Microbeam Facility Research Articles

A review of external microbeams for ion beam analyses

Ion microbeam facilities are analytical tools with high spatial resolution exploiting MeV ion beams. The interactions of beam particles with atoms and nuclei of the target induce the emission of characteristic radiation, the energy of which provides signatures of the compositional and/or structural properties of the target; Ion-Beam Analysis (IBA) techniques, based on the detection of such radiation, enable characterisation of samples of interest, e.g. in material and earth sciences, cultural heritage, biology, medicine, and environmental studies. External beams, obtained by extracting the particles into the atmosphere through a thin window, have many attractive features, e.g. non-destructive/non-invasive analysis and ease of working, so many laboratories have dedicated beam-lines to ex-vacuo IBA analyses. External microprobes have made it possible to obtain probes in the micron range by adopting strong focusing lenses, ultra-thin windows for beam extraction, and short/ultra-short external path of beam particles in light gases; they have also made possible the use of new external IBA techniques, e.g. BS, ERDA, STIM, and IBIC. By adopting systems to raster scan the beam over the sample, imaging capabilities have also become available for ex-vacuo analysis. External scanning microprobes + IBA techniques have enabled the characterisation of samples with high spatial resolution, comparable with that achievable in-vacuum for thick samples, avoiding sample damage.

High energy heavy ion microbeam irradiation facility at IMP

A high energy heavy ion microbeam irradiation facility was designed and installed in the Institute of Modern Physics (IMP) of Chinese Academy of Sciences (CAS). It can deliver well-focused ions into the targets at exact locations. With the advantage of allowing vertical irradiation as well as focusing ions from Carbon to Uranium with high energies in a broad energy range (7MeV/u to 100MeV/u for Carbon ions), this setup is able to deliver a preset number of ions into pre-selected target positions. Material specimens in vacuum and living cells in air can also be irradiated by this facility. Detailed description of this microbeam facility as well as its beam optics is presented in this paper. Some preliminary test results are also given.

Development of the Jyväskylä microbeam facility

A new microbeam facility is being constructed at the 1.7 MV Pelletron Accelerator in Jyväskylä. The facility is designed for easy upgrading and incorporates a number of innovative features. Initially, it is based on a Heidelberg doublet with a design capability of a 3 × 5 μm beamspot at PIXE intensities and later upgraded to nanobeam performance. A thermal-expansion compensated rigid frame mounted on a mechanically isolated floor section is used to support the ion optical components. A compact-post focusing electrostatic deflector is used for high linearity beam scanning. This together with a novel time-stamped data collection (TDC) allows dynamic effects in IBIC, fluorescence bleaching to be studied as well as facilitating multi-resolution image support for low-fluence imaging of cells. The target chamber is fitted with a novel low-cost large working distance optical microscope, extremely compact large solid angle photon detectors as well as conventional secondary electron, PIXE and Scanning Transmission Ion Microscopy (STIM) detectors.

Online imaging of initial DNA damages at the PTB microbeam

In an inter-disciplinary collaboration of Physikalisch-Technische Bundesanstalt (PTB), German Collection of Microorganisms and Cell Cultures (DSMZ) and Heinrich-Heine University, live-cell imaging has been established at the charged-particle microbeam facility of PTB. Candidate genes participating in DNA strand-break repair pathways such as PARP-1, MRE11, MSH2, MDC1 and p53BP1 have been modified to generate fluorescent fusion proteins. Using multi-cistronic expression vectors, stable genomic integration was achieved in HT-1080 fibroblasts. The aim of this study is to characterise and use these highly reliable cell lines for studying initial steps of DNA damage responses and kinetics of repair after microbeam irradiation with high- and low-linear energy transfer (LET) particles in living cells at physiological conditions.

External scanning micro-PIXE for the characterization of a polycapillary lens: Measurement of the collected X-ray intensity distribution

Abstract We developed a PIXE detection system for the analysis of medium-light elements which exploits a weakly focusing polycapillary lens for the transmission of the X-rays emitted from the target material to a Silicon Drift Detector. The polycapillary lens efficiently collects X-rays, while prevents back-scattered protons from impinging on the detector chip, thus avoiding electronics perturbation and consequent quality loss of PIXE spectra. The system is optimized for the detection of X-rays in the energy range 1–10 keV, when the emission from the target is induced by MeV proton beams with size of the order of a few hundreds of micrometers. This work reports the results of the lens characterization in terms of X-ray collection spot, i.e. the area of the sample actually “seen” by the lens, and its dependence on the X-ray energy. The lens properties have been measured using the external scanning microbeam facility of the Tandetron accelerator at LABEC-INFN in Florence. The detection system was used to detect X-rays from a set of pure elemental standards with an incident 3 MeV proton beam focused to a size of about 30 μm scanning an area of 1.9 × 1.6 mm 2 . By measuring the spatial distribution of characteristic X-rays from each given material, the collection profile of the lens at the corresponding X-ray energy was obtained. Using several standards, the behaviour throughout the range 1–10 keV was examined. The sensitivity of the lens collection profile on the lens-sample out-of-focus distance was also investigated.

An investigation of the potential of optical computed tomography for imaging of synchrotron-generated x-rays at high spatial resolution

X-ray microbeam radiation therapy (MRT) is a novel form of treatment, currently in its preclinical stage, which uses microplanar x-ray beams from a synchrotron radiation source. It is important to perform accurate dosimetry on these microbeams, but, to date, there has been no accurate enough method available for making 3D dose measurements with isotropic, high spatial resolution to verify the results of Monte Carlo dose simulations. Here, we investigate the potential of optical computed tomography for satisfying these requirements. The construction of a simple optical CT microscopy (optical projection tomography) system from standard commercially available hardware is described. The measurement of optical densities in projection data is shown to be highly linear (r2 = 0.999). The depth-of-field (DOF) of the imaging system is calculated based on the previous literature and measured experimentally using a commercial DOF target. It is shown that high quality images can be acquired despite the evident lack of telecentricity and despite DOF of the system being much lower than the sample diameter. Possible reasons for this are discussed. Results are presented for a complex irradiation of a 22 mm diameter cylinder of the radiochromic polymer PRESAGE™, demonstrating the exquisite ‘dose-painting’ abilities available in the MRT hutch of beamline ID-17 at the European Synchrotron Radiation Facility. Dose distributions in this initial experiment are equally well resolved on both an optical CT scan and a corresponding transmission image of radiochromic film, down to a line width of 83 µm (6 lp mm−1) with an MTF value of 0.40. A group of 33 µm wide lines was poorly resolved on both the optical CT and film images, and this is attributed to an incorrect exposure time calculation, leading to under-delivery of dose. Image artefacts in the optical CT scan are discussed. PRESAGE™ irradiated using the microbeam facility is proposed as a suitable material for producing phantom samples for quantitative characterization of optical CT microscopy systems.

X-ray Microbeams for Radiobiological Studies: Current Status and Future Challenges

Recent technological advances and new radiobiology challenges are behind the great interest in the use of microirradiation techniques for radiobiological studies. Radiobiolog- ical microbeams are facilities able to deliver precise doses of radiation to preselected individual cells (or part of them) in vitro and assess their biological consequences on a single cell base. They are therefore uniquely powerful tools to address speciflc problems where very precise targeting accuracy and dose delivery are required. The majority of radiobiological microbeams are centred on particle accelerators in order to irradiated biological samples with an exact number of ions. Currently there are only three microbeam facilities in routine use which employ focused X-rays: two are based on laboratory bench X-ray sources (Queen's University Belfast and Nagasaki Uni- versity) and one developed using synchrotron X-ray beams (Photon Factory in Tsukuba, Japan). While low dose rates limit laboratory bench sources to a few keV, micronsize X-ray probes of a few tens of keV are achievable using synchrotron sources. Each facility has however their own beneflts and draw back points. Techniques for focusing X-rays are well established and contin- uously improving with focal spots down to 50nm achievable for ultrasoft X-rays using circular difiraction gratings known as \zone plates. Re∞ection X-ray optics such as Kirkpatrick-Baez mirrors and polycapillary systems are also used to produce micron size X-ray spots. Combined with nano-positioning accuracy of the new generation of stages, improved optics and image anal- ysis algorithms, X-ray microbeams are able to address radiobiological issues in an unprecedented way. Microbeams have contributed signiflcantly to the discovery and characterization of important new flndings regarding the mechanisms of interaction of ionizing radiation with cells and tissues. In particular, they have played a fundamental role in the investigation of non-targeted efiects where radiation response is induced in samples whose DNA has not been directly exposed. The exquisite resolution ofiered by focused X-ray probes has allowed important questions regarding the locations and mechanisms of subcellular targets to be precisely addressed. Evidences of the critical role played by the cytoplasm have been collected and radio-sensitivity across the cell nucleus itself is attracting considerable interest. Moreover using the microbeam single cell approach, it has been possible to study the mechanisms underpinning the bystander efiect where radiation damage is expressed in cells which have not been directly irradiated but were in contact or shared medium with directly exposed samples. As a result, microbeam facilities are regarded as a main tool for the formulation of a new radiobiological paradigm where direct damage to cellular DNA is not a requirement. Additionally, the implications of the non-targeted efiects in in vivo systems and ultimately humans have still to be fully understood. The new generation of X-ray microbeams equipped with 3D image stations and higher X-ray energies ofiers the perfect approach to extend targeted studies to complex biological models. Finally, the single-cell approach and the high spatial resolution ofiered by the microbeam provide the perfect tool to study and quantify the dynamic processes associated with the production and repair of DNA damage. Using green ∞uorescent protein (GFP), it is now possible to follow the spatio-temporal development of the DNA damage sites which is currently of great interest in order to monitor the remodelling of chromatin structure that the cell undergoes to deal with DNA damage.

No Evidence for DNA and Early Cytogenetic Damage in Bystander Cells after Heavy-Ion Microirradiation at Two Facilities

The occurrence of bystander effects has challenged the evaluation of risk for heavy ions, mainly in the context of space exploration and the increasing application of carbon ions in radiotherapy. In the present study, we addressed whether heavy-ion-induced DNA and cytogenetic damage is detectable in bystander cells. The formation of gamma-H2AX foci, sister chromatid exchanges and micronuclei were used as markers of damage to DNA. Normal human fibroblasts were exposed to low fluences of carbon and uranium ions, and alternatively single cells were targeted with heavy ions using the GSI microbeam. We did not observe a significant increase in the bystander formation of gamma-H2AX foci, sister chromatid exchanges or micronuclei. In addition, we performed for the first time parallel experiments at two microbeam facilities (GSI, JAEA) using the same cell line, culture conditions and irradiation protocols. No significant enhancement of the micronucleus frequencies in bystander cells was detected after targeted carbon-ion irradiation, confirming the results. Details regarding the history, culture conditions or support of the cells might be affecting the detection of bystander effects. On the other hand, the potential X-ray- and heavy-ion-induced bystander effects investigated herein clearly do not exceed the experimental error and thus are either lacking or are less pronounced than the effects reported in the literature for similar end points after alpha-particle and X-ray exposure.

The single-particle microbeam facility at CEA-Saclay

Low dose and non-targeted effect studies continue to attract the attention of a growing number of radiobiologists. Experimental setups based on light ion microbeams constitute a tool of choice for this kind of investigations. However, a careful attention must be given to experimental conditions, as setup-induced stress levels should be well below those induced by the irradiation itself. Here, we present the current status of the single-particle microbeam facility that has been developed these last years at the nuclear microprobe of Saclay. The driving idea was to build a facility in which local irradiation studies are performed in an environment close to cellular biology standards. This facility includes unique features, such as (i) a compact setup that allows easy access and vertical irradiation mode, (ii) a collimated beam that can be mechanically positioned under the desired cells at a very fast speed, avoiding the requirement of a focusing element and (iii) a controlled environment (temperature, CO 2, humidity) that allows performing of very long term experiments on cultured cells. Fluorescent techniques are implemented and permit in situ monitoring of cellular responses to irradiations. Several radiobiological studies are already underway and this will be illustrated with recent results regarding DNA damage and reactive oxygen species signaling time courses following targeted irradiations.

Heavy ion microbeam vacuum requirements

Transport of heavy ions through an ion microbeam focusing system can be affected by insufficient vacuum within the beam transport tube. Due to interactions of heavy ions with atoms of residual gas in the vacuum tube of a microbeam facility, the angular, lateral and energy spreading of an ion beam increases prior to focusing, creating a beam halo. This beam halo can produce undesirable effects in some applications of ion microbeam techniques. In order to model this effect, the ion beam angular spread in residual gas has been approximated by Sigmund’s theoretical predictions for small-angle ion multiple scattering (MS), while ion energy loss straggling distributions have been applied for studying the energy spread. The extent of the beam halo has been estimated by combining the results of these calculations with ion optics calculations. Recommendations concerning microbeam focusing due to the vacuum conditions are given for different heavy ions in the MeV energy range.

Differential and scanning-mode external PIXE for the analysis of the painting “Ritratto Trivulzio” by Antonello da Messina

Extensive PIXE investigations have been performed at the LABEC accelerator laboratory in Florence on the painting “Ritratto Trivulzio” by Antonello da Messina, one of the great Italian masters of XV Century and a pioneer in modern oil painting. It is well known that a complete and unambiguous characterisation of materials in paintings is often difficult, owing to their complex structure. For the “Ritratto Trivulzio”, the combination of advanced variants of PIXE, such as differential and scanning-mode analysis, provided a relevant contribution to the characterisation of paint layers, in terms of composition and structure, in a totally non-invasive and non-destructive way. Single-spot mode PIXE with external proton beams of about half millimetre diameter was first used for a general characterisation of different areas of the painting. Differential PIXE performed in many spots led to reconstruct the sequence of paints employed by the artist and to estimate in some cases the local paint layer thickness. A second run carried out with the external scanning microbeam facility, using a 3 MeV proton beam of about 80 μm size, was then crucial to clarify some issues raised by the first analysis. In particular, elemental maps from selected areas helped to understand the way some colour shades had been obtained on the red mantle of the portrayed gentleman, using different pigments irregularly distributed on the surface.

Monte Carlo dosimetry for targeted irradiation of individual cells using a microbeam facility

Microbeam facilities provide a unique opportunity to investigate the effects of ionising radiation on living biological cells with a precise control of the delivered dose. This paper describes dosimetry calculations performed at the single-cell level in the microbeam irradiation facility available at the Centre d'Etudes Nucléaires de Bordeaux-Gradignan in France, using the object-oriented Geant4 Monte Carlo simulation toolkit. The cell geometry model is based on high-resolution three-dimensional voxelised phantoms of a human keratinocyte (HaCaT) cell line. Such phantoms are built from confocal microscopy imaging and from ion beam chemical elemental analysis. Results are presented for single-cell irradiation with 3 MeV incident alpha particles.

Ionizing Radiation Microbeam Facilities for Radiobiological Studies in Europe

A growing body of experimental evidence gathered in the last 10-15 years with regard to targeted and non-targeted effects of low doses of ionizing radiation (hyper-radiosensitivity, induced radio-resistance, adaptive response, genomic instability, bystander effects) has pushed the radiobiology research towards a better understanding of the mechanisms underlying these phenomena, the extent to which they are active in-vivo, and how they are inter-related. In such a way factors could be obtained and included in the estimation of potential cancer risk to the human population of exposure to low levels of ionizing radiation. Different experimental approaches have been developed and employed to study such effects in-vitro (medium transfer experiments; broad-field irradiation at low doses also with insert or shielding systems...). In this regard, important contributions came from ionizing radiation microbeam facilities that turn to be powerful tools to perform selective irradiations of individual cells inside a population with an exact, defined and reproducible dose (i.e. number of particles, in case of charged particle microbeams). Over the last 20 years the use of microbeams for radiobiological applications increased substantially and a continuously growing number of such facilities, providing X-rays, electrons, light and heavy ions, has been developing all over the world. Nowadays, just in Europe there are 12 microbeam facilities fully-operational or under-development, out of more than 30 worldwide. An overview of the European microbeam facilities for radiobiological studies is presented and discussed in this paper.

Microbeam Irradiation Facilities for Radiobiology in Japan and China

In order to study the radiobiological effects of low dose radiation, microbeam irradiation facilities have been developed in the world. This type of facilities now becomes an essential tool for studying bystander effects and relating signaling phenomena in cells or tissues. This review introduces you available microbeam facilities in Japan and in China, to promote radiobiology using microbeam probe and to encourage collaborative research between radiobiologists interested in using microbeam in Japan and in China.

Analysis and Optimization of Stability of CAS-LIBB Single Ion Microbeam

Single ion microbeam is the most advanced technology which can emit a single ion for precise localization. A single-ion microbeam facility has been constructed at the Key Laboratory of Ion Beam Bioengineering (LIBB), Chinese Academy of Sciences (CAS), with a spatial resolutions of about 5 μm. Based on CAS-LIBB microbeam, three key elements affecting the quality of the system are assessed: the size of beam spot, the energy range and the counting accuracy of implanting ions. Various contributions to the ion beam stability, including the ion source, the terminal voltage of electrostatic accelerator and the components in beam pipeline, are discussed. Analysis shows that the improvement of terminal voltage stability is the most important issue for future optimization of CAS-LIBB facility. Some preliminary investigations and project aimed at optimization and development are proposed as well.

Heavy ion microprobes: a unique tool for bystander research and other radiobiological applications

The risk assessment for low doses of high linear energy transfer (LET) radiation has been challenged by a growing body of experimental evidence showing that non-irradiated bystander cells can receive signals from irradiated cells to elicit a variety of cellular responses. These may be significant for radiation protection but also for radiation therapy using heavy ions. Charged particle microbeams for radiobiological application provide a unique means to address these issues by allowing the precise irradiation of single cells with a counted numbers of ions. Here, we focus specifically on heavy ion microbeam facilities currently in use for biological purposes, describing their technical features and biological results. Typically, ion species up to argon are used for targeted biological irradiation at the vertically collimated microbeam at JAEA (Takasaki, Japan). At the SNAKE microprobe in Munich, mostly oxygen ions have been used in a horizontal focused beam line for cell targeting. At GSI (Darmstadt), a horizontal microprobe with a focused beam for defined targeting using ion species up to uranium is operational. The visualization of DNA damage response proteins relocalizing to defined sites of ion traversal has been accomplished at the three heavy ion microbeam facilities described above and is used to study mechanistic aspects of heavy ion effects. However, bystander studies have constituted the main focus of biological applications. While for cell inactivation and effects on cell cycle progression a response of non-targeted cells has been described at JAEA and GSI, respectively, in part controversial results have been obtained for the induction of DNA damage measured by double-strand formation or at the cytogenetic level. The results emphasize the influence of the cellular environment, and standardization of experimental conditions for cellular studies at different facilities as well as the investigation of bystander effects in tissue will be the aims of future research. At present, the most important conclusion of radiobiology studies at heavy ion microbeams is that bystander responses are not accentuated for increasing ionizing density radiation.

The ionoluminescence apparatus at the LABEC external microbeam facility

In this paper, we describe the main features of the ionoluminescence (IL) apparatus recently installed at the external scanning microbeam facility of the 3MV Tandetron accelerator of the INFN LABEC Laboratory in Firenze. The peculiarity of this IL set-up resides in the fact that the light produced by the ion irradiation of the specimen is collected by a bifurcated optical fiber, so that photons are shunted both to a CCD spectrometer, working in the 200–900nm wavelength range, and to a photomultiplier (PMT). The accurate focusing of the optical system allows high photon collection efficiency and this results in rapid acquisition of luminescence spectra with low ion currents on luminescent materials; simultaneously, luminescence maps with a spatial resolution of 10μm can be acquired through the synchronization of PMT photon detection with the position of the scanning focused ion beam. An optical filter with a narrow passband facing the photomultiplier allows chromatic selectivity of the luminescence centres.The IL apparatus is synergistically integrated into the existing set-up for ion beam analyses (IBA). The upgraded system permits simultaneous IL and PIXE/PIGE/BS measurements. With our integrated system, we have been studying raw lapis lazuli samples of different known origins and precious lapis lazuli artworks of the Collezione Medicea of Museum of Natural History, University of Firenze, aiming at characterising their composition and provenance.

Scanning-PIXE analysis of gold lace embroideries in a relic of St. Francis

In this work, we describe the compositional analysis performed by scanning-mode PIXE on the metal threads of a XIII century embroidery. The precious work analysed is the pillow-case used to cover the pillow, on which – according to tradition – St. Francis of Assisi was resting his head when he died. Measurements were performed in order to characterise the embroideries of the two sides and the passementerie in the lateral hems. Several areas, each of the order of two square millimetres, were scanned with a 3MeV proton external beam of 20μm size on target, using the external micro-beam facility of our laboratory, with list-mode acquisition. Analysis of elemental maps and spectra from selected homogeneous sub-areas allowed us to extract the quantitative composition of the gilded tape and estimates of its thickness.

Micro-SR-XRF and micro-PIXE studies for archaeological gold identification – The case of Carpathian (Transylvanian) gold and of Dacian bracelets

Trace-elements are more significant for provenancing archaeological metallic artifacts than the main components. For gold, the most promising elements are platinum group elements (PGE), Sn, Te, Sb, Hg and Pb. Several small fragments of natural Transylvanian gold – placer and primary – were studied by using micro-PIXE technique at the Legnaro National Laboratory AN2000 microbeam facility, Italy and at the AGLAE accelerator, C2RMF, Paris, France and by using micro synchrotron radiation X-ray fluorescence (micro-SR-XRF) at BESSY synchrotron, Berlin, Germany. The goal of the study was to identify the trace-elements, especially Sn, Sb and Te. A spectacular application to five Dacian gold bracelets authentication is presented (Sn and Sb traces).

A Review: Development of a Microdose Model for Analysis of Adaptive Response and Bystander Dose Response Behavior

Prior work has provided incremental phases to a microdosimetry modeling program to describe the dose response behavior of the radio-protective adaptive response effect. We have here consolidated these prior works (Leonard 2000, 2005, 2007a, 2007b, 2007c) to provide a composite, comprehensive Microdose Model that is also herein modified to include the bystander effect. The nomenclature for the model is also standardized for the benefit of the experimental cellular radio-biologist. It extends the prior work to explicitly encompass separately the analysis of experimental data that is 1.) only dose dependent and reflecting only adaptive response radio-protection, 2.) both dose and dose-rate dependent data and reflecting only adaptive response radio-protection for spontaneous and challenge dose damage, 3.) only dose dependent data and reflecting both bystander deleterious damage and adaptive response radio-protection (AR-BE model). The Appendix cites the various applications of the model. Here we have used the Microdose Model to analyze the, much more human risk significant, Elmore et al (2006) data for the dose and dose rate influence on the adaptive response radio-protective behavior of HeLa x Skin cells for naturally occurring, spontaneous chromosome damage from a Brachytherapy type (125)I photon radiation source. We have also applied the AR-BE Microdose Model to the Chromosome inversion data of Hooker et al (2004) reflecting both low LET bystander and adaptive response effects. The micro-beam facility data of Miller et al (1999), Nagasawa and Little (1999) and Zhou et al (2003) is also examined. For the Zhou et al (2003) data, we use the AR-BE model to estimate the threshold for adaptive response reduction of the bystander effect. The mammogram and diagnostic X-ray induction of AR and protective BE are observed. We show that bystander damage is reduced in the similar manner as spontaneous and challenge dose damage as shown by the Azzam et al (1996) data. We cite primary unresolved questions regarding adaptive response behavior and bystander behavior. The five features of major significance provided by the Microdose Model so far are 1. Single Specific Energy Hits initiate Adaptive Response. 2. Mammogram and diagnostic X-rays induce a protective Bystander Effect as well as Adaptive Response radio-protection. 3. For mammogram X-rays the Adaptive Response protection is retained at high primer dose levels. 4. The dose range of the AR protection depends on the value of the Specific Energy per Hit, 1 >. 5. Alpha particle induced deleterious Bystander damage is modulated by low LET radiation.