In-air micro-PIXE Research Articles

Comparison of in-vacuum micro-PIXE and in-air micro-PIXE for unfixed plant sample

PIXE analysis is very useful for the simultaneous determination of various elements. Typically, when micro-PIXE analysis of biological samples with high water content is performed in a vacuum, freeze-drying methods are used to fix the structure and remove any ice crystals present in a controlled manner to prevent changes or redistribution of elemental content in the sample. This method is essential for cell size analysis. However, we considered that fixation is not always necessary for macroscopic analysis of plant tissue size. Therefore, we investigated the extent to which elemental density changes with vacuum extraction from elemental density ratios obtained from micro-PIXE analysis of Arabidopsis thaliana leaves in-air and in-vacuum. The deformation caused by beam irradiation and the quantitative changes of C, O, and H in the samples were also evaluated by STIM and RBS analyses of Arabidopsis thaliana petal.

In-air micro-PIXE chemical state analysis of phosphorus and sulfur using a new parallel-beam wavelength-dispersive X-ray emission spectrometer

The proton induced K X-ray emission spectra of several different phosphorus and sulfur compounds were recorded with the new parallel-beam wavelength dispersive X-ray emission spectrometer. The spectrometer was installed recently at the external proton microbeam at the J. Stefan Institute to perform high energy resolution in-air micro-PIXE analysis in the tender X-ray range. Reported measurements are used to probe the capabilities of the spectrometer to perform also chemical state analysis. For phosphorus measurements, where the energy resolution of the spectrometer is the highest, a clear correlation between measured energy position of the Kα emission line and the phosphorus formal oxidation state is obtained. Chemical contrast is increased further in the Kβ emission spectra reflecting the structure of occupied valence molecular orbitals defined by the first coordination shell around the central phosphorus atom. A reasonable chemical contrast is maintained also in the sulfur Kβ emission spectra, despite the fact that the energy resolution of the spectrometer declines with increasing X-ray energy. Finally, an example of phosphorus speciation in reference biological material is presented, demonstrating the feasibility of the new setup to extend the capabilities of in-air micro-PIXE analysis towards chemical speciation.

Characterization of hourly-collected airborne particulate matters from an automated sampling unit of the atmospheric environmental regional observation system by in-air micro-PIXE analysis

We investigated element composition ratios in two sets of sequentially collected airborne particulate matter (PM) samples obtained through the automatic sampler of the Atmospheric Environmental Regional Observation System (AEROS). A series of micro-PIXE analyses and imaging were performed on a PTFE (Polytetrafluoroethylene) membrane tape filter corresponding to two hourly time slots through a high-concentration air pollution event from 26th to 27th March 2020 sampled at Ota, Japan (36.290° N, 139.381° E) by utilizing an external 3 MeV proton microprobe. Latent variables of sequentially collected PM samples were analyzed by the non-linear least square method and classified by those clustering driven by house-made data analysis software. Micro-PIXE imaging effectively confirmed the presence of large elemental agglomerations on the PTFE membrane tape filter of AEROS that often appeared as outliners in data analysis.

A parallel-beam wavelength-dispersive X-ray emission spectrometer for high energy resolution in-air micro-PIXE analysis

A new parallel-beam wavelength dispersive (PB-WDS) X-ray emission spectrometer was constructed at the external proton beamline at the Microanalytical Centre of the Jožef Stefan Institute in Ljubljana.

Progression of Idiopathic Pulmonary Fibrosis Is Associated with Silica/Silicate Inhalation

Recently, environmental dust exposure has emerged as a possible risk factor for interstitial pneumonia. Inhaled silica/silicates and titanium are associated with the progression of rapidly progressive pneumoconiosis. However, the relationship between inhaled elements and idiopathic pulmonary fibrosis (IPF) remains unclear. In this study, we focused on IPF presenting with histological usual interstitial pneumonia and investigated the relationship between inhaled elements and IPF progression. We examined the elements in the lungs using in-air micro-particle-induced X-ray emission analysis (PIXE). Lung tissue samples with IPF were analyzed and compared with control lung samples. There was a significant difference between IPF and the control lungs in terms of silicon, assumed to form silica/silicates. Inhaled silicon in the lungs significantly correlated with the annual decline in forced vital capacity in patients with IPF. Furthermore, IPF patients with higher accumulation of silicon showed a significantly poorer prognosis than those with lower accumulation of silicon in the lungs. These significant differences were also seen in multivariate analyses, including smoking history, IPF severity, and/or antifibrotic treatment. In-air micro-PIXE can be beneficial for detecting inhaled elements in the lung. These results indicate that inhaled silica/silicates in the lung may be partly involved in the progression of IPF.

Preliminary Study on the Elemental Quantification of in Ambient Liquid Samples of Microliter Volume Using the In-air Micro-PIXE Technique

Quantifying the trace elements in infinitesimal ambient liquid samples (e.g., single raindrop, cloud/ fog water, and the soluble fraction extracted from the particles collected for a short time) is an important task for understanding formation processes, heating/cooling rates, and their health hazards. The purpose of this study is to employ an in-air micro PIXE system for quantitative analysis of the trace elements in a thimbleful of reference liquid sample. The bag type liquid sample holder originally designed with 10 μm thick Mylar ® film retained the original shape without any film perforation and apparent peaks of film blank by the end of the analysis. As one of tasks to be solved, the homogeneity of the elemental distribution in liquid reference species was verified by the X-ray line profiles for several references. It was possible to resolve the significant peaks for whole target elements corresponding to the channel number of micro-PIXE spectrum. The calibration curves for the six target elements (Si, S, Cl, Fe, Ni, and Zn) in standard solutions were successfully plotted by concentration (ppm) and ROI of interest net counts/dose (nC).

Elemental and immunohistochemical analysis of the lungs and hilar lymph node in a patient with asbestos exposure, a pilot study.

Studies have shown that inhaled mine dust, such as asbestos, can be translocated to various organs including the lymph nodes. Recently, we have established a protocol that enables us to identify inhaled elements using paraffin embedded lung specimens by in-air microparticle-induced X-ray emission (micro-PIXE). However, little research has examined the concentration of these inhaled fibers in various organs or the mechanisms of their translocation. In this study, we compared the concentration of inhaled fibers in the lung parenchyma to the concentration in the hilar lymph node as well as to determine the elemental spatial distribution of the inhaled fibers in a patient with occupational asbestos exposure. Lung tissues and hilar lymph node in a patient with asbestos exposure were used in this study. Elemental analysis was performed by in-air micro-PIXE. Immunohistochemical analysis was performed using anti CD163, smooth muscle actin, vimentin and β-catenin antibody. The analysis revealed that the amount of inhaled silicon was approximately 6 times higher in the lymph node than in the lungs. The spatial analysis showed that silicon, iron and aluminium were co-localized in the hilar lymph node. The immunohistochemical analysis showed localized agreement of the inhaled fibers with macrophages, smooth muscle actin, and vimentin in the hilar lymph node. This study showed that in-air micro-PIXE could be useful for analyzing the elemental distribution and quantification of inhaled fibers in the human body. Furthermore, immunohistochemistry in combination with in-air micro-PIXE analyses may help to determine the mechanism of mine dust distribution in vivo.

Analysis of Erythrocyte Elements in Chronic Hepatitis C Patients Receiving PegIFN Monotherapy, Dual Therapy, and Triple Therapy using in-air MicroPIXE

Analysis of Erythrocyte Elements in Chronic Hepatitis C Patients Receiving PegIFN Monotherapy, Dual Therapy, and Triple Therapy using in-air MicroPIXE

Development of a new in-air micro-PIXE set-up with in-vacuum charge measurements in Atomki

A new external microbeam set-up has recently been installed as the extension of the existing microprobe system at the Laboratory of Ion Beam Applications of Atomki, Debrecen, Hungary. The external beam set-up, based on the system of Oxford Microbeams (OM), is equipped with two X-ray detectors for PIXE analysis, a digital microscope, two alignment lasers and a precision XYZ stage for easy and reproducible positioning of the sample. Exit windows with different thicknesses and of different materials can be used according to the actual demands, currently silicon-nitride (Si3N4) film with 200nm thickness is employed in our laboratory. The first application was demonstrated in the field of archaeometry, on Bronze Age hoards from Hungary.

Elemental analysis of erythrocytes in patients with chronic liver diseases positive for hepatitis C virus

The present study aimed to evaluate the relationship between elemental changes in erythrocytes and hepatogenous anemia. Five chronic hepatitis C, five liver cirrhosis (HCV positive) and three healthy controls were enrolled in this study. Using in-air micro-PIXE, we determined that K rods formed small nodule and Cl rods distributed diffusely in all over erythrocytes, resulting in disappearance of the donut-like pattern of erythrocytes. These findings may reect the alternation of ery- throcyte membrane structures. Fe dots were divided into two to four compartments in erythrocytes of control group, and the similar distribution of Fe rods was shown in those of chronic hepatitis and liver cirrhosis. Na concentrations analyzed by in-vacuum PIXE were significantly higher in liver cirrhosis than in chronic hepatitis and control, possibly because of the disturbance of Na–K pump. Present study suggests that the alternation of erythrocyte membrane and Na–K pump failure may participate in hepatogenous anemia accompanied with liver cirrhosis.

Co-localization of iron binding on silica with p62/sequestosome1 (SQSTM1) in lung granulomas of mice with acute silicosis.

The cellular mechanisms involved in the development of silicosis have not been fully elucidated. This study aimed to examine influence of silica-induced lung injury on autophagy. Suspensions of crystalline silica particles were administered transnasally to C57BL/6j mice. Immunohistochemical examination for Fas and p62 protein expression was performed using lung tissue specimens. Two-dimensional and quantitative analysis of silica deposits in the lungs were performed in situ using lung tissue sections by an in-air microparticle induced X-ray emission (in-air micro-PIXE) analysis system, which was based on irrradiation of specimens with a proton ion microbeam. Quantitative analysis showed a significant increase of iron levels on silica particles (assessed as the ratio of Fe relative to Si) on day 56 compared with day 7 (p<0.05). Fas and p62 were expressed by histiocytes in granulomas on day 7, and the expressions persisted for day 56. Fas- and p62-expressing histiocytes were co-localized in granulomas with silica particles that showed an increase of iron levels on silica particles in mouse lungs. Iron complexed with silica induces apoptosis, and may lead to dysregulations of autophagy in histiocytes of granulomas, and these mechanisms may contribute to granuloma development and progression in silicosis.

Particle Induced X-ray Emission (PIXE): A Tool of Qualitative Elemental Analysis for Biological Sample

The facility of ion beam laboratory at Kochi University of Technology (KUT) has been extended by installing the Particle Induced X-ray Emission (PIXE) technique, in order to provide qualitative and quantitative elemental analysis and in-air micro-PIXE analysis. This paper is a description of PIXE setup and its application in biological sample for qualitative elemental analysis. The energy calibration of the system shows linearity. The minimum detection limit indicates that the system has good detection limit. The homogeneity shows uniformity of the sample itself and the internal standard within the sample. A 4 MeV He++ ion beam was used to analyze shellfish samples. Analyzing samples, it was found that the lower Z elements as well as some trace elements were detected. DOI: http://dx.doi.org/10.3329/rujs.v39i0.16538 Rajshahi University J. of Sci. 39, 01-10 (2011)

Proton Ion-Microbeam Elemental Analysis for Inhaled Particle-Induced Pulmonary Diseases: Application for Diagnosis and Assessment of Progression

Elemental analysis can be applied in the medical field to investigate the causes of disease. In patients with some pulmonary diseases, elements can be found in the exogenous dust deposited in the lungs and are also accumulated through the loss of cell homeostasis. Diseases induced by inhalation of dust typically affect the lungs. Although there are many pulmonary diseases induced by dust inhalation, it is often difficult to clarify the exact cause. In-air microparticle induced X-ray emission (in-air micro-PIXE) analysis is a method of elemental analysis that employs a proton ion-beam to directly measure the content of elements and their distribution in frozen sections or paraffin sections of tissue. We constantly inhale particles while breathing, but most of us do not develop pulmonary disease. Because in-air micro-PIXE analysis can determine the two-dimensional localization and content of particles in tissue, we can clarify the relationship between inhaled particles and diseases based on such analysis and the immunohistochemical expression of disease-related proteins. Elemental analysis methods like in-air micro-PIXE analysis may be useful for making precise diagnosis amd assesing disease progression to overcome threat such as occupational or environmental exposure.

Determining aerosol particles by in-air micro-IL analysis combined with micro-PIXE

A new external ion microbeam ion luminescence (micro-IL) imaging system was developed on a microbeam line of a 3MV single-ended accelerator at the TIARA facility of the Japan Atomic Energy Agency. Micro-IL was combined with an in-air micro-PIXE (particle-induced X-ray emission) system to determine the chemical composition and structures of microscopic airborne particles of several micrometers in size. The hardware and software for the combined in-air micro-IL analysis system, called ion luminescence microscopic imaging and spectroscopy (ILUMIS), were studied. Wavelength-dispersive optics, including a collimator lens, a monochromator, and a photon-counting photomultiplier, were installed on the beam line. The signal processing of the IL photon signals, which were collected as spectra and two-dimensional microscopic images, was examined. Several aerosol particles were characterized to demonstrate the ILUMIS/PIXE combined analysis. The external microbeam ILUMIS analysis method provided a variety of information on the chemical and elemental composition of the micrometer-sized aerosol targets under ambient atmospheric conditions.

ESTABLISHMENT OF ERYTHROCYTE SAMPLES FOR IN-AIR MICRO-PIXE ANALYSIS AND ITS APPLICATION TO ERYTHROCYTES IN HEMODIALYSIS PATIENTS

The present study aims to analyze the elemental distribution in erythrocytes from five hemodialysis patients treated with erythropoietin in comparison to four healthy controls. Using in-air micro-PIXE we determined that iron dots were distributed in the peripheral region of control erythrocytes, which were made up of two to four compartments. Iron dots tend to aggregate partially in hemodialysis patients. Calcium was rendered to form small dense nodules in erythrocytes of controls, and small dense nodules became marked in some hemodialysis patients. Nodular formation of phosphorous was weakened in both control and hemodialysis patients. Potassium aggregated focally and formed small dense granules in erythrocytes of healthy controls. On the other hand, hemodialysis patients showed enrichment of potassium, with diffuse spreading partially to all over erythrocytes. These findings indicate that in-air micro-PIXE is a useful tool for analyzing the elemental distributions in erythrocytes of hemodialysis patients.

Elemental analysis of lung tissue particles and intracellular iron content of alveolar macrophages in pulmonary alveolar proteinosis

BackgroundPulmonary alveolar proteinosis (PAP) is a rare disease occurred by idiopathic (autoimmune) or secondary to particle inhalation. The in-air microparticle induced X-ray emission (in-air micro-PIXE) system performs elemental analysis of materials by irradiation with a proton microbeam, and allows visualization of the spatial distribution and quantitation of various elements with very low background noise. The aim of this study was to assess the secondary PAP due to inhalation of harmful particles by employing in-air micro-PIXE analysis for particles and intracellular iron in parafin-embedded lung tissue specimens obtained from a PAP patient comparing with normal lung tissue from a non-PAP patient. The iron inside alveolar macrophages was stained with Berlin blue, and its distribution was compared with that on micro-PIXE images.ResultsThe elements composing particles and their locations in the PAP specimens could be identified by in-air micro-PIXE analysis, with magnesium (Mg), aluminum (Al), silicon (Si), phosphorus (P), sulfur (S), scandium (Sc), potassium (K), calcium (Ca), titanium (Ti), chromium (Cr), copper (Cu), manganase (Mn), iron (Fe), and zinc (Zn) being detected. Si was the major component of the particles. Serial sections stained by Berlin blue revealed accumulation of sideromacrophages that had phagocytosed the particles. The intracellular iron content of alveolar macrophage from the surfactant-rich area in PAP was higher than normal lung tissue in control lung by both in-air micro-PIXE analysis and Berlin blue staining.ConclusionThe present study demonstrated the efficacy of in-air micro-PIXE for analyzing the distribution and composition of lung particles. The intracellular iron content of single cells was determined by simultaneous two-dimensional and elemental analysis of paraffin-embedded lung tissue sections. The results suggest that secondary PAP is associated with exposure to inhaled particles and accumulation of iron in alveolar macrophages.

Influence of tissue particles on Fas expression in idiopathic interstitial pneumonia

Idiopathic interstitial pneumonia (IIP) is a progressive fibrosing interstitial pneumonia of unknown etiology with a poor prognosis. The aim of this study is to prove the occurrence of particle deposition and particle-induced tissue damage in IIP by examining proapoptotic Fas expression with in-air microparticle induced X-ray emission (in-air micro-PIXE) analysis. A total of 21 patients were enrolled. Lung tissues from 12 IIP patients and nontumorous lung tissues from 9 lung cancer patients (as a control) were subjected to in-air micro-PIXE analysis. The distribution of particles in lung tissue was compared with the localization of Fas expression by immunohistochemistry. Silicon (Si) was identified in 58.3% of IIP samples and 44.4% of control samples. Iron (Fe) was identified 25% in IIP samples and 11.1% in control samples. The mean lung tissue content of Si and Fe relative to S did not differ between IIP and control patients. Only two IIP patients showed the co-localization of Si and Fe deposition with Fas expression. Adaptation of this method would contribute to assess the influence of particles on IIP.

FLUORIDE UPTAKE INTO HUMAN TEETH FROM A FLUORIDE-RELEASING THIN LAYER AFTER A LONG PERIOD OF FILLING

Fluoride ( F ) distribution in human teeth was measured using an in-air micro-PIGE and micro-PIXE system. Class V cavities in the extracted teeth were prepared with Fluoro-Bond Shake One to provide a thin layer of an F-releasing material (FRM). The cavities were then filled with Beautifil Flow F10 (FRM, Group I) or LITE FIL IIP (non-FRM, Group II). Following a four-year period, specimens were cut longitudinally perpendicular to the cavity floor. The F distribution was measured at the floor of the cut surface. The position including 90% of the intact Ca level was defined as the wall surface. Based on this demarcation, indices of F distribution (surface F concentration and F penetration depth) were determined. Thickness of FRM thin layers varied (≈339 μm) and did not affect F distribution. Both values of F distribution indices in Group I [821–8763 (mean 3797) ppm, 34–668 (mean 241) μm] were significantly larger than those in Group II [0–7064 (mean 1865) ppm, 0–143 (mean 21) μm]. The F distribution in Group I was affected more by the filling material than by the FRM thin layer during the four-year exposure. Methodologies using this system may give insightful information for the development of new dental materials.

Quantitative analysis of cisplatin sensitivity of human esophageal squamous cancer cell lines using in‐air micro‐PIXE

Cisplatin is a key chemotherapeutic agent for the treatment of esophageal cancer. We examined the intracellular localization of cisplatin in esophageal cancer cell lines and determined their sensitivity to cisplatin using in-air micro-PIXE (particle induced X-ray emission). Two human esophageal squamous cell carcinoma (ESCC) cell lines, TE-2 and TE-13, were examined for their response to cisplatin using MTT assay, flow cytometry, and DNA fragmentation assays. Real-time reverse transcription-polymerase chain reaction was also used to evaluate the mRNA expression of multidrug resistance protein 2 (MRP2) in both cell lines. Platinum localizations of intracellular and intranuclear were measured using in-air micro-PIXE. TE-2 cells were more sensitive to cisplatin than TE-13 cells (IC(50): 37.5 mum and 56.3 mum, respectively). Flow cytometry analysis confirmed that more TE-2 than TE-13 cells were in the sub-G1 phase. DNA fragmentation assay was analyzed to confirm the MTT assay and flow cytometry results. The expression of MRP2 mRNA in TE-13 cells was stronger than in TE-2 cells. In-air micro-PIXE showed that TE-2 cells had higher intracellular cisplatin concentrations than TE-13 cells and the ratio of intranuclear to intracellular cisplatin in individual cells was not significantly different. We observed the intracellular and intranuclear localization of cisplatin using in-air micro-PIXE. The results of this study suggest that in-air micro-PIXE could be a useful quantitative method for evaluating the cisplatin sensitivity of individual cells. Finally, we speculate that MRP2 in the cell membrane may play an important role in regulating the cisplatin sensitivity of ESCC cells.

Analysis on the Co-Localization of Asbestos Bodies and Fas or CD163 Expression in Asbestos Lung Tissue by in-Air Micro-Pixe

To prevent and control disease caused by exposure to various agents, it is necessary to determine the harmful level of intervention and to establish a method for measuring that level. In-air microparticle-induced X-ray emission (in-air micro-PIXE) analysis is based on irradiation of specimens with a proton ion microbeam, and has been modified for biological application. Two-dimensional analysis and quantitative analysis using the system confirmed that asbestos induced apoptosis by upregulating Fas expression and also revealed the accumulation of CD163-expressing macrophages in the lungs of patients with asbestosis. By quantitative comparison of the area of Fas or CD163 expression and the Fas- or CD163-negative area in asbestos lung tissue, the harmful levels which caused the expression of Fas or CD163 could be estimated on Silica, Ferrous iron, and Magnesium (the components of asbestos) deposition. These results indicate that the system could be useful for investigating the pathogenesis of inhaled particle-induced immune reactions and for determining harmful levels of exogenous agents.