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Characterizing proton beam damage in biological materials is of interest to enable the integration of proton microprobe elemental mapping techniques with other imaging modalities. It is also of relevance to obtain a deeper understanding of mechanical damage to lipids in tissues during proton beam cancer therapy. We have developed a novel strategy to characterize proton beam damage to lipids in biological tissues based on mass spectrometry imaging. This methodology is applied to characterize changes to lipids in tissues ex vivo, irradiated under different conditions designed to mitigate beam damage. This work shows that performing proton beam irradiation at ambient pressure, as well as including the application of an organic matrix prior to irradiation, can reduce damage to lipids in tissues. We also discovered that, irrespective of proton beam irradiation, placing a sample in a vacuum prior to desorption electrospray ionization imaging can enhance lipid signals, a conclusion that may be of future benefit to the mass spectrometry imaging community.

Protein aggregation is a widespread process leading to deleterious consequences in the organism, with amyloid aggregates being important not only in biology but also for drug design and biomaterial production. Insulin is a protein largely used in diabetes treatment and its amyloid aggregation is at the basis of the so-called insulin-derived amyloidosis. Here we uncover the major role of zinc in both insulin dynamics and aggregation kinetics at low pH, where the formation of different amyloid superstructures (fibrils and spherulites) can be thermally induced. Amyloid aggregation is accompanied by zinc release and the suppression of water-sustained insulin dynamics, as shown by particle-induced X-ray emission and X-ray absorption spectroscopy and by neutron spectroscopy, respectively. Our study shows that zinc binding stabilizes the native form of insulin by facilitating hydration of this hydrophobic protein and suggests that introducing new binding sites for zinc can improve insulin stability and tune its aggregation propensity.Statement of SignificanceLocalized amyloidosis occurs at insulin injection sites for diabetes treatment, leading to deleterious inflammations known as insulin-derived amyloidosis. Amyloid superstructures are also promising candidates in the field of biomaterials. Here we revealed that zinc, coordinated to insulin in the native form, is released upon amyloid aggregation, when insulin forms superstructures known as fibrils and spherulites. Zinc release leads to a full suppression of functionally essential protein dynamics through a modification of the protein’s hydration properties and completely modifies insulin amyloid kinetics. The results suggest that changes in protein hydration upon zinc binding/release modifies both stability and dynamics of insulin and might then be a general strategy to control protein stability and tune protein aggregation into amorphous and ordered superstructures.

The self-consistent Ion Beam Analysis (IBA) of cultural heritage samples using the external beam is technically demanding. We report on the calibration of an analysis of glass samples from the Rosslyn Chapel where the interest will ultimately be in the full characterisation of the weathered glass. Such an analysis requires a comprehensive Total-IBA approach using p-PIGE and He-PIXE to obtain ”bulk” and surface Na, with H-PIXE/EBS for multielemental depth profiling to 10 μm and He-PIXE/EBS for higher depth resolution near the surface; also with two PIXE detectors as usual for the high and low energy parts of the spectrum. A revised NDF v.10 code capable of a self-consistent handling of all these signals at state-of-the-art accuracy is described, together with the calibration protocols required for such an analysis. Other capabilities of the NDF code not previously discussed are also reviewed.

Understanding mechanisms of antibody synergy is important for vaccine design and antibody cocktail development. Examples of synergy between antibodies are well-documented, but the mechanisms underlying these relationships often remain poorly understood. The leading blood-stage malaria vaccine candidate, CyRPA, is essential for invasion of Plasmodium falciparum into human erythrocytes. Here we present a panel of anti-CyRPA monoclonal antibodies that strongly inhibit parasite growth in in vitro assays. Structural studies show that growth-inhibitory antibodies bind epitopes on a single face of CyRPA. We also show that pairs of non-competing inhibitory antibodies have strongly synergistic growth-inhibitory activity. These antibodies bind to neighbouring epitopes on CyRPA and form lateral, heterotypic interactions which slow antibody dissociation. We predict that such heterotypic interactions will be a feature of many immune responses. Immunogens which elicit such synergistic antibody mixtures could increase the potency of vaccine-elicited responses to provide robust and long-lived immunity against challenging disease targets.

Ion Beam Analysis (IBA) consists of a set of analytical techniques addressing elemental composition of inorganic material normally conducted using ion beams in the MeV kinetic energy range. Secondary Ion Mass Spectrometry using MeV ions (MeV SIMS) is the only IBA technique which can provide extensive molecular information about organic materials. MeV ions can be extracted into air hence offering the potential to apply MeV SIMS under atmospheric pressure. At the University of Surrey Ion Beam Centre, a fully ambient MeV SIMS setup has been developed and termed “Ambient Pressure MeV SIMS”. This AP MeV SIMS can be optimized for analysis and imaging of organic molecules. MeV SIMS relies upon electronic sputtering of the target material and this is much more efficient in insulating or organic targets, and less efficient in conducting metallic materials. PIXE, on the other hand, is efficient at providing good signals from elemental metallic systems, but does not readily provide molecular information from organics. The combination of the two techniques – preferably simultaneously with the same beam – provides useful complementary information which can readily be combined. Here we present pioneering preliminary work in simultaneous molecular and elemental imaging of a complex sample comprising of two organic species and two metallic species by combining AP MeV SIMS with Heavy Ion Particle Induced X-ray emission (HIPIXE).

In this work we report about the layout, assembly and the first resolution tests of the nuclear nanoprobe at MTA Atomki, Debrecen, Hungary. The nanoprobe setup was installed on the recently inaugurated 2 MV Tandetron accelerator manufactured by High Voltage Engineering Europa B.V. (HVEE). Although the complete Tandetron Laboratory was planned at the first project proposal stage, due to funding limitations the major components were purchased step by step, and so the present layout is only temporary. The necessary building reconstruction was also performed in phases. We present the preliminary experimental results and compare them what to expect in the final configuration using WinTRAX calculations. These calculations include focusing at different working distances, the performance at the planned final length (object distance), as well as scanning simulations using ‘dog-leg’ scan. At the present status it is already worth reporting that the preliminary nanoprobe setup has reached a satisfactory experimental performance of around 200 nm spot size for low current mode, and below 600 nm for high current mode.

We demonstrate that the simultaneous combination of ion beam analysis (IBA) and ion beam induced luminescence (IL) can reveal valuable information concerning the performance of strained doped silica fibre thermoluminescence microdosimeters. The micron scale spatial resolution and low detection limits of IBA allow the lateral distribution of dopant elements to be mapped and then correlated with the distribution of prompt radioluminescence. Measurement of the decay of the IL signal with dose provide information concerning the saturation of the subsequent TL signal at high doses. MeV ion beams can deposit relatively high energy in localized, well-quantified small volumes and so this method is valuable for studying high dose effects in TL dosimeters. We describe a simple modification of the target chamber microscope which enables sensitive low background light detection in two wavelength bands and present preliminary results from three types of germanium doped silica fibre dosimeter.

Porphyry-type deposits are the world’s main source of copper and molybdenum and provide a large proportion of gold and other metals. However, the mechanism by which mineralising fluids are extracted from source magmas and transported upwards into the ore-forming environment is not clearly understood. Here we use field, micro-textural and geochemical techniques to investigate field relationships and samples from a circa 8 km deep cross-section through the archetypal Yerington porphyry district, Nevada. We identify an interconnected network of relatively low-temperature hydrothermal quartz that is connected to mineralised miarolitic cavities within aplite dykes. We propose that porphyry-deposit-forming fluids migrated from evolved, more water-rich internal regions of the underlying Luhr Hill granite via these aplite dykes which contained a permeable magmatic crystal mush of feldspar and quartz. The textures we describe provide petrographic evidence for the transport of fluids through crystal mush dykes. We suggest that this process should be considered in future models for the formation of porphyry- and similar-type deposits. Hydrothermal mineralising fluids migrated from relatively deep, evolved and water-rich magmas via crystal mush dykes to reach the ore-forming environment in the Yerington porphyry system, according to field, petrographic and geochemical investigations.

“Total-IBA” implies the synergistic use of multiple IBA techniques. It has been claimed that Total–IBA inherits the accuracy of the most accurate IBA technique used. A specific example is now given of this where (in vacuo) EBS/PIXE of a glass sample uniform in depth is validated against absolutely calibrated EPMA of the same sample. The EPMA results had a mass closure gap of 2.0 ± 0.6 wt%; the full PIXE analysis determined the composition of this missing 2 wt%. The PIXE calibration was against a single certified glass sample, with uncertainties per line ~10%. Benchmarking also demonstrates ~10% underestimation of the Si scattering cross-section at proton energies ~3 MeV. But the Total-IBA determination of the silica content had a low standard uncertainty of about 2%. This is due to the strong constraints of both the chemical prior and also the mass closure properties of the EBS. Irradiation-induced sodium migration in this soda-lime glass is explored.

Metalloproteins comprise over one-third of proteins, with approximately half of all enzymes requiring metal to function. Accurate identification of these metal atoms and their environment is a prerequisite to understanding biological mechanism. Using ion beam analysis through particle induced X-ray emission (PIXE), we have quantitatively identified the metal atoms in 30 previously structurally characterized proteins using minimal sample volume and a high-throughput approach. Over half of these metals had been misidentified in the deposited structural models. Some of the PIXE detected metals not seen in the models were explainable as artifacts from promiscuous crystallization reagents. For others, using the correct metal improved the structural models. For multinuclear sites, anomalous diffraction signals enabled the positioning of the correct metals to reveal previously obscured biological information. PIXE is insensitive to the chemical environment, but coupled with experimental diffraction data deposited alongside the structural model it enables validation and potential remediation of metalloprotein models, improving structural and, more importantly, mechanistic knowledge.

The material characteristics of doped SiO2 fibre are studied, the electron traps in the product medium creating a situation attractive for their application in thermoluminescence (TL) radiation dosimetry. To date, rather limited research has been conducted towards gaining an essential understanding of the magnitude of TL signal and material characteristics of doped fibres. Characterization is being sought to ensure that the mechanism of TL yield in optical fibres is well understood, allowing a favourable well controlled production situation to be established. The intended end point is to specify dosimeters, not only for clinical dosimetry but also for their application in industrial/energy–industry settings. Investigation of the surface oxidation state of the Ge-doped SiO2 optical preform has been carried out using the X-ray photoelectron spectroscopy technique. In a further development using the fibre forming technology, particle-induced X-ray emission/Rutherford back scattering measurements have been employed to ascertain dopant concentrations of Ge-doped-cladding photonic crystal fibres (PCFs) with a view to improving TL yield. Present results concern uncollapsed and collapsed-hole-PCFs.

The proton beam writing (PBW) technique was used to fabricate microfluidic structures in SU-8 resist. A network of the buried channels was fabricated as part of a project to develop functional microfluidic device for neuronal studies and self-powered microfluidics. Protons with energies between 2.5 MeV and 0.75 MeV were used to fabricate the buried channels with a minimum feature size of around 1 μm and depths of 40–55 μm. Roughness of channels sidewalls was around 2.5 nm rms. Exposure regime and examples of functional networks fabricated using PBW are described. COMSOL Multiphysics® software was used to model the flow characteristics of fluid in the SU-8 microchannels structured by PBW. The results obtained using PBW are compared with the structures fabricated by UV-lithography.

In humans and other mammals it is known that calcium and phosphate ions are secreted from the distal small intestine into the lumen. However, why this secretion occurs is unclear. Here, we show that the process leads to the formation of amorphous magnesium-substituted calcium phosphate nanoparticles that trap soluble macromolecules, such as bacterial peptidoglycan and orally fed protein antigens, in the lumen and transport them to immune cells of the intestinal tissue. The macromolecule-containing nanoparticles utilize epithelial M cells to enter Peyer's patches, small areas of the intestine concentrated with particle-scavenging immune cells. In wild-type mice, intestinal immune cells containing these naturally formed nanoparticles expressed the immune tolerance-associated molecule 'programmed death-ligand 1', whereas in NOD1/2 double knockout mice, which cannot recognize peptidoglycan, programmed death-ligand 1 was undetected. Our results explain a role for constitutively formed calcium phosphate nanoparticles in the gut lumen and show how this helps to shape intestinal immune homeostasis.

Sample elemental concentrations can be determined using the microbeam proton-induced X-ray emission (PIXE) technique, providing non-destructive simultaneous low-background multi-element analysis. Present interest concerns analysis of Ge-doped SiO2 fibres intended as high spatial-resolution thermoluminescence (TL) dosimeters for radiation measurements in place of their more typical applications in telecommunications. During fibres fabrication, defined amounts of the Ge dopant are added, the dopant more usually having a determining role in the transmission properties of the fibre. Characteristic X-rays produced in PIXE analysis provide information on the relative distribution of elements within a sample, as in for instance Ge and Si concentrations, the Ge acting as point defect centres that promote TL. With the dopant tending to diffuse in and away from the fibre core, it is essential to define the sample matrix composition in order to accurately evaluate the X-ray yield. This is determined in part using simultaneous Rutherford Back Scattering analysis. In present work, PIXE/Rutherford Back Scattering measurements have been employed to ascertain dopant concentrations of fibres that have been fabricated at the University of Malaya with a view to improving TL yield. Present results concern cylindrical fibres, nominally with 4%, 6% and 8% weight peak Ge concentrations and flat fibres of nominal 6% weight Ge concentration. For the cylindrical fibres, Ge dopant concentration has been found to be in the range of 2.41–4.56%, 6.44–8.29% and 10.27–12.25% weight, respectively, while for the flat fibres, the Ge concentration range is broader, at 0.07–6.55% weight. Copyright © 2014 John Wiley & Sons, Ltd.

Proton-induced X-ray emission (PIXE) in combination with 3D depth profiling with Rutherford backscattering spectrometry (RBS) was used to establish the distribution and concentration of trace elements within individual corneal and retinal areas in frozen sections from adult male Wistar rats (n = 6). The distribution of endogenous trace elements in the cornea and retina is non-homogenous. The most abundant metal in the cornea is calcium followed by zinc. Iron and copper are present in small amounts localised particularly to the epithelium. Iron is also identified in keratocytes. Relatively high levels of calcium occur in the corneal epithelial cell bodies. Zinc has a wide intense distribution across the corneal epithelium (with greater levels in the basal part) and posterior stroma. In the retina, zinc is the most common metal followed by iron and copper. Relatively high levels of zinc exist in the retinal pigment epithelium (RPE), photoreceptor inner segments (RIS) and inner nuclear layer (INL). Chelatable zinc was localised with fluorescent TSQ in the RPE, RIS and plexiform layers. It is interesting to note that the highest levels of total zinc and the greatest intensity of chelatable zinc staining do not coincide. In the RPE and corneal epithelium, zinc co-localised with the zinc-containing metallothioneins (MT). However, there was a clear mismatch between the localisation of the most intense levels of zinc in the neuroretina (i.e. INL) and corneal posterior stroma with that reported for MT. For example, the presence of zinc is not particularly associated with the retinal ganglion cells, retinal area that contains MTs in significant amounts. While high amounts of zinc are present in the INL and corneal posterior stroma, which are largely devoid of MTs. This probably represents pools of static, catalytic and structural zinc associated with substances other than the MTs.

The quality of the structures fabricated using proton beam writing (PBW) and other direct-write microfabrication methods is strongly influenced by the path followed by the writing beam during the exposure. In particular, it is necessary to avoid paths in which the beam makes large jumps or changes in direction close to the edges of the structure, and ideally the scan path should follow the outline of the pattern to be exposed (sometimes referred to as turtle scanning). While this is relatively easy to implement when the patterns to be created can be built up from simple geometric shapes (circles, rectangles, etc), it has not been possible to do this in the case of arbitrarily complex images, at least using software available to the PBW community. This paper describes a simple edge-following algorithm (EFA) which uses a method of spiral searching around each pixel to determine a scan path which not only optimizes the conformity of the scan path to the edges of the required pattern, but also minimizes jumps (and hence blanking time) and scan reversals which can cause artefacts due to scanning system transients. The EFA operates on a 1-bit BMP format input image file and has been implemented in the OMDAQ-3 software package (Oxford Microbeams Ltd). The paper is illustrated with examples of complex structures written using the EFA at the University of Surrey Ion Beam Centre which demonstrate enhanced edge smoothness compared with simple blanked raster scanning.

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 4 mm 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 2 MeV protons and 4 MeV 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 2 MV 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. © 2013 Springer-Verlag Berlin Heidelberg.

The software package TRAX was a simulation tool for modelling the path of charged particles through linear cylindrical multipole fields described by analytical expressions and was a development of the earlier OXRAY program (Grime and Watt, 1983; Grime et al., 1982) [1] , [2] . In a 2005 comparison of raytracing software packages (Incerti et al., 2005) [3] , TRAX/OXRAY was compared with Geant4 and Zgoubi and was found to give close agreement with the more modern codes. TRAX was a text-based program which was only available for operation in a now rare VMS workstation environment, so a new program, WinTRAX, has been developed for the Windows operating system. This implements the same basic computing strategy as TRAX, and key sections of the code are direct translations from FORTRAN to C++, but the Windows environment is exploited to make an intuitive graphical user interface which simplifies and enhances many operations including system definition and storage, optimisation, beam simulation (including with misaligned elements) and aberration coefficient determination. This paper describes the program and presents comparisons with other software and real installations.

Porous silicon (PS) has been prepared using a microwave-assisted hydrofluoric acid (HF) etching method from a silicon wafer pre-implanted with 5 MeV Cu ions. The use of microbeam proton-induced X-ray emission (micro-PIXE) and microbeam Rutherford backscattering techniques reveals for the first time the capability of these techniques for studying the formation of micropores. The porous structures observed from micro-PIXE imaging results are compared to scanning electron microscope images. It was observed that the implanted copper accumulates in the same location as the pores and that at high implanted dose the pores form large-scale patterns of lines and concentric circles. This is the first work demonstrating the use of microwave-assisted HF etching in the formation of PS.

The suite of techniques which are available with the small accelerators used for MeV ion beam analysis (IBA) range from broad beams, microbeams or external beams using the various particle and photon spectrometries (including RBS, EBS, ERD, STIM, PIXE, PIGE, NRA and their variants), to tomography and secondary particle spectrometries like MeV-SIMS. These can potentially yield almost everything there is to know about the 3-D elemental composition of types of samples that have always been hard to analyse, given the sensitivity and the spacial resolution of the techniques used. Molecular and chemical information is available in principle with, respectively, MeV-SIMS and high resolution PIXE. However, these techniques separately give only partial information – the secret of “Total IBA” is to find synergies between techniques used simultaneously which efficiently give extra information. We here review how far “Total IBA” can be considered already a reality, and what further needs to be done to realise its full potential.

Plaggen soils are man-made agricultural soils which were created in historical times by using peat or turf as animal bedding. This bedding was then spread onto fields to create a rich and unusually deep topsoil. The plaggen system was used in Northern Europe from the Middle Ages up to, in some cases, the 1960s. Plaggen soils can be difficult to distinguish from soils which have simply had manure added, and in this paper we report the use of Proton Induced X-Ray Emission (PIXE) to investigate the levels of nutrient elements, particularly phosphorus, in soils from a farm in Shetland which is known to have used plaggen soils until 1967. Elemental maps obtained in air using the University of Surrey External Beam system allow us to identify peat fragments which contain significantly elevated levels of phosphorus relative to fresh peat and relative to the surrounding soil matrix. A further finding is that the peat fragments also contain elevated levels of bromine which can be used to distinguish plaggened peat from manure. This technique will be applied to a study of the origins of the plaggen method. Copyright © 2011 John Wiley & Sons, Ltd.

This paper evaluates the lens aberrations in microbeam and nanobeam systems caused by stray DC magnetic fields. Stray DC fields are far less influential on focussed beam spots than stray AC fields, but in order to achieve good beam-spot resolution the beamline must be aligned to the stray DC fields in the laboratory. The relative thickness of the optical elements compared to the curvature of the beam in such fields causes aberration where the beam axis differs from the optical axis of the lens system. In this paper numerical ray tracing has been used to study the influence of stray DC magnetic fields on beam resolution at the sub-micron level using typical field strengths for the Earth’s magnetic field as a case study.

Hard α-keratins such as hair, nail, wool and horn are stiff epidermal appendages used by mammals in a variety of functions including thermoregulation, feeding and intraspecific competition. Hard α-keratins are fibre-reinforced structures consisting of cytoskeletal elements known as ‘intermediate filaments’ embedded in an amorphous protein matrix. Recent research has shown that intermediate filaments are soft and extensible in living keratinocytes but become far stiffer and less extensible in keratinized cells, and this stiffening may be mediated by air-drying. Baleen, the keratinous plates used by baleen whales during filter feeding, is an unusual mammalian keratin in that it never air dries, and in some species, it represents the most heavily calcified of all the hard α-keratins. We therefore tested the hypothesis that whale baleen is stiffened by calcification. Here, we provide, to our knowledge, the first comprehensive description of baleen material properties and show that calcification contributes to overcoming the shortcomings of stiffening this hard α-keratin without the benefit of air-drying. We also demonstrate striking interspecies differences in the calcification patterns among three species of baleen whales and provide novel insights into the function and evolution of this unusual biomaterial.

The Ca:P ratio in a certified standard of hydroxyapatite was determined by Xray spectrometry (XRS), with the X-rays excited by both electrons and ions using energy dispersive spectroscopy on the scanning electron microscope (SEM-EDS) and particle induced X-ray emission (PIXE). The certified value of Ca:P was accurately verified by 3 MeV 4 He + Rutherford Backscattering Spectrometry (RBS). We show that the demonstrably rough surface of this sample does not cause perturbation of the Ca:P ratio within the uncertainties of each of the XRS measurements.

Proton beam writing (PBW) is a direct write technique that employs a focused MeV proton beam which is scanned in a pre-determined pattern over a target material which is subsequently electrochemically etched or chemically developed. By changing the energy of the protons the range of the protons can be changed. The ultimate depth of the structure is determined by the range of the protons in the material and this allows structures to be formed to different depths. PBW has been successfully employed on etchable glasses, polymers and semiconductor materials such as silicon (Si) and gallium arsenide (GaAs). This study reports on PBW in p–type GaAs and compares experimental results with computer simulations using the Atlas© semiconductor device package from SILVACO. It has already been proven that hole transport is required for the electrochemical etching of GaAs using Tiron (4,5-dihydroxy-m-benzenedisulfonic acid, di-sodium salt). PBW in GaAs results in carrier removal in the irradiated regions and consequently minimal hole transport (in these regions) during electrochemical etching. As a result the irradiated regions are significantly more etch resistant than the non-irradiated regions. This allows high aspect ratio structures to be formed.

A new initiative to build a vertical scanning focussed nanobeam is outlined. This is a collaboration between the Gray Cancer Institute and the University of Surrey. The new beam line will operate in both single ion and full current modes and will enable the irradiation of single cells in vitro with precisely counted numbers of ions, it will also enable the analysis of cells in vitro. The beam will be focussed and scanned and should be capable of irradiating 100,000 cells per hour. A new end station will enable the cells to be irradiated in an environmentally controlled environment and will enable the cells to be imaged both on-line and off-line. The beam line will be housed in its own purpose built building, with the area around the end station comprising a biological clean room. © 2007 Elsevier B.V. All rights reserved.

Since the construction of the first ion microprobe at Harwell in the early 1970s, there has been a steady improvement in the resolution of ion microprobes. However, in recent years the rate of improvement has slowed dramatically, with few systems able routinely to achieve resolutions less than 1 μm. There are many reasons for this lack of progress relating both to engineering at the nanometer scale and to fundamental physics. One crucial factor in the achievement of sub-micrometer resolution is the beam optical performance of the focusing system. This requires a high demagnification to permit the use of larger object apertures giving lower slit scattering, but this usually results in a correspondingly large aberration. We are investigating the use of two-stage lens systems with an intermediate focus. Such systems are capable of far greater demagnification than single-stage systems, but the challenge is to find a two-stage system with an acceptable ratio between demagnification and aberration. This paper presents preliminary the results of a systematic survey of two-stage lenses for nanobeam design. The scope of the survey is bounded by a number of practical limitations for nanobeam design. The survey encompasses systems of up to 8 quadrupole lenses and 4 independent power supplies arranged as two groups of up to four lenses constrained to form an intermediate image. The parameter space for this survey is vast, and even restricting the quadrupole lengths to those commercially available and to the 9 m beam length available in our laboratory, several million system geometries have been considered. A matrix-based beam optics software package has been developed which surveys the parameter space to determine the optimum value of a figure of merit based on the ratio of demagnification to spherical aberration. This uses the analytical approximations for spherical aberration in quadrupole lenses derived by Dymnikov et al. [A.D. Dymnikov, T.Ya. Fishkova, S.Ya. Yavor, Nucl. Instr. and Meth. 37 (1965) 268]. The performance of selected systems with good figures of merits have been further investigated using numerical raytracing software and the results are presented.

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.

Microscopic particles (0.5–2 μm diameter), rich in calcium and phosphorus, are found in the lumen of the mid-distal gut of all mammals investigated, including humans, and these may play a role in immuno-surveillance and immune regulation of antigens from food and symbiotic bacteria that are contained in the gut. Whether these particles can cross in to tissue of the intestinal mucosa is unclear. If so, characterising their morphology and chemical composition is an important task in elucidating their function. The analysis of calcium phosphate in biological tissues has been approached in several ways including optical microscopy, scanning electron microscopy and, most recently in this work, with nuclear microscopy. In this paper, we describe the use of microPIXE and microRBS to locate these particles and to determine, accurately, the ratio of phosphorus to calcium using the information on sample thickness obtained from RBS to allow the PIXE ratios to be corrected. A commercial sample of hydroxy apatite was used to demonstrate accuracy and precision of the technique. Then, in a pilot study on intestinal tissue of mice, we demonstrated the presence of calcium phosphate microparticles, consistent with confocal microscopy observations, and we identified the average molar P:Ca molar ratio as 1.0. Further work will confirm the exact chemical speciation of these particles and will examine the influence of differing calcium containing diets on the formation of these microparticles.

The precision and accuracy of four different X-ray analytical techniques at the micrometre level, PIXE–RBS, PIXE–PIXE, μ-SRXRF and WDS, have been investigated and compared. A range of different reference materials was considered for this purpose, and the glass BCR 126A was selected for the inter-comparison study, due to its homogeneity, chemical stability and elemental composition. The experimental protocols were tailored to employ the standard, optimal measurement conditions for each technique and material, but still retaining comparable measurement parameters between the different methods. The results show that the different techniques can complement each other without compromising in accuracy, and that they agree with each other to better than 5%.

The identification and quantification of metals bound to proteins is a crucial problem to be solved in structural biology. This paper describes the technique of particle induced X-ray emission with a microfocused beam (microPIXE) as a tool for analysing the elemental composition of liquid and crystalline protein samples. The proton beam induces characteristic X-ray emission from all elements in the protein, which can be interpreted in terms of the metal content of the protein molecule with a relative accuracy of between 10% and 20%. The compelling advantage of this method is that the sulphur atoms in the methionines and cysteines of the protein provide an internal calibration of the number of protein molecules present so that systematic errors are minimised and the technique is entirely internally self-consistent. This is achieved by the simultaneous measurement of the energy of backscattered protons (Rutherford backscattering), to enable us to determine the matrix composition and thickness, and so correct the PIXE data for the self-absorption of X-rays in the sample. The theoretical background to the technique is described, and the technical and experimental procedures are outlined. Examples of recent measurements are given which have informed a range of investigations in structural biology. The use of the technique is increasing and we envisage that future developments will enable it to become a routine high-throughput method.

This report describes how proton beam writing can be used to produce direct write, high resolution three dimensional structures on the nano and micron scales in semiconductor materials such as p-type (1 0 0) bulk silicon and gallium arsenide. The lattice damage caused by the proton irradiation increases the electrical resistance of the semiconductors resulting in a raised structure of the scanned area after an electrochemical etch. Advances in this field over the past few years and its relevance to future technology mean that it is now a powerful contender for direct write technology for future nodes 45 nm and below.

We have investigated the mechanism of high rate erosion which is observed when polytetrafluoroethylene (PTFE) is exposed to MeV protons in an oxygen rich atmosphere (e.g. air). Using currents of the order of nA, it is possible to create holes with depths of millimetres and diameter defined by the beam area on the surface with exposure times of minutes. This is not observed in atmospheres of nitrogen, helium or argon nor in vacuum. We propose that the erosion is a result of the formation of a stable gaseous compound following beam induced decomposition of the PTFE (possibly an acyl fluoride) which does not re-deposit in the hole. We present the results of experiments leading to this hypothesis and propose a method for fabricating three-dimensional structures in PTFE with micrometre feature size. This process is the subject of an international patent application.

The University of Surrey Ion Beam Centre has recently commissioned a new in-air scanning microbeam. The new external beam uses a magnetic quadrupole doublet to focus a beam-spot of μm dimensions on the vacuum exit window so that there are no beam defining apertures close to the analysis region. A pre-lens magnetic dipole allows beam scanning for measuring two dimensional elemental distributions. The scanned area is limited by the diameter of the exit window to 3 mm diameter at present. The sample is positioned using a front viewing video microscope and an alignment laser, and detectors for X-rays (high energy and low energy), backscattered particles and gamma rays are installed. This paper will detail the ion optical characteristics of the in-air scanning microbeam, and the methods used in the characterisation. A comparison between calculated optical properties of the in-air scanning microbeam, using the ray-tracing software TRAX, and the measured properties of the beam will be presented.

Investigations using MeV protons for lithography applications are being performed at the Ion Beam Centre of the University of Surrey, UK. High aspect ratio three dimensional structures have been produced by protons in gallium arsenide (GaAs) and Poly(methyl methacrylate) (PMMA). Structures produced in PMMA require significantly lower fluences than GaAs and behave positively to etching, whereas GaAs shows a negative behaviour. Variable fluence studies on GaAs show a transition from low to high aspect ratio three dimensional structures as the fluence increases.

A new proton lithography mechanism has been identified and investigated: the micropatterning of photosensitive etchable glass based on the crystallization of the glass after irradiation with MeV protons and heat treatment. The use of MeV protons results in a significantly reduced minimum feature size compared with reported results using ultraviolet (UV) irradiation and the threshold dose for etching is very low (4000 protons µm−2), offering the potential for creating complex microstructures by direct writing using very short exposures. The depth of the structures is determined only by the range of the protons in the glass, which allows structures with different depths to be fabricated. This technique appears to be very valuable for the rapid fabrication of high aspect ratio microstructures such as fluid networks and micro-optical devices in a material with useful optical and mechanical properties.

Polytetrafluoroethylene (PTFE) displays an unusual behaviour when exposed to energetic light ions. Whereas other polymers tend to go black and degrade when exposed to MeV proton beams in air, rapid ablation of PTFE is observed. This rapid direct etching of PTFE with protons was investigated in a series of experiments and it was found that the presence of oxygen was required to obtain the high etch rates observed. The phenomenon described here offers a mechanism for fabricating high aspect ratio PTFE microstructures.

The tenth meeting in the series of triennial International Conferences on Particle Induced X-ray Emission (PIXE) and its Applications marks a milestone for the field. Not only is there the significance of the tenth occurrence of the meeting, reflecting perhaps a symbolic coming of age of the technique, but a new policy on publishing the proceedings has been introduced. Extended abstracts of all conference submissions have been published as a CD-ROM, while contributions deemed by a panel of experienced PIXE scientists to represent the current status of PIXE development and applications were selected for publication in this journal, and so to be presented to a wider scientific audience than would normally be the case. The aim of this short introduction is to present an overview of the background and current state of development of PIXE to enable the succeeding papers to be seen in perspective. Copyright © 2005 John Wiley & Sons, Ltd.

This paper describes the new external microbeam on the 15° beamline of the 5 MV Tandetron accelerator recently installed at the CMAM in Madrid. The focusing and beam extraction system was supplied by Oxford Microbeams Ltd. and consists of a high precision quadrupole doublet with an interchangeable Kapton window exit nozzle and front-viewing video microscope. The sample is positioned in the beam using a stepper motor stage. The beam current and beam profile have been determined under different experimental conditions. A simple method based on the signal processing of ion-induced luminescence from quartz targets has been used to determine the beam profile in two dimensions simultaneously, without scanning. This is the first step in the development of a real time beam profile monitoring system, which could be used as part of an automated beam focusing procedure. The beam line will be primarily devoted to archaeometry and cultural heritage studies. As an example we report the characterisation of two Tang appearance antique porcelains.

One of the fundamental limitations in the performance of MeV ion microbeam focusing systems is the effect of ion scattering at the edges of the object aperture. As the aperture is reduced in the search for smaller spot sizes, the fraction of scattered to unscattered beam increases. The scattered beam contains lower energy particles which can be transmitted through the system to create a halo of over-focused particles surrounding the final image. Removal of this halo is critical to achieving small spot sizes, especially in single ion applications. In this paper, we discuss the use of a Wien filter (crossed magnetic and electrostatic fields) to deflect the reduced energy scattered particles and ensure that only ions with the correct energy are accepted into the lens. This paper reviews the beam optics of Wien filter systems and presents calculations of the parameters required to obtain useful energy dispersion.

RES and PIXE microbeam analysis is used to investigate the trace element concentrations as well as the surface corrosion of counterfeited copper halfpennies from the mid 18th century. PIXE elemental mapping was used to identify regions with a minimum of light element contamination which could be used for accurate trace element determinations. RES analysis of selected points on the map could be used to confirm the absence of corrosion or contamination, and in addition yielded valuable information on the composition and structure of the corrosion layers. (C) 1999 Elsevier Science B.V. All rights reserved.

The Oxford Scanning Proton Microprobe continues to be used in the field of trace element measurement in biological systems, exploiting the unique advantages of sensitive, quantitative trace element analysis using PIXE, high spatial resolution and the long penetrating power of MeV protons. This paper outlines a number of recent applications which highlight these advantages. These include: (a) Analysing the distribution of metals in the pupae of leaf-cutting ants to determine the storage sites and transport mechanism of metals used to harden the edges of the mandibles. (b) A study of the distribution of zinc in the retina of rats to determine the role of zinc in light and dark adaptation of the eye. (c) The analysis of crystals of proteins and other large organic molecules prepared for structure determination using x-ray diffraction. These often contain metal atoms, and the identity and concentration of the metal is an important diagnostic for determining the nature of the protein and the quality of the crystallisation. The crystals are normally small (~100μm) and so microPIXE is being used to characterise them. This technique has wide ranging applications, including qualitative and quantitative identification of metals in reaction centres, in active sites and in metal binding proteins, and of DNA or RNA bound to proteins.

PIXE analysis has been used for many years for the characterization of environmental samples, especially aerosol particles. The low detection limits, speed, quantitative accuracy and multielemental capability give this technique a unique role in determining low levels of trace elements in samples. The recent extension of the PIXE technique to use focused beams gives the additional capability of microscopic analysis and so the distribution of trace elements in samples can be determined. This paper describes the technique of nuclear microscopy and reviews its use in the analysis of individual environmental particles such as aerosols, fly ash and lake sediments. © 1998 John Wiley & Sons, Ltd.

Sensors such as electrodes and optical fibre devices, optrodes, can be used to determine steep concentration gradients of chemical species in aquatic microenvironments, such as in the pore waters of surface sediments1 and microbial mats2,3,4, but are limited to a restricted range of determinands. The highest-resolution measurements of trace-metal concentrations in pore waters, at about 1.25 mm, have been provided by a recently developed thin-film gel technique5,6, but the resultant metal distributions suggest that sub-millimetre-scale gradients need to be determined if the fluxes and cycling of the metals are to be fully quantified and understood. Here we report the development of this thin-film gel technique to measure Zn, Mn, Fe and As fluxes and concentrations at a resolution of 100 μm, and demonstrate the utility of the method in situ within the surface sediments and overlying microbial mat of a stream. Vertical profiles through the mat and sediments, and horizontal two-dimensional mapping just below the sediment–water interface, reveal the contrasting gradients, fluxes and remobilization niches of the four metal species at a sub-millimetre scale. The microbial mat appears to be an important regulator of the cycling of these metals. This technique has the potential to be extended to other chemical species and applied to other microenvironments with steep concentration gradients, such as redox boundaries, plant roots, animal burrows and sites of precipitation/dissolution in soils and sediments.

Many disparate methods of compositional analysis of materials are underpinned by the same fundamental atomic processes: the excitation of the electronic system of the atoms followed by its subsequent relaxation. These methods include the electron spectroscopies (XPS, AES) used for surface studies, the electron microscopies used for elemental and structural characterisation (SEM using EDS and WDX; TEM using EELS), the X-ray methods (XRF, XAS) and ion beam analysis (PIXE) used for elemental and chemical characterisation. All rely on measuring the characteristic energy absorbed or emitted by the unknown target atom when its electronic system is excited by ionisation due to charged particles or electromagnetic radiation. This excitation is defined by the energy levels of the atomic electrons, determined primarily by the atomic number of the atom. (Atoms can also be excited without ionisation, as in optical and infra-red spectroscopy: this is outside the scope of this article.) The theoretical description of the electronic structure of atoms is a major intellectual triumph of the twentieth century and this body of knowledge is exploited in the theoretical description of each of these methods, but the treatment of any particular method is usually presented by specialists in that method in isolation from all others. In this chapter we present a brief synthetic overview of materials analysis using atomic excitation, highlighting those features and physical concepts which underpin all these apparently disparate analysis methods. We hope to encourage modern analysts to appreciate the truly complementary nature of the powerful methods at their disposal.

Polyacrylamide gel probes provide a rapid, simple procedure for measuring iron and manganese in pore waters at sub-millimeter resolution by diffusional equilibration in thin films (DET). Fe(II) and Mn(II) diffuse freely within the gel matrix, and with a 0.4 mm thick gel, complete equilibration is achieved in 6 min. Gels must be deoxygenated prior to deployment, and exposure to air limited to 15 s to avoid overestimation of iron. Treatment with 10 mmol L -1 NaOH or KOH quantitatively immobilizes Fe and Mn as their insoluble oxyhydroxides and brings about a diffusional transfer of Fe and Mn to the edges of the gel. The time from removal from the sediment to fixing or slicing the gel is the greatest limit to resolution