Microbeam Facility Research Articles

Microirradiation techniques in radiobiological research.

The aim of this work is to review the uses of laser microirradiation and ion microbeam techniques within the scope of radiobiological research. Laser microirradiation techniques can be used for many different purposes. In a specific condition, through the use of pulsed lasers, cell lysis can be produced for subsequent separation of different analytes. Microsurgery allows for the identification and isolation of tissue sections, single cells and subcellular components, using different types of lasers. The generation of different types of DNA damage, via this type of microirradiation, allows for the investigation of DNA dynamics. Ion microbeams are important tools in radiobiological research. There are only a limited number of facilities worldwide where radiobiological experiments can be performed. In the beginning, research was mostly focused on the bystander effect. Nowadays, with more sophisticated molecular and cellular biological techniques, ion microirradiation is used to unravel molecular processes in the field of radiobiology. These include DNA repair protein kinetics or chromatin modifications at the site of DNA damage. With the increasing relevance of charged particles in tumour therapy and new concepts on how to generate them, ion microbeam facilities are able to address unresolved questions concerning particle tumour therapy.

'BioQuaRT' project: design of a novel in situ protocol for the simultaneous visualisation of chromosomal aberrations and micronuclei after irradiation at microbeam facilities.

The aim of the 'BioQuaRT' (Biologically weighted Quantities in RadioTherapy) project is to develop measurement techniques for characterising charged particle track structure on different length scales, and to correlate at the cellular level the track structure properties with the biological effects of radiation. This multi-scale approach will allow characterisation of the radiation qualities used in radiotherapy and the related biological effects. Charged-particle microbeam facilities were chosen as the platforms for all radiobiology experiments in the 'BioQuaRT' project, because they allow targeting single cells (or compartments of a cell) with a predefined number of ionising particles and correlating the cell-by-cell induced damage with type and energy of the radiation and with the number of ions per cell. Within this project, a novel in situ protocol was developed for the analysis of the misrepaired and/or unrepaired chromosome damage induced by charged-particle irradiations at the Physikalisch-Technische Bundesanstalt (PTB) ion microbeam facility. Among the cytogenetic biomarkers to detect and estimate radiation-induced DNA damage in radiobiology, chromosomal aberrations and micronuclei were chosen. The characteristics of the PTB irradiation system required the design of a special in situ assay: specific irradiation dishes with a base made from a biofoil 25-µm thick and only 3000-4000 cells seeded and irradiated per dish. This method was developed on Chinese hamster ovary (CHO) cells, one of the most commonly used cell lines in radiobiology in vitro experiments. The present protocol allows the simultaneous scoring of chromosome aberrations and micronuclei on the same irradiated dish. Thanks to its versatility, this method could also be extended to other radiobiological applications besides the single-ion microbeam irradiations.

Live cell imaging at the Munich ion microbeam SNAKE - a status report.

Ion microbeams are important tools in radiobiological research. Still, the worldwide number of ion microbeam facilities where biological experiments can be performed is limited. Even fewer facilities combine ion microirradiation with live-cell imaging to allow microscopic observation of cellular response reactions starting very fast after irradiation and continuing for many hours. At SNAKE, the ion microbeam facility at the Munich 14 MV tandem accelerator, a large variety of biological experiments are performed on a regular basis. Here, recent developments and ongoing research projects at the ion microbeam SNAKE are presented with specific emphasis on live-cell imaging experiments. An overview of the technical details of the setup is given, including examples of suitable biological samples. By ion beam focusing to submicrometer beam spot size and single ion detection it is possible to target subcellular structures with defined numbers of ions. Focusing of high numbers of ions to single spots allows studying the influence of high local damage density on recruitment of damage response proteins.Electronic supplementary materialThe online version of this article (doi:10.1186/s13014-015-0350-7) contains supplementary material, which is available to authorized users.

The first interdisciplinary experiments at the IMP high energy microbeam

The high energy beam of tens to hundred MeV/u ions possesses mm-to-cm penetration depth in materials and can be easily extracted into air without significant energy loss and beam scattering. Combination of high energy ions and microbeam technology facilitates the microprobe application to many practical studies in large scale samples. The IMP heavy ion microbeam facility has recently been integrated with microscopic positioning and targeting irradiation system. This paper introduced the first interdisciplinary experiments performed at the IMP microbeam facility using the beam of 80.5MeV/u carbon ions. Bystander effect induction via medium transferring was not found in the micro-irradiation study using HeLa cells. The mouse irradiation experiment demonstrated that carbon irradiation of 10Gy dose to its tuberomammillary nucleus did not impair the sleep nerve system. The fault injection attack on RSA (Rivest–Shamir–Adleman) decryption proved that the commercial field-programmable gate array chip is vulnerable in single event effect to low linear-energy-transfer carbon irradiation, and the attack can cause the leakage of RSA private key. This work demonstrates the potential of high energy microbeam in its application to biology, biomedical, radiation hardness, and information security studies.

Wide area scanning system and carbon microbeams at the external microbeam facility of the INFN LABEC laboratory in Florence

Recently, developments have been made to the external scanning microbeam of INFN-LABEC laboratory in Florence. A new system for mechanical sample scanning was implemented. This system allows us to acquire large maps (up to 20×20cm2), of great interest in the Cultural Heritage field.In parallel, the possibility of using carbon microbeams for experiments, such as, for example, ion beam modification of materials and MeV Secondary Ion Mass Spectrometry, has been investigated.As a test application, Particle Induced X-ray Emission with carbon microbeams has been performed on a lapis lazuli stone.First results for both wide area imaging and external carbon microbeams are briefly reported.

The set-up for forward scattered particle detection at the external microbeam facility of the INFN-LABEC laboratory in Florence

In the last few years some new implementations and upgrades have been made to the external scanning microbeam of INFN-LABEC laboratory in Florence, enriching the existing PIXE, PIGE, BS, IBIL set-up with complementary techniques, when possible allowing for simultaneous multi-technique analyses.We developed a system, compatible with the existing set-up, for the out-of-vacuum detection of the forward scattered particles. This system makes feasible the external-STIM (Scanning Transmission Ion Microscopy) and external-FS (Forward Scattering), now both available at our beamline. Test measurements are shortly presented.

Ore petrography and chemistry of the tellurides from the Dongping gold deposit, Hebei Province, China

The Dongping gold deposit is a mesothermal lode gold deposit hosted in syenite. The ore petrography and chemistry of the tellurides from the alteration zone of the deposit have been studied in detail using optical microscopy, scanning electron microscopy, electron probe micro-beam and X-ray diffraction facilities. The tellurides, consisting mostly of calaverite, altaite, petzite tellurobismuthite and tetradymite, are hosted irregularly in pyrite fractures and voids. In the ore bodies, the species and quantity of tellurides decrease from the top downwards, accompanied with lowering of gold fineness, and the existence of tellurides exhibits a positive correlation with gold enrichment. Mineral paragenesis and chemical variations suggest that during evolution of the ore-forming fluids Te preferably incorporated with Pb to form altaite, followed in sequence by precipitation of petzite, and calaverite when Ag has been exhausted, and the residue fluids were enriched in Au, giving rise to formation of native gold. Calculation with reference of the fineness of native gold coexisting with the tellurides indicates that at 300°C, log f (Te2) varied between −8.650 and −7.625. Taking account of the Au–Ag–Te mineral paragenesis, it is inferred that log ƒ (Te2) varies from −9.12 to −6.43, log ƒ (S2) −11.47 to −8.86. In consideration of the physicochemical conditions for formation of tellurides, with comparison to some known telluride deposits, it is suggested that high log ƒ (Te2) is a key factor for high fineness of native gold as well as precipitation of abundant tellurides.

Development of a TOF SIMS setup at the Zagreb heavy ion microbeam facility

We describe a new Time-of-flight Secondary Ion Mass Spectrometry (TOF SIMS) setup for MeV SIMS application, which is constructed and installed at the heavy ion microbeam facility at the Ruđer Bošković Institute in Zagreb. The TOF-SIMS setup is developed for high sensitivity molecular imaging using a heavy ion microbeam that focuses ion beams (from C to I) with sub-micron resolution. Dedicated pulse processing electronics for MeV SIMS application have been developed, enabling microbeam-scanning control, incoming ion microbeam pulsing and molecular mapping. The first results showing measured MeV SIMS spectra as well as molecular maps for samples of interest are presented and discussed.

Bystander signaling in C. elegans: proton microbeam studies

Biological model: In this project, we investigated the control of radiation-induced genotoxic damage expression in somatic cells of the nematode Caenorhabditis elegans. We measured genotoxic damage in the C. elegans intestine by irradiating young larvae with 20 intestinal cells. Fourteen of these cells undergo exactly one nuclear division without cytoplasmic division leading to 14 binucleate cells. This nuclear division is synchronized and occurs at the first larval molt. Irradiation induces chromosome aberrations including dicentrics which we can quantify as stable anaphase bridges in the binucleate cells of young adult intestines. The endpoint is dose- and LET-dependent and we have demonstrated that individual intestinal cells have unique radiosensitivities.Results: The project has two components, a genetic screen for genes that control cell sensitivity and a microbeam component to directly probe individual cells. The genetic screen has identified several genes in NHEJ repair and telomere metabolism that modulate overall bridge frequency. Knockout mutants of cku-70, cku-80 and lig-4 greatly sensitize animals for anaphase bridge induction. A statistical method was used to determine whether induction of bridges was strictly random and cell autonomous and we determined that expression of bridges in pairs of cells was, in fact, non-random which suggested that signaling between cells affected the pattern of bridge expression. This allowed us to conduct an RNAi and mutation screen for genes that control the signaling (block non-random distributions) and several candidates have been identified.To directly test the notion that signaling of genotoxic damage occurs, we conducted experiments with alpha particles collimated through slits in metal foils and showed that genotoxic damage could be expressed many cell diameters away from a partial body exposure site. Thus, an in vivo bystander effect was demonstrated. Dose targeting was then improved to small regional exposures and eventually to individual cell targeting using 2 MeV protons from the microbeam facility at Texas A&M University. We now employ a green fluorescent protein (GFP)-expressing transgenic worm (rrIs1[elt-2::GFP]) to target GFP-positive gut cells via the gut-specific transcription factor elt-2. This allows alignment of the cell of interest over the microbeam aperture under appropriate fluorescence illumination.Microbeam irradiation experiments for many pairwise combinations of cell signal transmission and reception (observed as expression of anaphase bridges) have been conducted and several interesting patterns emerge. (i) The signaling pattern is cell-specific and does not simply reflect cell–cell distance or require direct contact between cell pairs. (ii) The signal range can be as far as from cell pair 2 to cell pair 8 (>100 µm). (iii) There appears to be a functional compartment boundary at the pharynx/intestine valve as even high-dose exposures to the posterior pharyngeal bulb fail to induce bridges in nearby intestinal cells. (iv) The frequency of signal transmission and reception corresponds broadly to the overall frequency of bridges observed during whole-body irradiations which suggests that direct irradiation and ‘out-of-field’ effects may be additive. These patterns have been analyzed in terms of a cellular logic circuit map for signal transmission and reception.A dose–response for a subset of microbeam-targeted cells was measured over the range of 5–20 Gy. Controlled cell pair targeting was used to test the potential additivity of signals and we found that effects were supra-additive. Finally, preliminary measurements were conducted on GFP-expressing transgenic strains that bore cku-70(tm1524) III and smk-1(mn156) V mutations which confer enhanced radiosensitivity. Cku-70 is a Ku-70 ortholog while smk-1 is orthologous to the mammalian and Dictyostelium discoideum SMEK (suppressor of MEK null) protein. In the cku-70(0/0) strain, the severity of the bridges in bystander cells was enhanced, suggesting that signal recipient cells employ NHEJ repair pathways in the expression of anaphase bridges.Clinical trial registration number: Not applicable.

Space Radiation Research Unit, International Open Laboratory in NIRS

The radiation environment encountered by astronauts during spaceflight is far more complex than any radiation field existed on Earth. Space crew living and working in the International Space Station (ISS) are exposed to a mixed radiation field comprises primary high-energy cosmic rays, including energetic protons and heavy ions, and to secondary radiations, including energetic neutrons, produced when the primary radiation interacts with the mass of the space station and its contents. The doses of ionizing radiation received by astronauts and cosmonauts aboard the ISS are many times greater than those received by radiation workers on the ground. Exposure to ionizing radiation in space includes high LET events than can produce significant biological damage in human cells and tissues, and thus represents an important risk to space crew health and safety.The Space Radiation Research Unit at the National Institute of Radiological Sciences (NIRS) includes both physicists and radiation biologists and there is extensive collaboration between these two groups. This provides us with the expertise needed to investigate the effects of space crew exposure to the highly complex, mixed radiation environment encountered in space. In addition, NIRS is home to a heavy ion accelerator, HIMAC and the Medical Cyclotron that can be used to simulate various components of the space radiation environment. Recently, we have developed a medium energy proton radiation field using the NIRS Medical Cyclotron. [How about a sentence or two on the significance of this proton facilities.] In addition, NIRS has also developed a high precision tool, the Single Particle Irradiation System to Cell (SPICE) microbeam facility, for use in investigating various radiobiological endpoints, including the bystander effect and the adaptive response of various cell types, Caenorhabditis elegans and in Medaka fish. Some of these research activities are described in these proceedings [1, 2].The Space Radiation Research Unit at NIRS has extensive international collaborations with researchers in China, Germany, Russia, USA, Sweden, Austria and other countries. In particular, collaborations with our Russian colleagues has led to opportunities to expose our dosimeters aboard the ISS and to numerous other experiments in the frame of the IOL, as described more fully elsewhere in these proceedings. We believe that the framework of the IOL is very powerful and productive and we plan to continue our collaborative research with our international colleagues.

Research and development of focused proton microbeam irradiation system, SPICE for radio-biological studies.

There is continuing interest for the use of microbeam irradiation systems designed to deliver a defined number of charged particles on a single cell with a resolution of a few micrometers. Irradiation of an exact number of charged particles on a single cell means that the limitations of the Poisson distribution of the number of charged particles can be overcome. Moreover, microbeams are particularly useful for the field of radiation-induced non-targeted effects, so-called bystander effects that are considered to be one of the major effects in the low-dose region. Thus, microbeam technique is one of the powerful tools for investigating studies related to radiation effect and risk of low dose in space radiation for astronauts and cosmonauts.Our microbeam irradiation system, the Single-Particle Irradiation system to CEll (SPICE) provides a 3.4 MeV proton microbeam focused with a quadrupole magnetic lens on an upward vertical beam line. SPICE was severely damaged by the Tohoku-oki Earthquake on 11 March 2011, and was out of operation for about a year and a half. We have successfully reconstructed the facility, and it is now operational with system refinements. At present, SPICE is the only proton microbeam facility in Japan at which a single-ion single-cell irradiation can be performed on mammalian cells with stability and high throughput using an upward vertical beam of below 2-μm diameter, focused with a magnetic quadrupole triplet lens [ 1]. A variety of irradiation modes have been established for radiation-induced bystander effects, cytoplasm irradiation etc. An example of cells targeted with a multi-position targeting mode is shown in Fig. 1, which cells were targeted with five different positions in the nucleus with 50 protons per position. SPICE has been administrated as a ‘Joint-use facility for Collaborative Research’, and thus researchers outside NIRS can apply for beam time of SPICE after their research proposals are approved.Fig. 1.(A) Nuclei of HCT116 cells were targeted with five different positions (3-µm apart), and 50 protons were delivered to each position. (B) Cells were incubated for 1 h and then fixed for immunostaining against γ-H2AX.

Study of DSSSD detecto response in the inter-strip region using a proton micro-beam

Double Sided Silicon Strip Detectors (DSSSDs) are segmented silicon detectors widely used for the detection of charged particles. When a particle hits the inter-strip gap between two adjacent strips, energy measurement can be affected by different effects reducing the efficiency of output signal generation. With the purpose to study this behaviour, an experiment was performed at the scanning proton micro-beam facility of the Ruđer Boskovic Institute (RBI). Response of two detectors of thickness 75 and 998 µm is examined as a function of the beam impact position and bias voltage. Results show that the effective width of the inter-strip gap, which is linked to the efficiency for full energy detection, depends on the experimental conditions and detector settings.

“Broadbeam” irradiation of mammalian cells using a vertical microbeam facility

A "broadbeam" facility is demonstrated for the vertical microbeam at Surrey's Ion Beam Centre, validating the new technique used by Barazzuol et al. (Radiat Res 177:651-662, 2012). Here, droplets with a diameter of about 4mm of 15,000 mammalian cells in suspension were pipetted onto defined locations on a 42-mm-diameter cell dish with each droplet individually irradiated in "broadbeam" mode with 2MeV protons and 4MeV alpha particles and assayed for clonogenicity. This method enables multiple experimental data points to be rapidly collected from the same cell dish. Initially, the Surrey vertical beamline was designed for the targeted irradiation of single cells with single counted ions. Here, the benefits of both targeted single-cell and broadbeam irradiations being available at the same facility are discussed: in particular, high-throughput cell irradiation experiments can be conducted on the same system as time-intensive focused-beam experiments with the added benefits of fluorescent microscopy, cell recognition and time-lapse capabilities. The limitations of the system based on a 2MV tandem accelerator are also discussed, including the uncertainties associated with particle Poisson counting statistics, spread of linear energy transfer in the nucleus and a timed dose delivery. These uncertainties are calculated with Monte Carlo methods. An analysis of how this uncertainty affects relative biological effect measurements is made and discussed.

Supply Voltage Dependent On-Chip Single-Event Transient Pulse Shape Measurements in 90-nm Bulk CMOS Under Alpha Irradiation

Direct on-chip pulse shape measurements of single-event transients (SETs) in a single inverter in 90-nm bulk CMOS have been performed at the microbeam facility at the Physikalisch-Technische Bundesanstalt (PTB), Braunschweig, Germany. Alpha particles with an energy of 8 MeV were used as projectiles, and the supply voltage dependence of the arising SETs was investigated. A strong dependence of the resulting pulse heights, widths, and shapes on the supply voltage could be observed.

Focusing giga-electronvolt heavy ions to micrometers at the Institute of Modern Physics

To study the radiation effect of cosmic heavy ions of low fluxes in electronics and living samples, a focusing heavy ion microbeam facility, for ions with energies of several MeV/u up to 100 MeV/u, was constructed in the Institute of Modern Physics of the Chinese Academy of Sciences. This facility has a vertical design and an experiment platform for both in-vacuum analysis and in-air irradiation. Recently, microbeam of (12)C(6+) with energy of 80.55 MeV/u was successfully achieved at this interdisciplinary microbeam facility with a full beam spot size of 3 μm × 5 μm on target in air. Different from ions with energy of several MeV/u, the very high ion energy of hundred MeV/u level induces problems in beam micro-collimation, online beam spot diagnosis, radiation protection, etc. This paper presents the microbeam setup, difficulties in microbeam formation, and the preliminary experiments performed with the facility.

The data acquisition and beam control system at the IMP microbeam facility

To perform routine irradiation and spatial analysis using ion microbeam, a compact data acquisition and beam control system was developed at the high energy heavy ion microbeam facility in the Institute of Modern Physics (IMP), CAS. The system comprises PXI modules from National Instruments. Together with the in-house developed Cell Exposure and Nuclear Application (CENA) software using LabVIEW, the system implements beam scanning, single ion targeting, ion counting, energy spectrum measurement, spectrum mapping and sample irradiation. This paper introduces the hardware configuration, software design and preliminary tests of the data acquisition and beam control system.

Review of doped silica glass optical fibre: Their TL properties and potential applications in radiation therapy dosimetry

Review is made of dosimetric studies of Ge-doped SiO2 telecommunication fibre as a 1-D thermoluminescence (TL) system for therapeutic applications. To-date, the response of these fibres has been investigated for UV sources, superficial X-ray beam therapy facilities, a synchrotron microbeam facility, electron linear accelerators, protons, neutrons and alpha particles, covering the energy range from a few eV to several MeV. Dosimetric characteristics include, reproducibility, fading, dose response, reciprocity between TL yield and dose-rate and energy dependence. The fibres produce a flat response to fixed photon and electron doses to within better than 3% of the mean TL distribution. Irradiated Ge-doped SiO2 optical fibres show limited signal fading, with an average loss of TL signal of ∼0.4% per day. In terms of dose response, Ge-doped SiO2 optical fibres have been shown to provide linearity to x and electron doses, from a fraction of 1Gy up to 2kGy. The dosimeters have also been used in measuring photoelectron generation from iodinated contrast media; TL yields being some 60% greater in the presence of iodine than in its absence. The review is accompanied by previously unpublished data.

Position dependent measurement of single event transient voltage pulse shapes under heavy ion irradiation

A CMOS inverter in 90 nm CMOS bulk technology was exposed to heavy ion radiation (Au-197) at a micro-beam facility. The targeted inverter occupies an area of 6×3 µm2. The resulting single event transient (SET) voltage pulses at the output were measured using an on-chip analogue sense amplifier. The output of the amplifier was recorded with a 15 GHz real-time oscilloscope. Depending on the location of the ion hits strongly different pulse shapes were observed.

Movements of the tongue, lips, and jaw for selected vocalic nuclei in speakers with dysarthria

Speech movement data in persons with dysarthria are relatively rare, and much of this sparse data is restricted to the lips and jaw. The purpose of this presentation is to provide a survey of speech movements for selected vocalic nuclei, in speakers with dysarthria due to Parkinson's disease and Amyotrophic Lateral Sclerosis, and to compare these movements to those of healthy controls. The speech materials used in this analysis are those collected over a ten-year period at the x-ray microbeam facility at the University of Wisconsin-Madison. The selected vocalic nuclei are extracted from connected speech samples, including both read sentences and an extended reading passage; multiple repetitions of sentence data are available to estimate the variability of the selected movements. Both qualitative and quantitative analyses of these speech movements, with emphasis on lingual motions, will be employed to generate hypotheses concerning the speech movement deficit in the dysarthrias. This inductive analysis approach will pay special attention to across-speaker variability, among both healthy controls and speakers with dysarthria. A working hypothesis is that the lingual motions for speakers with dysarthria are no more variable across repetitions than those in healthy controls, but show deficits in scale and form.

The Use of Electron-Beam Lithography for Localized Micro-Beam Irradiations

A controlled microbeam facility based on electron-beam lithography is demonstrated as a means of observing total dose induced compensation effects on circuit parameters and understanding degradation mechanisms. The system uses electron-beam lithography techniques to perform on-chip microbeam irradiations on individual transistors or sub-circuits without any irradiation of the neighboring transistors. An experimental validation of the mechanisms proposed to explain the sensitivity of the input current of the LM139 as an illustration of this technique is provided.