pieces with a full-field XRF scanner based on a 2D-THCOBRA detector

In this work, we present a novel application of the full-field energy-dispersive X-ray fluorescence (EDXRF) imaging system based on a MicroPattern Gaseous Detector (2D-THCOBRA) in the cultural heritage field. The detector has an intrinsic imaging capability with spatial resolution of $$400\,{\upmu }\mathrm { m}\ \mathrm {FWHM}$$ , and is energy sensitive, presenting an energy resolution of approximately $$1\ \mathrm {keV\ FWHM}$$ at $$5.9\ \mathrm {keV}$$ . The full-field XRF scanner based on the 2D-THCOBRA detector allows mapping the distribution of elements in large area samples with high detection efficiency ( $$75\%$$ at $$5.9\ \mathrm {keV}$$ ), being a very promising choice for elemental mapping analysis of large area cultural heritage samples. In this work, we have demonstrated the imaging capabilities of the full-field XRF scanner and used it to assess the restoration of a Portuguese faience piece.

Multiscale Synchrotron-Based Imaging Analysis for the Transfer of PEGylated Gold Nanoparticles In Vivo.

High spatial resolution imaging analysis is urgently needed to explore the biodistribution, transfer and clearance profiles, and biological impact of nanoparticles in the body, which will be helpful to clarify the efficacy of nanomedicine in clinical applications. Herein, by combination with multiscale synchrotron-based imaging techniques, including X-ray fluorescence (XRF) spectrometry, Fourier transform infrared (FTIR) spectroscopy, and micro X-ray phase contrast computed tomography (micro-XPCT), we visually displayed the transfer patterns and site-specific distribution of PEGylated gold nanoparticles (PEG-GNPs) in the suborgans of the liver, spleen, and kidney after an intravenous injection in mice. A combination of XRF and FTIR imaging analysis showed that the PEG bands presented similar distribution patterns with Au in the intraorgans, suggesting the stability of PEGylation on GNPs. We show that the PEG-GNPs presented heterogeneous distribution in the hepatic lobules with a large amount around the portal vein zone and then a gradient decrease in the sinusoidal region and the CV zone; in the spleen, it gradually accumulated in the splenic red pulp over time; and in the kidney, it quickly transported via the bloodstream to the renal pyramids and renal pelvis, and parts of PEG-GNPs finally accumulated in the renal medulla and renal cortex. Multidimensional micro-XPCT images further show that the PEG-GNP transfer in the liver induced hepatic blood vessel dilatation while they transferred in the liver, providing evidence of GNP transport across the blood vessel endothelial barrier.

Nondestructive Mineral Imaging of Chinese Chive Leaves Withered by Physiological Damage Using Microbeam Synchrotron Radiation X-Ray Fluorescence Analysis.

A significant problem encountered in Chinese chives (Allium tuberosum) grown in greenhouses is the reduction in the yield and quality due to symptoms of withered leaf tips. Withered leaf tips of three Chinese chive cultivars were nondestructively analyzed by microbeam synchrotron radiation X-ray fluorescence (μ-SR-XRF) imaging. Dead, wilting, and healthy parts of the leaves exhibited significant variations in the mineral composition. The Ca/K X-ray intensity ratios were significantly increased with the degree of withering.

Direct evidence for eoarchean iron metabolism?

Metasedimentary rocks from Isua, West Greenland (> 3,700 million years old) contain carbonaceous compounds, compatible with a biogenic origin (Hassenkam, Andersson, Dalby, Mackenzie, & Rosing, 2017; Ohtomo, Kakegawa, Ishida, Nagase, & Rosing, 2014; Rosing, 1999). The metamorphic mineral assemblage with garnet and quartz intergrowths contains layers of carbonaceous inclusions contiguous with carbon-rich sedimentary beds in the host rock. Previous studies (Hassenkam et al., 2017; Ohtomo et al., 2014; Rosing, 1999) on Isua rocks focused on testing the biogenic origin of the carbonaceous material, but here we searched for evidence which could provide new insights into the nature of the life that generated this carbonaceous material. We studied material trapped in inclusions armoured within quartz grains inside garnet porphyroblasts by non-destructive ptychographic X-ray nanotomography (PXCT). The 3D electron density maps generated by PXCT were correlated with maps from X-ray fluorescence tomography and micro-Raman spectroscopy. We found that the material trapped inside inclusions in the quartz grains consist of disordered carbon material encasing domains of iron-rich carbonaceous material. These results corroborate earlier claims (Hassenkam et al., 2017; Ohtomo et al., 2014; Rosing, 1999) for biogenic origins and are compatible with relics of metamorphosed biological material originally containing high iron/carbon ratios, comparable to ratios found in most extant organisms. These iron-rich domains represent the oldest evidence for organic iron complexes in the geologic record and are consistent with Fe-isotopic evidence for metabolic iron fractionation in>3,700Ma Isua banded iron formation (Czaja et al., 2013; Whitehouse & Fedo, 2007).

Imaging spectroscopies to characterize a 13th century Japanese handscroll, The Miraculous Interventions of Jiz\u014d Bosatsu

Scientific imaging of a large fragile work of art can be especially challenging, but especially rewarding to better grasp the complexity and changes that have occurred during its creation and lifetime. Here, noninvasive imaging, macro X-ray fluorescence (MAXRF) imaging spectroscopy and reflectance imaging spectroscopy, from the visible to the near infrared spectral range, are utilized to document a 14-m-long Japanese narrative handscroll, The Miraculous Interventions of Jizō Bosatsu. Due to the scroll’s age and its handling during past use as a teaching tool, it has a number of conservation needs and shows evidence of past repairs. The scroll has extensive and severe creasing, breaks and tears, as well as unstable and powdering pigments. Microscopic observation and scientific analyses were performed both to document the current condition of the scroll and to better understand its long history. Combining RIS and MAXRF allowed for pigment characterization through elemental and molecular information. While RIS and MAXRF previously have been applied to the study of other painted materials, their application to East Asian paintings is rare. The obstacles of the scroll’s length and fragile uneven surface were overcome by optimizing the setups of the two imaging systems. The MAXRF and RIS analyses, here focused on a select scene of the scroll, found certain original pigments common in early Japanese scroll paintings were used frequently, such as vermilion, iron-based compounds (yellow and red ochres), and copper-containing greens, while others occurred sparingly, such as azurite and red lead. A chloride-containing lead-based white pigment was employed. Faded organic colorants, notably indigo as well as an organic yellow/brown, could be detected but their vibrancy has been muted over the centuries. In the case of indigo, it may be visibly observed in some areas; however, analysis revealed its previously unknown presence mixed with a copper green in a select area. This focused study sets a foundation for further studies on both this object and other Asian works of art.

Monte Carlo study of a convergent X-ray beam for high resolution X-ray fluorescence imaging

The traditional X-ray tube design is built upon the impact of energetic electrons on high atomic number absorbers producing the X-ray output, consisting of photons due to Bremsstrahlung and fluorescence. Typically, electrons current hits the target within a limited area of a few millimeters square stopping the electrons, which lose their energy and produce the X rays constituting an inherently divergent beam. This geometrical property of traditional X-ray beams is responsible for several undesirable effects when trying to optimize applications requiring high incident fluence spatial concentration, like X-ray fluorescence imaging. This work presents a Monte Carlo study about a novel X-ray tube design, based on a cylindrical target that is capable of producing a convergent X-ray beam aimed at improving overall performance and spatial resolution in certain applications, like X-ray fluorescence imaging. Main design characteristics for relevant parts, like target/anode, filter, and collimator, have been carefully investigated by means of Monte Carlo simulation using two independent codes: FLUKA and PENELOPE. The obtained results suggest the feasibility of the proposed design remarking that high fluence concentration can be achieved, which can be particularly useful for further applications, like tumor targeting by X-ray fluorescence imaging by means of high atomic number nanoparticle infusion, as reported in this work.

Rhenium carbonyl complexes bearing methylated triphenylphosphonium cations as antibody-free mitochondria trackers for X-ray fluorescence imaging

A convenient rhenium-based multimodal mitochondrial-targeted probe compatible with Synchrotron Radiation X-ray Fluorescence nano-imaging.

Micro-beam X-ray Fluorescence Imaging for Indium Distributed at Cross Section of Hair Contamination Characteristics of Indium in Hair of Workers

Micro-beam X-ray Fluorescence Imaging for Indium Distributed at Cross Section of Hair Contamination Characteristics of Indium in Hair of Workers

Review of in vivo optical molecular imaging and sensing from x-ray excitation.

.Significance: Deep-tissue penetration by x-rays to induce optical responses of specific molecular reporters is a new way to sense and image features of tissue function in vivo. Advances in this field are emerging, as biocompatible probes are invented along with innovations in how to optimally utilize x-ray sources.Aim: A comprehensive review is provided of the many tools and techniques developed for x-ray-induced optical molecular sensing, covering topics ranging from foundations of x-ray fluorescence imaging and x-ray tomography to the adaptation of these methods for sensing and imaging in vivo.Approach: The ways in which x-rays can interact with molecules and lead to their optical luminescence are reviewed, including temporal methods based on gated acquisition and multipoint scanning for improved lateral or axial resolution.Results: While some known probes can generate light upon x-ray scintillation, there has been an emergent recognition that excitation of molecular probes by x-ray-induced Cherenkov light is also possible. Emission of Cherenkov radiation requires a threshold energy of x-rays in the high kV or MV range, but has the advantage of being able to excite a broad range of optical molecular probes. In comparison, most scintillating agents are more readily activated by lower keV x-ray energies but are composed of crystalline inorganic constituents, although some organic biocompatible agents have been designed as well. Methods to create high-resolution structured x-ray-optical images are now available, based upon unique scanning approaches and/or a priori knowledge of the scanned x-ray beam geometry. Further improvements in spatial resolution can be achieved by careful system design and algorithm optimization. Current applications of these hybrid x-ray-optical approaches include imaging of tissue oxygenation and pH as well as of certain fluorescent proteins.Conclusions: Discovery of x-ray-excited reporters combined with optimized x-ray scan sequences can improve imaging resolution and sensitivity.

A feasibility study on the application of separable coded masks to X-ray fluorescence imaging

The coded images acquired by the XRF imaging system can be reconstructed with a neural network and an iterative algorithm.

Benchtop X-ray fluorescence imaging as a tool to study gold nanoparticle penetration in 3D cancer spheroids.

The use of nanomaterials to improve medical diagnostics and therapeutics has been rapidly increasing. Among these materials are gold nanoparticles, which can be functionalized to target specific cells, acting as nanovectors for drug delivery, enhanced contrast agents as well as other targeted therapies. Au nanoparticles are very useful as they selectively accumulate in tumour sites due to the enhanced permeability-retention effect. There is however little information about the spatial distribution of the nanoparticles within tumours, which might hinder efficient therapies. In this study, X-ray fluorescence was used to investigate the diffusion of gold nanoparticles in cancer cell spheroids mimicking true tumour growth. Functionalization of the nanoparticles has the effect of allowing better diffusion into and out of the spheroid, while those nanoparticles that are only partially covered rapidly formed aggregates. This clustering led to size exclusion during transport within the tumour, changing its distribution profile while greatly increasing the nanoparticle concentration.

Characterization of a Pixelated Cadmium Telluride Detector System Using a Polychromatic X-Ray Source and Gold Nanoparticle-Loaded Phantoms for Benchtop X-Ray Fluorescence Imaging.

Pixelated semi-conductor detectors providing high energy resolution enable parallel acquisition of x-ray fluorescence (XRF) signals, potentially leading to performance enhancement of benchtop XRF imaging or computed tomography (XFCT) systems utilizing ordinary polychromatic x-ray sources. However, little is currently known about the characteristics of such detectors under typical operating conditions of benchtop XRF imaging/XFCT. In this work, a commercially available pixelated cadmium telluride (CdTe) detector system, HEXITEC (High Energy X-ray Imaging Technology), was characterized to address this issue. Specifically, HEXITEC was deployed into our benchtop cone-beam XFCT system, and used to detect gold Kα XRF photons from gold nanoparticle (GNP)-loaded phantoms. To facilitate the detection of XRF photons, various parallel-hole stainless steel collimators were fabricated and coupled with HEXITEC. A pixel-by-pixel spectrum merging algorithm was introduced to obtain well-defined XRF + scatter spectra with parallel-hole collimators. The effect of charge sharing addition (CSA) and discrimination (CSD) algorithms was also investigated for pixel-level CS correction. Finally, the detector energy resolution, in terms of the full-width at half-maximum (FWHM) values at two gold Kα XRF peaks (~68 keV), was also determined. Under the current experimental conditions, CSD provided the best energy resolution of HEXITEC (~1.05 keV FWHM), compared with CSA and no CS correction. This FWHM value was larger (by up to ~0.35 keV) than those reported previously for HEXITEC (at ~60 keV Am-241 peak) and single-crystal CdTe detectors (at two gold Kα XRF peaks). This investigation highlighted characteristics of HEXITEC as well as the necessity for application-specific detector characterization.

In vivo percutaneous permeation of gold nanomaterials in consumer cosmetics: implication in dermal safety assessment of consumer nanoproducts

The increasing emergence of nano-cosmetics in the marketplace provokes safety concerns with respect to percutaneous permeation and toxicity of nanomaterials inside the human body. In this study, in vivo percutaneous permeation and dermal safety of cosmetic cream containing Au nanosheets and extracted Au nanosheets from cosmetic creams are investigated with guinea pigs. Quantitative percutaneous permeation data suggests that Au nanosheets in cosmetic creams permeate into the skin epidermis, dermis, and subcutaneous layer after 10 d cutaneous exposure, but cannot enter the systemic circulation. However, more Au nanosheets are accumulated in the skin and the permeation of Au nanosheets increased after embedded into the cream matrix. Synchrotron radiation X-ray fluorescence (SRXRF) imaging reveals that Au nanosheets in cosmetics penetrate mainly through hair follicles in a time-dependent manner. Cosmetic creams rather than extracted Au nanosheets decrease the cell viability of keratinocytes and slightly induce apoptosis/necrosis of keratinocytes and skin dermal fibroblasts. Intriguingly, the growth of hair is inhibited by the cosmetic cream and the extracted Au nanosheets revealed by HE staining and immunohistochemistry (IHC) assay. Altogether this study provides insights into the comprehensive understanding of percutaneous permeation and dermal safety of cosmetic creams containing Au nanosheets. This work provides reliable methods to study the skin permeation, biodistribution, and dermal safety of nano-cosmetics and reminds the community of the crucial need to combine the assays at molecular, cellular, and organ levels in nanotoxicology research.

Correlating STED and synchrotron XRF nano-imaging unveils cosegregation of metals and cytoskeleton proteins in dendrites.

Zinc and copper are involved in neuronal differentiation and synaptic plasticity but the molecular mechanisms behind these processes are still elusive due in part to the difficulty of imaging trace metals together with proteins at the synaptic level. We correlate stimulated-emission-depletion microscopy of proteins and synchrotron X-ray fluorescence imaging of trace metals, both performed with 40 nm spatial resolution, on primary rat hippocampal neurons. We reveal the co-localization at the nanoscale of zinc and tubulin in dendrites with a molecular ratio of about one zinc atom per tubulin-αβ dimer. We observe the co-segregation of copper and F-actin within the nano-architecture of dendritic protrusions. In addition, zinc chelation causes a decrease in the expression of cytoskeleton proteins in dendrites and spines. Overall, these results indicate new functions for zinc and copper in the modulation of the cytoskeleton morphology in dendrites, a mechanism associated to neuronal plasticity and memory formation.

Full-field X-ray fluorescence imaging with a straight polycapillary X-ray collimator

Due to the availability of X-ray imaging detectors, full-field X-ray fluorescence (FF-XRF) imaging technique has become achievable, which provides an alternative to scanning X-ray fluorescence imaging with a micro-focus X-ray beamline. In this paper, we present a setup based on straight capillary optics and an energy-dispersive hybrid pixel detector, which can perform simultaneous mapping of several chemical elements. The photon transmission efficiency and spatial resolution are compared between two X-ray collimation setups: one using pinhole optics and one using straight polycapillary optics. There is a tradeoff between the spatial resolution and transmission efficiency when considering X-ray optics. When optimizing the spatial resolution, using straight capillary optics achieved a higher intensity gain when comparing with the pinhole setup. Characterization of the polycapillary imaging setup is performed through analyzing various samples in order to investigate the spatial frequency response and the energy sensitivity. This developed setup is capable of FF-XRF imaging in characteristic energies below 20 keV, while for higher energies the spatial resolution is affected by photon transmission through the collimator. This work shows the potential of the FF-XRF instrument in the monitoring of toxic metal distributions in environmental mapping measurements.

Self-Assembly of Plasmonic Nanoantenna-Waveguide Structures for Subdiffractional Chiral Sensing.

Spin-momentum locking is a peculiar effect in the near-field of guided optical or plasmonic modes. It can be utilized to map the spinning or handedness of electromagnetic fields onto the propagation direction. This motivates a method to probe the circular dichroism of an illuminated chiral object. In this work, we demonstrate local, subdiffraction limited chiral coupling of light and propagating surface plasmon polaritons in a self-assembled system of a gold nanoantenna and a silver nanowire. A thin silica shell around the nanowire provides precise distance control and also serves as a host for fluorescent molecules, which indicate the direction of plasmon propagation. We characterize our nanoantenna-nanowire systems comprehensively through correlated electron microscopy, energy-dispersive X-ray spectroscopy, dark-field, and fluorescence imaging. Three-dimensional numerical simulations support the experimental findings. Besides our measurement of far-field polarization, we estimate sensing capabilities and derive not only a sensitivity of 1 mdeg for the ellipticity of the light field, but also find 103 deg cm2/dmol for the circular dichroism of an analyte locally introduced in the hot spot of the antenna-wire system. Thorough modeling of a prototypical design predicts on-chip sensing of chiral analytes. This introduces our system as an ultracompact sensor for chiral response far below the diffraction limit.

(Invited) Direct Observation of the Sulfur-Based Chemical Species in Li-S Batteries By Spatially-Resolved X-Ray Fluorescence Microscopy and X-Ray Absorption Spectroscopy

The demands on low cost and high energy density rechargeable batteries for both transportation and large-scale stationary energy storage are attracting more and more research attention toward new battery systems such as metal, metal-sulfur, metal-air, and multivalent batteries. Since sulfur is an earth-abundant material with low cost and has a high theoretical capacity, Li-S battery chemistry has attracted significant interest during the past decade.The Li-S battery utilizes electrochemical conversion of sulfur (S8) to lithium sulfide (Li2S), going through multiple electron transfer processes associated with long- and short-chain polysulfide (Li2Sx) intermediates. It is well known that the long-chain polysulfides can be dissolved into electrolyte with aprotic organic solvents and migrated to the Li anode side. This so-called “shuttle effect” is considered as the main reason for the capacity loss and low coulombic efficiency of the Li-S system. A lot of efforts have been made on suppressing the polysulfide dissolution through new sulfur-based material and electrolyte, as well as cell engineering. Although the Li-S battery performance has been improved over the past decades, the long‐term cycling stability is still one of the major barriers for the practical application of Li–S batteries. More in‐depth studies on the fundamental understanding of the sulfur reaction mechanism and interactions among the different polysulfide species, the electrolyte, and the electrodes are still greatly needed.In this study, we utilize the spatially-resolved X-ray fluorescence (XRF) microscopy combined with X-ray absorption spectroscopy (XAS) to directly probe the inhomogeneous electrochemical/chemical reactions of the sulfur cathode, and chemical reactions of sulfur contained chemical species. The morphology changes and the redistribution of sulfur and polysulfides in both the S cathode and lithium metal anode were monitored through the XRF images, while the oxidation state of sulfur and changes of the sulfur-containing interfacial layer (i.e., SEI) were characterized through the XAS spectra. Our characterization studies on the sulfur-based cathode materials in Li-S batteries using spatially resolved XRF/XAS techniques provides critical information enabling us to understand the reaction processes and their correlation to cell cycling behavior and failure mechanisms. More details of the result will be discussed at the meeting. Acknowledgement The work done at Brookhaven National Laboratory was supported by Assistant Secretary for Energy Efficiency and Renewable Energy, Vehicle Technology Office of the U.S. DOE, through the Advanced Battery Materials Research (BMR) Program, including Battery500 Consortium under contract DE-SC0012704. This research used beamline 8-BM (TES) of the National Synchrotron Light Source II, U.S. DOE Office of Science User Facilities operated for the DOE Office of Science by Brookhaven National Laboratory under Contract No. DE-SC0012704.

PBT2 acts through a different mechanism of action than other 8-hydroxyquinolines: an X-ray fluorescence imaging study.

8-Hydroxyquinolines (8HQs) comprise a family of metal-binding compounds that have been used or tested for use in numerous medicinal applications, including as treatments for bacterial infection, Alzheimer's disease, and cancer. Two key 8HQs, CQ (5-chloro-7-iodo-8-hydroxyquinoline) and PBT2 (2-(dimethylamino)methyl-5,7-dichloro-8-hydroxyquinoline), have drawn considerable interest and have been the focus of many studies investigating their in vivo properties. These drugs have been described as copper and zinc ionophores because they do not cause metal depletion, as would be expected for a chelation mechanism, but rather cellular accumulation of these ions. In studies of their anti-cancer properties, CQ has been proposed to elicit toxic intracellular copper accumulation and to trigger apoptotic cancer cell death through several possible pathways. In this study we used synchrotron X-ray fluorescence imaging, in combination with biochemical assays and light microscopy, to investigate 8HQ-induced alterations to metal ion homeostasis, as well as cytotoxicity and cell death. We used the bromine fluorescence from a bromine labelled CQ congener (5,7-dibromo-8-hydroxyquinoline; B2Q) to trace the intracellular localization of B2Q following treatment and found that B2Q crosses the cell membrane. We also found that 8HQ co-treatment with Cu(ii) results in significantly increased intracellular copper and significant cytotoxicity compared with 8HQ treatments alone. PBT2 was found to be more cytotoxic, but a weaker Cu(ii) ionophore than other 8HQs. Moreover, treatment of cells with copper in the presence of CQ or B2Q resulted in copper accumulation in the nuclei, while PBT2-guided copper was distributed near to the cell membrane. These results suggest that PBT2 may be acting through a different mechanism than that of other 8HQs to cause the observed cytotoxicity.

X-Ray Fluorescence Imaging Based on CdTe Detector Array for Analysis of Various Materials

An X-ray fluorescence (XRF) imaging system was developed to analyze various materials based on characteristic X-rays emitted from objects exposed to X-ray flux. The XRF system can be used in various industrial fields, such as nuclear fuel analysis, to guarantee combustion stability in the nuclear reactor, glazed material analysis to preserve and restore ceramic cultural assets, impurity measurements in electronic circuits, and so on. In this study, we built an XRF imaging system and performed several material analyses using a lookup table, which contained energy and channel information of the characteristic X-ray peaks of each element. Ceramic specimens coated in various glazes were analyzed and imaged for oriental pottery research. Pigments of various colors were also analyzed and imaged for picture assessment, as were electronic circuits and gold plates. Ceramic, pigment, and metal components in the samples were discriminated by comparing characteristic X-ray peak data on the lookup table. Two-dimensional material images, which showed the material distribution of each sample, were successfully obtained.