In-air micro-PIXE Research Articles

PIXE STUDY ON ARSENIC ACCUMULATION BY A FERN (PTERIS VITTATA)

Pteris vittata is a fern reported to be an arsenic hyper-accumulator. To develop the practical application of the fern to a phytoremediation technique, it is necessary to explicate the effective accumulation mechanism. In this study, the arsenic distribution and the elemental correlation in the cellular level were examined in the fronds supplied with arsenate and arsenite separately via xylem vessel using an in-air micro-PIXE system at Tohoku University. The difference in transportation rate between arsenate and arsenite as well as the translocation of elements necessary for plant metabolism was revealed in different tissues of the fronds accumulating arsenic in high concentration. Hence, the in-air micro-PIXE analysis is an effective measure for undertaking phytoremediation research of hyper-accumulator plants.

Three-dimensional measurement of elemental distribution in minute samples by combination of in-air micro-PIXE and STIM

A technique of measuring three-dimensional (3D) distribution of trace elements in a minute sample was studied using the in-air micro-PIXE (Particle Induced X-ray Emission) system. A 3D distribution was acquired by means of reconstructing numbers of projection images of the sample obtained by micro-PIXE analyses from different directions. This is basically the same technique as X-ray CT (Computed Tomography). However, when this technique was applied to the in-air micro-PIXE analysis, the X-ray yields should be corrected in taking account of such parameters as the X-ray production cross section, the X-ray attenuation coefficient, the energy of an incident particle, and the 3D densities of major elements in the analysis sample. In this study, STIM (Scanning Transmission Ion Microscopy)-CT was used to obtain the 3D density map of the sample. Corrected 3D distributions of sulfur and iron in a minute test sample were successfully measured with this technique in combination of STIM-CT and in-air micro-PIXE.

IN-AIR MICRO-PIXE ANALYSIS FOR METAL ELEMENTS IN RAT HEPATOCYTES TREATED WITH FUCOIDAN

This study was undertaken to elucidate physiological properties of fucoidan extracted from Cladosiphon okamuranus TOKITA (C. okamuranus) and Fucus vesiculosus (F.vesilurosus). According to the data on MTT assay and TUNEL staining, F. vesiculosus possessed more anti-proliferate activity than C. okamuranus through apoptosis induction. We determined the metal contents of fucoidan to evaluate the relevance of metal elements in its biological effects. Al , Fe and Zn levels were significantly increased in F. vesiculosus than in C. okamuranus. The intracellular changes of metal elements following the administration of fucoidan was analyzed by in-air micro-PIXE in TRL1215 cells (normal rat liver cell line). At 24 h after 5-bromo-2'-deoxyuridine ( BrdU ) dosing, TRL1215 cells were treated with 1.0 mg/mL of each fucoidan for 9 h. The X-ray spectra showed that F. vesiculosus-treated cells markedly increased the yield of aluminum ( Al ) compared to those of C. okamuranus -treated cells and the control. In F. vesiculosus -treated cells, the focal accumulation of Br was spatially correlated with Al map, suggesting that Al was localized within the nucleus. These findings suggest that F. vesiculosus fucoidan increases the accumulations of Al in rat hepatocytes, which may participate in anti-proliferate activity of this fucoidan.

DEVELOPMENT OF A SYSTEM FOR MEASURING FLUORIDE DISTRIBUTION IN TEETH USING A NUCLEAR REACTION

Fluoride ( F ) distributions in a synthesized hydroxyl apatite ( HAp ) block of uniform structure and in teeth were measured using in-air micro-PIGE (particle induced gamma-ray emission) and micro-PIXE system, which was developed at the Japan Atomic Energy Agency (JAEA) in Takasaki. We used a nuclear reaction 19 F ( p ,αγ)16 O to measure F density. The characteristic important feature of this technique is that it can measure F quantitatively in a microscopic area of the specimen placed in air. A surface of the HAp , the enamel buccal surface of a human molar, and a class V cavity wall in dentin were applied a sodium fluoride solution ( NaF ) four times and immersed in a normal saline solution. After one month, specimens were cut longitudinally. The F distributions were measured from the surface toward the inner part of the cut surface. The F penetration into specimens following NaF application was quantitatively configured in a two-dimensional mapping form. This method is quite useful for characterizing F distribution in a microscopic area of a tooth.

In-Air Micro-Particle Induced X-ray Emission Analysis of Asbestos and Metals in Lung Tissue

Inhalation of asbestos increases the risk of lung cancer and pulmonary fibrosis. It is difficult to directly assess the distribution and content of inhaled particles in lung tissue sections. The purpose of this study is to employ an in-air micro particle induced X-ray emission (in-air micro-PIXE) system for assessment of the spatial distribution and content of asbestos and other metals in lung tissue. A proton ion-microbeam from this system was applied to irradiate lung tissue of patients with or without asbestosis, tumor tissue from both groups, and asbestos fibers (in vitro). The content of each element composing asbestos and those of other metals were calculated and their distribution was assessed from the characteristic X-ray pattern for each element obtained after irradiation. This in-air micro-PIXE system could identify the location of asbestos bodies composed of Si, Mg, and Fe in lung tissue sections. Macrophage and lymphocytes accumulated in that area. This new system also revealed deposits of titanium, nickel, and cobalt in the lung tissues, in addition to asbestos bodies. The Si and Fe content were higher in lungs with asbestosis than in lungs without asbestosis or in tumor tissue. Analysis of asbestos fibers composed of chrysotile, crocidolite, and amosite showed that the ratios of Si, Fe, and Mg corresponded with those for the chemical structures. In-air micro-PIXE analysis is useful for assessing the distribution and quantities of asbestos bodies and also other metals in lung tissue comparing to immune-related cell localizations, and is also useful for analysis of standard asbestos fibers.

Direct visualization and quantification of the anticancer agent, cis‐diamminedichloro‐platinum(II), in human lung cancer cells using in‐air microparticle‐induced X‐ray emission analysis

The authors designed an elemental analysis system using an ion microbeam combined with a microparticle-induced X-ray emission (micro-PIXE) method for the analysis of biomedical samples in air with a spatial resolution of 1 microm (in-air micro-PIXE system). This system was used to develop an imaging and quantification method for intracellular cis-diamminedichloro-platinum(II) (CDDP) in a human lung cancer cell line. A human lung adenocarcinoma cell line, A549, was cultured and nuclear labeling was carried out by incubating the cells with BrdU. The cells were then exposed to CDDP at concentrations ranging from 1 micromol to 1 mmol, for 30 min to 24 h. After drug treatment, samples were washed and frozen with liquid nitrogen, and freeze-dried for 24 h. Standard samples were made using agar containing several concentrations of CDDP. Experiments using standard samples showed a linear correlation between CDDP concentration and platinum signal strength. No clear platinum signal was detected after exposure to CDDP for 24 h at doses between 1 and 100 micromol. However, significant platinum signals were observed at 1 mmol. When nucleus and cytoplasm visualization was sufficiently clear to efficiently use in-air micro-PIXE, the platinum image quality was considered satisfactory. The detected signals of CDDP were stronger in the nucleus than in the cytoplasm. A time-course study showed increased CDDP uptake in cells after longer drug exposure periods. The present study demonstrates the application of element analysis using in-air micro-PIXE to biomedical samples. The use of this system enables the high-resolution visualization of intracellular CDDP distribution and measurement of intracellular CDDP concentrations.

PIXE STUDY ON ABSORPTION OF ARSENATE AND ARSENITE BY ARSENIC HYPERACCUMULATING FERN (PTERIS VITTATA)

Pytoremediation using an arsenic hyperaccumulator, Petris vittata L., has generated an increasing interest worldwide due to both environmentally sound and cost effectiveness. However the mechanism of arsenic accumulation by this fern is not clear at this time. This study examined the uptake of arsenate (As(V)) and arsenite (As(III)) by a hydroponic culture of Pteris vittata using both in-air submilli-PIXE for different parts of the fern and in-air micro-PIXE for the tissue cells. These PIXE analysis systems used 3 MeV proton beams from a 4.5-MV single-ended Dynamitron accelerator at Tohoku University, Japan. The fern took up both arsenate and arsenite from hydroponic solutions which were spiked with 50 mg of arsenic per litter. Final amount of arsenic accumulation in the fern is 1,500 mg per kg (wet weight) of the plant biomass in arsenite treatment and 1,100 mg per kg in arsenate treatment. Arsenic accumulation was not observed at the root parts of the ferns. The in-vivo mapping of elements by submilli-PIXE analyses on the fern laminas showed the arsenic accumulation in the edges of a pinna. The micro-PIXE analyses revealed arsenic maps homogeneously distributed in cells of the lamina, stem and rhizome of the fern. These results indicate that arsenic, both arsenate and arsenite in a contaminated medium are translocated quickly from roots to fronds of Pteris vittata, and distributes homogeneously into tissue cells of the fern laminas.

Analysis of tissue cadmium distribution in chronic cadmium-exposed mice using in-air micro-PIXE

This study undertook the analysis of tissue cadmium (Cd) distribution using in-air micro-particle-induced X-ray emission (PIXE) and the examination of the involvement of metal ions in parenteral Cd toxicity. A mouse was injected intraperitoneally with 3 mg/kg body weight of CdCl2 thrice weekly. After 27 wk, the liver and kidney were excised and fixed in 10% formalin solution for 4 h and then embedded in paraffin. Thin paraffin sections were used to analyze trace elements with in-air micro-PIXE and to examine metallothionein protein and histological changes. Cd distribution was determined by micro-PIXE in the liver and renal cortex of the Cd-exposed mouse, and the net Cd count was higher in the liver than in the renal cortex. The net iron (Fe) count was higher in the liver of the Cd-exposed mouse compared to the control, and an opposite tendency was observed in the renal cortex. Wide cellular Cd distribution was demonstrated in the liver and renal cortex of the chronic Cd-exposed mouse compared to the control. Metallothionein staining was increased by chronic exposure to Cd both in the liver and kidney, and nephrotoxicity was more apparent than hepatotoxicity. The modification of tissue Fe and calcium distribution by an intraperitoneal injection of Cd might be involved in Cd-induced toxicity.

Subcellular changes of essential metal shown by in-air micro-PIXE in oral cadmium-exposed mice

To clarify the relation of essential metals to cadmium (Cd) toxicity, we evaluated metallothionein expression and analyzed the subcellular distribution of essential metals using in-air micro-Particle-Induced X-ray Emission (PIXE). Four mice were dosed orally with 100 mg/L of Cd in drinking water for 1.5 or 2 years. Frozen samples of organs were used for micro-PIXE analysis and formalin-fixed samples were used for metallothionein staining. Immunohistochemically, metallothionein induction by 1.5y-Cd exposure was higher in the renal cortex than in the liver. Metallothionein expression was reduced after 2y-Cd administration compared to the 1.5y-Cd-exposed mice. Cd-induced tissue damage became marked in the 2y-Cd-exposed mice compared to the 1.5y-Cd-exposed mice, in which nephrotoxicity was more prominent than hepatotoxicity. Cd yield was higher in the renal cortex of the 2y-Cd-exposed mouse than in that of the 1.5y-Cd-exposed mouse, whereas no such increasing tendency was found in the liver. Compared to the control, the Cd-exposed mice markedly accumulated zinc in the liver and renal cortex. In the Cd-exposed mice, iron was mildly accumulated in the renal cortex and was slightly deprived in the liver. Elemental maps showed that a large amount of Cd was spatially combined with zinc in the 1.5y-Cd mouse. Free Cd became abundant in the 2y-Cd-exposed mouse. In addition, a small amount of Cd was colocalized with iron. The data suggest that zinc may contribute to protect against oral-administrated Cd toxicity, and impaired induction of MT may participate in hepato-nephrotoxicity of the 2y-Cd-exposed mouse.

RESEARCH OF DISEASE ONSET MECHANISM BY DETERMINING THE DISTRIBUTION OF INTRACELLULAR TRACE ELEMENTS USING AN IN-AIR MICRO-PIXE ANALYZER SYSTEM

A program was started to create a new medical scientific field, which involves radiation oncology and nuclear medicine, utilizing advanced accelerator and ion beam technology. An in-air micro-PIXE analyzer system, which is among the most important technical aspects of the program, was upgraded to improve the accuracy of elemental mapping for samples with thickness variation in the scope of microbeam scanning. In order to address important bio-medical problems in cancer, the intracellular dynamics of trace elements according to the development mechanism of diseases were studied using this system. This paper outlines the program, showing correction of micro-PIXE elemental map by STIM analysis and the preliminary application results.

New in-air micro-PIXE system for biological applications

Two unique apparatuses have been installed at in-air micro-PIXE system at JAERI Takasaki. One is a multiple X-ray detection system that consists of three X-ray detectors. The efficiency below 2keV is greatly improved by a new high energy resolution Si(Li) detector, on the other hand higher energy region above 6keV is covered by a backward X-ray detector that is set just behind a sample. The X-ray detection efficiency is drastically improved by the system. Another improvement is a sample cooling system that blows cryogenic gas (∼100K) from liquid nitrogen onto the sample. This technique enables elemental mapping of a frozen raw biological specimen without serious irradiation damage. These improvements are very useful especially in biological applications.

JAERI Takasaki in-air micro-PIXE system for various applications

In JAERI Takasaki, an in-air micro-PIXE system has been developed. This system enables multi-elemental mapping of samples in atmospheric environment with spatial resolution of 1 μm. Various research programs, such as biomedical research, dental study, environmental science and geology, have been started in recent years. Several related techniques for these applications were developed. A FTP server has been operated for the remote collaborators to share experimental data over the internet. We have developed image processing methods for elemental concentration analysis and a new beam monitoring technique in thick target irradiation.

IN-AIR MICRO-PIXE ANALYSIS OF TISSUE SAMPLES

Micro-PIXE is capable of providing spatial distributions of elements in the micro-meter scale and its application to biology is useful to elucidate the cellular metabolism. Since, in this method, a sample target is usually irradiated with proton or α-particle beams in vacuum, beam heating results in evaporation of volatile elements and shrinking of the sample. In order to avoid these side effects, we previously developed a technique of in-air micro-PIXE analysis for samples of cultured cells. In addition to these, analysis of exposed tissue samples from living subjects is highly desirable in biological and medical research. Here, we describe a technique of in-air micro-PIXE analysis of such tissue samples. The target samples of exposed tissue slices from a Donryu rat, in which a tumor had been transplanted, were analyzed with proton micro-beams of 2.6 MeV. We report that the shape of cells and the distribution of volatile elements in the tissue sample remain uncharged when using a target preparation based on a freeze-drying method.

Elemental analysis of cellular samples by in-air micro-PIXE

We developed a micro-PIXE system for the analysis of frozen cellular samples at atmospheric pressure under helium gas cooling. We analyzed bovine aortic endothelial (BAE) cells previously cultured in a medium containing bromodeoxyuridine (BrdU). It is known that these cells take BrdU into their nuclei at the S phase of their metabolic cycle. We examined the Br uptake for incubation times of 6, 12 and 24 h, respectively. The Br maps showed an accumulation in the nuclei and the Br concentration was found strongly dependent on the incubation time. We have demonstrated that in-air micro-PIXE analysis is a relevant technique for examining the molecular uptake in cells.

DEVELOPMENT OF IN-AIR MICRO-PIXE ANALYSIS AND DATA SHARING SYSTEMS IN JAERI TAKASAKI

An external scanning ion microbeam system has been developed for in-air micro-PIXE analysis at JAERI Takasaki. The analysis system is widely used for various researches in recent years. The system consists of the external scanning ion microbeam system, a multi-parameter data acquisition system, a file transfer protocol (FTP) server and analysis software. The software of the system provides a graphical user interface for interaction between users and an experimental setup. The server is connected to the Internet and allows remote users to access the experimental data.