Dr Geoffrey Grime

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.

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, in which 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.

Antibodies can have synergistic effects, but mechanisms are not well understood. Here, Ragotte et al. identify three antibodies that bind neighbouring epitopes on CyRPA, a malaria vaccine candidate, and show that lateral interactions between the antibodies slow dissociation and inhibit parasite growth synergistically. 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.

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.

Sr/Ca and Mg/Ca ratios in biomineral CaCO3 have recently been regarded as more reliable than δ18O values as proxy for paleotemperature because they are less affected by salinity or polar ice volume. We argue, however, that vital effects can exert a greater control than paleotemperature over fossil Sr/Ca. Seasonal perturbations in isotopic data from the Eocene bivalve Venericardia planicosta reveal a gradually decreasing annual growth rate through ontogeny. High-resolution Sr/Ca ratios, analyzed with a new proton-microbe technique, increase markedly through ontogeny, however, suggesting that more Sr was incorporated as growth rate slowed rather than as a result of changing paleotemperature. Comparative δ18O and Sr/Ca data from the broadly coeval marine gastropod Clavilithes macrospira, which exhibits a linear growth rate through ontogeny, also shows a significant increase in Sr concentration with age as well as seasonal, possibly temperature-related variations. Our observations show that neither growth, calcification rate, nor temperature can be the sole factor controlling Sr incorporation into molluscan aragonite. Metabolic activity, related to factors such as temperature, salinity, age, and growth rate, is likely to exert the primary control over Sr/Ca ratios in aragonitic mollusks.

The Scanning proton microprobe uses a focused beam of MeV protons to give analytical information by exploiting two physical processes, particle induced X-ray emission (PIXE) and Rutherford Backscattering (RBS). Scanning transmission ion microscopy may also be included in the nuclear microscopy package to give imaging data. PIXE is particularly appropriate for biomedical research as signals from hydrogen, carbon, nitrogen and oxygen, which are the main components of a biological matrix, are not detected and therefore signals from inorganic elements with z > 10 will not be swamped by signals from the matrix. Elements with z < 10 can, however, be detected using RBS when present at higher concentrations. Apart from the biomedical field, the SPM has wide-ranging applications spanning diverse areas such as archaeology, materials science and environmental chemistry. This paper reviews the theory of ion beam analysis, the Oxford Scanning Proton Microprobe and outlines some of the work currently being carried out at Oxford.

The principles of proton microprobe analysis are outlined and compared to electron beam analysis. Sample preparation methods are summarized, and the types of suitable sample are enumerated. The information that can be achieved from the analysis in terms of elemental composition and images is also described. © 1999 Elsevier Ltd All rights reserved.

The use of high energy ion beams (mainly of protons or alpha particles) for chemical analysis is described. The equipment necessary for achieving this is summarized. The main detection systems are proton-induced X-ray emission (PIXE), Rutherford backscattering (RBS) and nuclear reaction analysis (NRA). © 1999 Elsevier Ltd All rights reserved.

“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.

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.

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.

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).

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.

The 12th International Conference on Proton Induced X-ray Emission (PIXE) and its Analytical Applications was held on the campus of the University of Surrey in Guildford, U.K. between 29 th June and 2nd July 2010. The PIXE conferences have been held at approximately three year intervals since the mid 1970s and have reflected the development of the PIXE technique and its expanding range of applications. This special issue of X-Ray Spectrometry contains papers based on conference contributions which have been selected to represent the latest developments in PIXE and its applications.

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.

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.

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.

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.

Return of materials from the Hubble Space Telescope (HST) during shuttle orbiter service missions has allowed inspection of large numbers of hypervelocity impact features from long exposure at about 615 km altitude in low Earth orbit (LEO) [1,2]. Here we describe the application of advanced X-ray microanalysis techniques on scanning electron microscopes (SEM), microprobes and a 2 MV Tandetron, to nearly 400 impacts on the painted metal surface of the Wide Field and Planetary Camera 2 (WFPC2) radiator shield [3,4]. We identified artificial Orbital Debris (OD) and natural Micrometeoroid (MM) origins for small [5] and even for larger particles [6], which usually may leave little or no detectable trace on HST solar arrays, as they penetrate through the full cell thickness [2,7].

This book presents the theory of quadrupole focusing with the practical development of microprobe systems in mind. The introductory chapter outlines the analytical processes and provides a background to the problems of designing quadrupole probe-forming systems. Following a description of the quadrupole lens and the mathematical models used to describe the fields within both magnetic and electrostatic quadrupole lenses, the authors describe two techniques for calculating the path of a charged particle through a beam-optical system; matrix methods and numerical raytracing. The minimum spot size achieved by a quadrupole system is influenced strongly by parasitic aberrations due to imperfections in the construction and alignment of the instrument. The authors assess the importance of the various effects by reference to their influence in three existing microprobe systems. Finally, they describe briefly some novel and interesting alternatives to the quadrupole lens as the focusing element. Refs.

This paper discusses the results of a pilot study using Proton Induced X-ray Emission (PIXE) analysis of gold granulated objects from the Bronze Age site of Tell el-Ajjul in Southern Palestine obtained with the Oxford University proton microprobe. Using modern granulated samples with known smithing processes as points of comparison, it is demonstrated how information on ancient joining practices may be accurately gauged. The importance of characterizing objects of known provenance is emphasized in a discussion of archaeological context. It is argued that the building of a large Mediterranean-wide database of granulation analyses can add new depths to our understanding of temporal, social, cultural and technological relationships in the past.

Silver-staining "senile" plaques occurring in the brain are a major part of the pathology of Alzheimer's disease. The elemental composition of these structures, and the possible presence of aluminum and silicon in these structures, has been the subject of an increasing research effort over the last decade. However, the results have often been contradictory. Using a scanning proton microprobe, the elemental composition of senile plaques has been determined. This instrument, similar to an electron probe, uses a focused beam of protons scanned across a sample to map the elements. The technique is absolutely quantitative and is sensitive down to the parts per million level. Tissue from six cases of clinically and pathologically characterized cases of Alzheimer's disease and two aged neurologically normal controls was scanned. It was found that aluminum and silicon occur at a level of 50 ppm or greater in the cores of 20% of senile plaques and that the total occurrence of aluminum or silicon in scans containing plaques was not above background. The major uncertainties affecting interpretation of results of this kind are discussed, and it is suggested that the least controllable factor is contamination in the reagents used to prepare and stain the tissue. This indicates that until plaques can be unambiguously identified and analyzed in untreated tissue, no conclusion can be reached on whether senile plaques contain aluminum and silicon.