Microbeam Facility Research Articles

CHARACTERIZATION OF AEROSOL PARTICLES IN A MECHANICAL WORKSHOP ENVIRONMENT

Indoor aerosols are directly affecting human lives. Especially aerosols in workshops, factories, and laboratories, where many chemical substances are used in treatment and production processes, might contain toxic elements: special care must be taken to alleviate air pollution and assure a clean breathing environment for the workers. For this study, size segregated aerosol particle sampling with a cascade impactor was performed in the machine workshop of Jožef Stefan Institute. The samples, collected during weekdays and weekend were analyzed with a microbeam facility at Tohoku University. Bulk PIXE analysis with scanning over the whole sample area was conducted along with multimodal microanalysis with microscopic scanning. Using bulk analysis, high concentrations of Pb and Ba were detected on weekend days, which was related to the removal of an old white paint from the furniture. On weekdays, concentrations of W and of soil origin elements increased, probably because of the machine operations and worker movements. At the same time high concentration of sulfur was detected. A microscopic multimodal analysis shows that it stems from a lubricant oil vapor. The combination of bulk and microanalysis of the size selected samples is an effective approach to aerosol characterization in the working environment.

Involvement of Oxidative Metabolism and Membrane Signaling in Bystander Response: A Microbeam Study.

Charged particles microbeam facilities are a unique tool that allows targeting of single cells and analysis of the induced damage on a cell-by-cell basis. The ability of these tools to discriminate between hit and non-hit cells, and to deliver a precise dose is of particular interest in the investigation of various phenomena related to low dose radiation responses and more particularly in bystander effects. Since spring 2004, the choice was made to develop a device specifically designed and dedicated to radiobiology studies, with for imperative to adapt the device to the ideal conditions for cell culture. Today the single-cell microbeam system is operational: it can deliver 10 individual alpha particles (3 MeV) to selected cell nuclei in a precisely known proportion of cells in a population. The investigated endpoint in radiobiology studies is the identification of signaling pathways implicated in the appearance of non targeted effects. Here, the induction of bystander effect was studied by assessing DNA double-strand breaks (DSB) formation with the γH2AX and 53BP1 foci formation assay. Then, a comparative kinetics was established between these foci formation assay and reactive oxygen species (ROS) generation. This kinetics was accomplished in a range of time going from 30 minutes till 24 hours post-irradiation. By this way, we showed the presence of ROS in the hit cells as well as in the bystander cells. This was noticed till 2 hours post irradiation and correlated in observation of foci in both populations of cells. Moreover, alpha particles induced generation of ROS in the hit cells by a mitochondria-dependent way. This ROS generation was partly involved in a process of radio-induced apoptosis (24h post-irradiation) in the hit cells. However, no apoptosis was detected in bystander cells. On the other hand, the cell membrane seemed to play a decisive role in the signaling leading to the generation of ROS in the hit and bystander cells. Indeed, the use of filipine, which destabilized the rafts of cholesterol in the membrane, annihilated the ROS generation in these two populations of cells. We thus support the hypothesis that ROS, generated by mitochondria as well as cell membrane, belongs to signaling pathways directly involved in bystander effect.

A composite microdose Adaptive Response (AR) and Bystander Effect (BE) model–application to low LET and high LET AR and BE data

Purpose: It has been suggested that Adaptive Response (AR) may reduce risk of adverse health effects due to ionizing radiation. But very low dose Bystander Effects (BE) may impose dominant deleterious human risks. These conflicting behaviors have stimulated controversy regarding the Linear No-Threshold human risk model. A dose and dose rate-dependent microdose model, to examine AR behavior, was developed in prior work. In the prior work a number of in vitro and in vivo dose response data were examined with the model. Recent new data show AR behavior with some evidence of very low dose BE. The purpose of this work is to supplement the microdose model to encompass the Brenner and colleagues BaD (Bystander and Direct Damage) model and apply this composite model to obtain new knowledge regarding AR and BE and illustrate the use of the model to plan radio-biology experiments.Materials and methods: The biophysical composite AR and BE Microdose Model quantifies the accumulation of hits (Poisson distributed, microdose specific energy depositions) to cell nucleus volumes. This new composite AR and BE model provides predictions of dose response at very low dose BE levels, higher dose AR levels and even higher dose Direct (linear-quadratic) Damage radiation levels.Results: We find good fits of the model to both BE data from the Columbia University microbeam facility and combined AR and BE data for low Linear Energy Transfer (LET) and high LET data. A Bystander Factor of about 27000 and an AR protection factor of 0.61 are obtained for the low LET in vivo mouse spleen exposures. A Bystander Factor of 317 and an AR protection factor of 0.53 are obtained for high LET radon alpha particles in human lymphocytes. In both cases the AR is activated at most by one or two radiation induced charged particle traversals through the cell nucleus.Conclusions: The results of the model analysis is consistent with a premise that both Bystander damage and Adaptive Response radioprotection can occur in the same cell type, derived from the same cell species. The model provides an analytical tool to biophysically study the combined effects of BE and AR.

Recent developments of ion beam induced luminescence at the external scanning microbeam facility of the LABEC laboratory in Florence

A new ionoluminescence (IL) apparatus has been successfully installed at the external scanning microbeam facility of the 3 MV Tandetron accelerator of the INFN LABEC in Firenze; the apparatus for photon detection has been fully integrated in the existing ion beam analysis (IBA) set-up, for the simultaneous acquisition of IL and PIXE/PIGE/BS spectra and maps.The potential of the new set-up is illustrated in this paper by some results extracted by the analysis of art objects and advanced semiconductor materials. In particular, the adequacy of the new IBA set-up in the field of cultural heritage is pointed out by the coupled PIXE/IL micro-analysis of a lapis lazuli stone; concerning applications in material science, IL spectra from a N doped diamond sample were acquired and compared with CL analyses to evaluate the relevant sensitivities and the effect of ion damage.

Quantifying a bystander response following microbeam irradiation using single-cell RT-PCR analyses

There is growing recognition that the effects of ionizing radiation may extend to more than those cells that directly suffer damage to DNA in the cell nucleus. Data from several investigators have indicated that cells neighboring those that are irradiated also demonstrate several responses seen in hit cells--the so-called bystander effect. The microbeam facility at the Center for Radiological Research is particularly well suited for the study of this bystander effect, since it has the ability to place known numbers of charged particles (protons or alpha-particles at LETs from 20 to 180 KeV/microm) at defined positions relative to individual cells. That is, some known fraction of cells in a population can be irradiated through the nucleus, or the cytoplasm or even adjacent to cells through the media. Therefore, using the microbeam it is possible to examine individual cell responses in both hit and nonhit cells in the same population. Alterations in the cyclin-dependent kinase inhibitor CDKN1a (p21/Cip1/WAF1) were quantified at the mRNA level in single normal human fibroblasts following precise delivery of 0 or 10 alpha-particles per cell at 90 KeV/microm to 50% of cells in a population. Semiquantitative RT-PCR of individual hit cells demonstrated increases in the levels of CDKN1A message that followed the kinetics previously described for irradiated populations. Furthermore, nonhit bystander cells also showed increased (though lesser) levels of CDKN1a message. Data presented here demonstrate the power of this approach, which combines the ability of the microbeam to irradiate specific cells in a population and the ability to quantify the response to the irradiation in individual targeted and bystander cells.

Micro-machining of resists on silicon by proton beam writing

We report micro-machining of resists on silicon by proton beam writing (PBW) at Takasaki Ion Accelerators for Advanced Radiation Application (TIARA), JAEA Takasaki, Japan. We studied the proton beam irradiation effects on typical positive and negative resists such as PMMA and SU-8, respectively, for application of the PBW technique to micro-machining. These resist materials were subjected to the scanning of a focused beam of protons accelerated using the microbeam facility of TIARA. Diameter of the proton beam was focused to about 1 μm. The fluence was varied to examine the irradiation effects on these resists as a function of the beam current and irradiation time. After exposure to proton beam, samples were developed and evaluated by a scanning electron microscope. Attempts to fabricate nickel stamps were also made by electroplating on the structures formed by PBW for application to imprint lithography.

A microPIXE investigation of the interaction of cells of Schizosaccharomyces pombe with the culture medium

Schizosaccharomyces pombe ( S. pombe) is a eucaryotic cell type similar to mammalian cells but much more simple. As it also executes its cell cycle rapidly it is very useful for investigating basic processes in cells. In this paper we report a feasibility study of the applicability of microPIXE to investigate the interaction between S. pombe cells and the surrounding culture medium. Cells were cultured in various growth medium prior to preparation for analysis. 1 μl drops of medium and cells were spotted onto polypropylene foils held in contact with a polished copper block previously cooled in liquid nitrogen. The samples were dehydrated by freeze-drying. Micro PIXE analysis was carried out with the IBC microbeam facility using a beam of 2.5 MeV protons focused to 1–2 μm diameter. Initially no elemental contrast was observed between the cells and the medium, but by modifying the dilution of the cell suspension, the cells could be distinguished from the surrounding medium through an increased concentration of P and reduced concentration of Cl. The distribution of Na in the medium around the cells showed evidence of the action of the Na pump. Sporulation appears to be induced in the cells by adding Cu to the growth medium and the uptake of Cu by the cells could be clearly observed. This study shows that it is possible to analyse the mass transport of elements in and out of cells In the future this will enable concentration gradients to be analysed and allow the rate of production or consumption of individual cells to be calculated. By observing these patterns for individual cells (not populations) at various known points in the cell cycle, fundamental data can be derived.

Transcriptional response of human cells to microbeam irradiation with 2.1 MeV α-particles

Within the next decades, an increasing number of human beings will be brought into space to carry out technical and scientific tasks. There, they will be exposed simultaneously to combined stimuli, especially microgravity and radiation. In the endeavour to assess the risks for humans during long-duration space missions, it is necessary to understand already at the cellular level the complex interplay of these parameters. Cellular stress protection responses lead to an increased transcription of several genes via the modulation of transcription factors. The activation of the nuclear factor κB (NF-κB) pathway as a possible anti-apoptotic route represents such an important cellular stress response. It is hypothesized that the activation of NF-κB and the subsequent expression of NF-κB-dependent genes is involved in the cellular response to components of the cosmic radiation. Irradiation of human embryonic kidney cells (HEK/293) with α-particles (2.1 MeV, LET ∼160 keV/μm) was performed at the PTB, Braunschweig, Germany. Using the microbeam facility, cells were exposed to nuclear hits or, for the purpose of comparison, to a diffuse irradiation of the whole cell. After irradiation the following biological endpoints were determined: (i) cell survival (by means of the colony forming ability test), and (ii) quantitative RT-PCR analysis of selected NF-κB target genes (IκBα GADD45β, bcl-2, and bcl-X L). One nuclear α-particle traversal reduces the probability to survive to ∼75%. Exposure to two α-particles per nucleus resulted in an upregulation of the expression of the GADD45β gene. After exposure of HEK cells to five nuclear hits, about 43% of the irradiated cells survived, and the transcriptional response was not significant. Ten nuclear hits activated the IκBα expression, this increased IκBα production might be involved in the termination of the radiation-induced NF-κB activation. Diffuse irradiation increased the transcription of IκBα and GADD45β. Expression of the antiapoptotic genes bcl-2 and bcl-X L remained almost unchanged after α-particle irradiation. These findings suggest a role for GADD45β in the cellular radiation response to low fluences, while IκBα is enrolled at higher fluences.

A comparative review of charged particle microbeam facilities

At the low doses (and low dose rates) relevant to environmental and occupational radiation exposure (0-50 mSv), which are of practical concern for radiation protection, very few cells in the human organism experience more than one traversal by densely ionising particles in their lifetime, the intervals between the tracks, if any, typically being months or years. The biological effects of exactly one particle are not well known and cannot be simulated in vitro by conventional broad-beam exposures, due to the random Poisson distribution of tracks. Charged particle microbeam facilities are a unique tool that allows targeting of single cells and analysis of the induced damage on a cell-by-cell basis. In the past few years, many charged particle microbeam facilities for radiobiology have come into operation or are under development worldwide. Different experimental designs have been adopted at various laboratories regarding the achievement of micrometre (or sub-micrometre) ion beam size, by mechanical collimation or focusing, particle detection, and cell recognition and positioning systems. The different approaches are reviewed and discussed in this paper.

Automatic Thresholding in the Control Software of CAS-LIBB Microbeam Facility

In this paper, two automatically calculated thresholds based on a statistical analysis of the histogram were used to apply binary segmentation to the bitmap. When the CCD and the microscope have been properly configured and the raw image is preprocessed, the Otsu's method can meet the need of the control program on the whole.

ATR-dependent radiation-induced γH2AX foci in bystander primary human astrocytes and glioma cells

Radiotherapy is an important treatment for patients suffering from high-grade malignant gliomas. Non-targeted (bystander) effects may influence these cells' response to radiation and the investigation of these effects may therefore provide new insights into mechanisms of radiosensitivity and responses to radiotherapy as well as define new targets for therapeutic approaches. Normal primary human astrocytes (NHA) and T98G glioma cells were irradiated with helium ions using the Gray Cancer Institute microbeam facility targeting individual cells. Irradiated NHA and T98G glioma cells generated signals that induced gammaH2AX foci in neighbouring non-targeted bystander cells up to 48 h after irradiation. gammaH2AX bystander foci were also observed in co-cultures targeting either NHA or T98G cells and in medium transfer experiments. Dimethyl sulphoxide, Filipin and anti-transforming growth factor (TGF)-beta 1 could suppress gammaH2AX foci in bystander cells, confirming that reactive oxygen species (ROS) and membrane-mediated signals are involved in the bystander signalling pathways. Also, TGF-beta 1 induced gammaH2AX in an ROS-dependent manner similar to bystander foci. ROS and membrane signalling-dependent differences in bystander foci induction between T98G glioma cells and normal human astrocytes have been observed. Inhibition of ataxia telangiectasia mutated (ATM) protein and DNA-PK could not suppress the induction of bystander gammaH2AX foci whereas the mutation of ATM- and rad3-related (ATR) abrogated bystander foci induction. Furthermore, ATR-dependent bystander foci induction was restricted to S-phase cells. These observations may provide additional therapeutic targets for the exploitation of the bystander effect.

Development of ERCS hydrogen analysis for melt inclusions in erupted magmatic samples

In geological science, hydrogen analysis of melt inclusions can provide important insights into the dynamic mechanism of magmatic activities. Melt inclusions are specimens of the magma trapped within capsules in crystals in the magma chamber that are subsequently expelled from volcano and end up in volcanic rocks. Since the sizes of the melt inclusions are very small, ranging from tens to several hundred micrometers in diameter, hydrogen analysis by means of elastic recoil coincidence spectrometry (ERCS) has been developed at a proton microbeam facility. This method of hydrogen analysis is simple, non-destructive, and rapid, especially when using a newly developed calibration method that employs Mylar films as hydrogen standards. The depth profile of hydrogen can be derived from the measured sum-energy spectrum using our ERCS analysis method.

The visigothic treasure of Torredonjimeno (Jaén, Spain): A study with IBA techniques

The visigothic treasure of Torredonjimeno (Jaén, Spain) was found by chance in the year 1926 buried in an olive grove. The finding consisted of some hundreds fragments of gold objects and gems coming from several votive crowns and crosses, some of them belonging to an unidentified visigothic king. The treasure may belong chronologically to the same period as the Guarrazar treasure or, possibly, to a somewhat later time, but the pieces are fragmented and of less technological workmanship than the latter. This is the reason why the Torredonjimeno treasure has not attracted as much attention from archeologists and art historians as that from Guarrazar. On the occasion of an exhibition showing together all the objects of the treasure, it is normally kept in three different museums in Barcelona, Cordoba and Madrid, a number of pieces were analyzed, using PIXE, PIGE and RBS, at the external microbeam facility installed at the Center for Micro-Analysis of Materials.

External Micro-PIXE Measurements: Preliminary Results on Volcanic Rocks from Nyiragongo Volcano

The external microbeam facility at the 3 MV Tandetron accelerator of the LABEC Laboratory of INFN in Florence has been used to determine, by PIXE, major, minor and trace element abundances of minerals and groundmass in the lavas from the active Nyiragongo Volcano (Democratic Republic of Congo), which is well known for its lava lake intra-crateric activity [e.g. 1, 2]. During the last eruption of this volcano (January, 2002), two main flows entered the Goma town producing major devastation, forcing the rapid exodus of about 300,000 people and causing the death of 150 persons [3]. After this eruption, the interest of the scientific community on this volcanic area suddenly increased. In this respect, determination of major and trace element abundances in mineral phases and groundmass of lavas may allow to constrain the evolution of magma within the volcanic system from a quantitative view point, thus helping to better understand the way this volcano works.

Preliminary Study of the CAS-LIBB Single-Particle Microbeam II Endstation: I. Proposed Multi-Dimensional Quantitative Fluorescence Microscopy

Single-particle microbeam as a powerful tool can open a research field to find answers to many enigmas in radiobiology. A single-particle microbeam facility has been constructed at the Key Laboratory of Ion Beam Bioengineering (LIBB), Chinese Academy of Sciences (CAS), China. However there has been less research activities in this field concerning the original process of the interaction between low-energy ions and complicated organisms. To address this challenge, an in situ multi-dimensional quantitative fluorescence microscopy system combined with the CAS-LIBB single-particle microbeam II endstation is proposed. In this article, the rationale, logistics and development of many aspects of the proposed system are discussed.

Single-ion microbeam as a tool for low-dose radiation effects investigations

Practical assessment of human radiation exposure risk deserves particular attention especially for low doses (and low dose rates), which concern environmental and occupational exposure. At these dose levels ionizing radiation exposures involve mainly isolated charged particle tracks, which strike individual cells at time intervals averaging from weeks to several years apart. Accelerator-based microbeam irradiation technique offers a unique tool to mimic such an exposure, allowing irradiating single cells individually with micrometer precision and with a preset number of charged particles down to one particle per cell. A horizontal single-ion microbeam facility for single-cell irradiations has been designed and set up at the INFN-LNL 7MV CN Van de Graaff accelerator. The light ion beam is collimated in air down to a section of 2-3µm in diameter by means of appropriate pinholes. Semi-automatic cell visualization and automatic cell positioning and revisiting system, based on an inverted phase contrast optical microscope and on X-Y translation stages with 0.1µm positioning precision, has been developed. An in-house-written software allows to control remotely the irradiation protocol. As a distinctive feature of the facility, cell recognition is performed without using fluorescent staining and UV light. Particle detection in air, behind the biological sample, is based on a silicon detector while in-air beam profile and precise hit position measurements are accomplished by a custom-made cooled-CCD camera and Solid State Nuclear Track detectors, respectively. A particle counting rate of less than 1 ion/sec can be reached.

A Microcollimated Ion Beam Facility for Investigations of the Effects of Low-Dose Radiation

Charged-particle microbeams are unique tools to mimic low-dose exposure in vitro by delivering a defined number of particles to single mammalian cells down to only one particle per cell or group of cells. A horizontal single-ion microbeam facility has been built at the INFN-Laboratori Nazionali di Legnaro 7 MV Van de Graaff accelerator. Different light ions (1H+, 2H+, 3He2+, 4He2+) are available covering a wide range of LET from 7 to 150 keV/microm. Collimators of different geometries and materials have been tested, and beam spots 2-3 microm in diameter have been obtained using a tantalum disc. Cell visualization and recognition are performed with a phase-contrast optical microscope coupled with dedicated software. One unique characteristic of such a system is that neither cell staining nor UV light is used. Cells are automatically positioned on the beam spot through remotely controlled precision XY translation stages. A particle detector is positioned downstream of a specially designed petri dish to perform energy measurements and count particles crossing the cell. A particle counting rate of less than 1 ion/s can be reached. This feature, combined with a fast beam deflection system, ensures high reproducibility in administering a preset number of particles per cell.

High-resolution, back-side illuminated monolithic active pixel sensor for low-energy electron imaging

A new domain of application of monolithic active pixel sensors, with respect to particle tracking, has been triggered by the innovative idea of a nondestructive beam monitoring system in the extraction lines of a hadron-therapy center. The beam monitoring exploits secondary electrons emitted from a submicrometer thick Al foil, intersecting the beam. Electrons are accelerated in an electrostatic field. The detection of low-energy electrons, up to 30 keV, is required. The sensitivity to these energies is obtained by thinning the detector, originally fabricated in a standard VLSI technology, down to the thickness of the radiation sensitive layer. A thin entrance window, in the order of 100 nm, is provided. Monolithic active pixel sensors for low-energy electron imaging can be prospectively used in several domains: in bioscience for cell process study using radiotracers, e.g., /sup 3/H(18 keV/spl beta//sup -/) or fluorescence imaging exploiting the Hybrid Photodiodes principle, in safety or environmental studies for neutron imaging with converters directly deposited or in micro-beam facilities for position resolving in studies of living cell irradiations. The low-energy electron imaging capabilities for installation inside an HPD test facility and the results obtained with a /sup 3/H marked source are shown. The detector used is the 1/spl times/10/sup 6/ pixel MIMOSA V chip. The back-thinning up to the epitaxial layer was applied, resulting in a high resolution, back-side illuminated imager.

Phase-based cell imaging techniques for microbeam irradiations

The microbeam facility at Columbia University is expanding current protocols for single-particle, single-cell irradiations, so experimenters can locate and irradiate nuclei and cytoplasm of unstained cells. The ion beamline is located directly under the dish, therefore, any new techniques must use reflection microscopy. Two approaches are being integrated and neither require the removal of the cell growth medium prior to irradiation.(1)A novel immersion-based Mirau interferometry lens which uses low-coherence light sources to inhibit unwanted fringing is under design. The process requires tens of nanometers or better precision of vertical stage motion, which will be accomplished with our custom high-precision z-stage.(2)Quantitative Phase microscopy is under testing, also using the z-stage. Future plans include optimization of software routines to decrease time between irradiations.Both methods will be compared further with the automated location routines which use nuclear and cytoplasm stains.

Applications of centroid coordinates for cell image analysis system in ASIPP microbeam

A single-particle microbeam facility has been constructed at the Key Laboratory of Ion Beam Bioengineering of Institute of Plasma Physics, Chinese Academy of Sciences (ASIPP). In the image analysis system, the centroid coordinates of each cell are important features of cell images. For irradiation of nuclei, the centroid coordinates of each nucleus are reference coordinates for the points (the centroid itself or some neighbouring points of the centroid at a specific distance from it) to be irradiated. After the coordinates have been calculated, the irradiation points are found relative to the position of the beam exit aperture, and each cell is then positioned over the exit aperture by stepping motors driving a sample plate. Furthermore, in image acquisition, because the size of an image is limited, cells on the cell plate cannot all be scanned in one image. Therefore, the cell dish is divided into several overlapping districts to be acquired. Centroid coordinates are also a useful application for acquisition and recognition of overlapped images. The scheme and operational procedures for acquiring images accurately and recognizing cells are described; the feasibility and validity of the scheme are also discussed using an example.