Proton Microprobe Research Articles

Does iron inhibit calcification during atherosclerosis?

Oxidative stress has been implicated in the etiology of atherosclerosis and even held responsible for plaque calcification. Transition metals such as iron aggravate oxidative stress. To understand the relation between calcium and iron in atherosclerotic lesions, a sensitive technique is required that is quantitatively accurate and avoids isolation of plaques or staining/fixing tissue, because these processes introduce contaminants and redistribute elements within the tissue. In this study, the three ion-beam techniques of scanning transmission ion microscopy, Rutherford backscattering spectrometry, and particle-induced X-ray emission have been combined in conjunction with a high-energy (MeV) proton microprobe to map the spatial distribution of the elements and quantify them simultaneously in atherosclerotic rabbit arteries. The results show that iron and calcium within the atherosclerotic lesions exhibit a highly significant spatial inverse correlation. It may be that iron accelerates the progression of atherosclerotic lesion development, but suppresses calcification. Alternatively, calcification could be a defense mechanism against atherosclerotic progression by excluding iron.

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Microcalcifications in Early Intimal Lesions of Atherosclerotic Human Coronary Arteries

Although calcium (Ca) precipitation may play a pathogenic role in atherosclerosis, information on temporal patterns of microcalcifications in human coronary arteries, their relation to expression of calcification-regulating proteins, and colocalization with iron (Fe) and zinc (Zn) is scarce. Human coronary arteries were analyzed post mortem with a proton microprobe for element concentrations and stained (immuno)histochemically for morphological and calcification-regulating proteins. Microcalcifications were occasionally observed in preatheroma type I atherosclerotic intimal lesions. Their abundance increased in type II, III, and IV lesions. Moreover, their appearance preceded increased expression of calcification-regulating proteins, such as osteocalcin and bone morphogenetic protein-2. In contrast, their presence coincided with increased expression of uncarboxylated matrix Gla protein (MGP), whereas the content of carboxylated MGP was increased in type III and IV lesions, indicating delayed posttranslational conversion of biologically inactive into active MGP. Ca/phosphorus ratios of the microcalcifications varied from 1.6 to 3.0, including amorphous Ca phosphates. Approximately 75% of microcalcifications colocalized with the accumulation of Fe and Zn. We conclude that Ca microprecipitation occurs in the early stages of atherosclerosis, inferring a pathogenic role in the sequel of events, resulting in overt atherosclerotic lesions. Microcalcifications may be caused by local events triggering the precipitation of Ca rather than by increased expression of calcification-regulating proteins. The high degree of colocalization with Fe and Zn suggests a mutual relationship between these trace elements and early deposition of Ca salts.

First direct-write lithography results on the Guelph high resolution proton microprobe

The recently completed high-resolution proton microprobe at the University of Guelph is Canada’s first one-micron nuclear microprobe, which represents the country’s state-of-the-art technology for various nuclear microprobe applications, e.g. direct-write microlithography. Its probe-forming system is comprised of a triplet Oxford Micro beams magnetic quadrupole lenses, along with high-precision objective slits. High energy protons coming off a 3MV particle accelerator can achieve a nominal resolution of one micro and a beam current of several hundred of picoamperes when arriving at the target. This proton probe is ideal for the use of direct-write lithography with the incorporation of a magnetic scanning system and motorized sample stage.Preliminary lithography results have been obtained using spin-coated PMMA photoresist as specimen. The beam spot size, beam range and straggling inside the substrate and the exposure conditions are investigated by using scanning electron microscopy. This facility is the first in Canada to perform focused direct-write ion beam lithography, which is ideal for modification and machining of polymer and semiconductor materials for biological, microfluidic and ultimate lab-on-chip applications.

Microcalcifications in atherosclerotic lesion of apolipoprotein E‐deficient mouse

Evidence is accumulating that calcium-rich microdeposits in the vascular wall might play a crucial role in the onset and progression of atherosclerosis. Here we investigated an atherosclerotic lesion of the carotid artery in an established murine model, i.e. the apolipoprotein E-deficient (APOE(-/-) ) mouse to identify (i) the presence of microcalcifications, if any, (ii) the elemental composition of microcalcifications with special reference to calcium/phosphorus mass ratio and (iii) co-localization of increased concentrations of iron and zinc with microcalcifications. Atherosclerosis was induced by a flow-divider placed around the carotid artery resulting in low and high shear-stress regions. Element composition was assessed with a proton microprobe. Microcalcifications, predominantly present in the thickened intima of the low shear-stress region, were surrounded by areas with normal calcium levels, indicating that calcium-precipitation is a local event. The diameter of intimal microcalcifications varied from 6 to 70 μm. Calcium/phosphorus ratios of microcalcifications varied from 0.3 to 4.8, mainly corresponding to the ratio of amorphous calcium-phosphate. Increased iron and zinc concentrations commonly co-localized with microcalcifications. Our findings indicate that the atherosclerotic process in the murine carotid artery is associated with locally accumulated calcium, iron and zinc. The calcium-rich deposits resemble amorphous calcium phosphate rather than pure hydroxyapatite. We propose that the APOE(-/-) mouse, in which atherosclerosis was evoked by a flow-divider, offers a useful model to investigate the pathophysiological significance of accumulation of elements such as calcium, iron and zinc.

Croatian Appoxiomenos alloy composition and lead provenance study

An ancient bronze statue of an athlete with a strigil (known as Apoxiomenos) – was raised from the north Adriatic Sea in 1999. In order to help to determine its place and date of manufacture various scientific techniques were used. 14C dating performed by the Accelerator Mass Spectrometry (AMS) on the organic material found inside the statue gave calibrated dates between 100 B.C. and 250 A.D. Non-destructive analysis performed by portable X-Ray Fluorescence (XRF) system and external beam Particle Induced X-Ray Emission (PIXE) spectroscopy could not provide representative results for metal alloy composition due to the electrochemical deterioration of the surface of bronze in seawater. This was observed by the analysis of alloy cross-sections performed by the proton microprobe. The analysis showed much higher lead concentrations on the surface of the bronze than inside, confirming that realistic alloy composition can be only performed on samples taken from at least 0.6 mm below the surface. Subsequent micro-PIXE analysis on metal cross-section showed inhomogeneous lead concentrations between 1 and 12%. Lead isotope analysis for provenance investigation has been done on 15 samples using Multi Collector Inductively Coupled Plasma Mass Spectrometry (MC-ICP-MS). Analysis showed that lead isotope composition is consistent with some of the lead ores originating either from the Eastern Alps or from Sardinia. Altogether, the results lead to the conclusion that the statue is not of a Greek origin, but most probable it is a Roman copy of the Greek original.

Nuclear Microscopy: A Novel Technique for Quantitative Imaging of Gadolinium Distribution within Tissue Sections

All clinically-approved and many novel gadolinium (Gd)-based contrast agents used to enhance signal intensity in magnetic resonance imaging (MRI) are optically silent. To verify MRI results, a "gold standard" that can map and quantify Gd down to the parts per million (ppm) levels is required. Nuclear microscopy is a relatively new technique that has this capability and is composed of a combination of three ion beam techniques: scanning transmission ion microscopy, Rutherford backscattering spectrometry, and particle induced X-ray emission used in conjunction with a high energy proton microprobe. In this proof-of-concept study, we show that in diseased aortic vessel walls obtained at 2 and 4 h after intravenous injection of the myeloperoxidase-sensitive MRI agent, bis-5-hydroxytryptamide-diethylenetriamine-pentaacetate gadolinium, there was a time-dependant Gd clearance (2 h = 18.86 ppm, 4 h = 8.65 ppm). As expected, the control animal, injected with the clinically-approved conventional agent diethylenetriamine-pentaacetate gadolinium and sacrificed 1 week after injection, revealed no significant residual Gd in the tissue. Similar to known in vivo Gd pharmacokinetics, we found that Gd concentration dropped by a factor of 2 in vessel wall tissue in 1.64 h. Further high-resolution studies revealed that Gd was relatively uniformly distributed, consistent with random agent diffusion. We conclude that nuclear microscopy is potentially very useful for validation studies involving Gd-based magnetic resonance contrast agents.

High resolution imaging and elemental analysis of PAGE electrophoretograms by scanning proton microprobe

Metal content of metalloproteins can be detected and even quantified by the PIXE–PAGE method. In this technique the proteins are separated by thin layer electrophoresis (by polyacrylamide gel electrophoresis (PAGE) in most cases) and the properly dried gel sections are analyzed by PIXE using “band-shaped” proton milli-beam. This PIXE–PAGE method was adapted for our scanning proton microprobe. The microPIXE–PAGE version provides two-dimensional elemental mapping of the protein bands. In addition, the fast continuous scanning reduces the risk of the thermal deterioration of the sample and the X-ray contribution from dust-impurities can be filtered out in the data evaluation process.

Micro-elemental analysis of some Transylvanian meteorites and lunar samples

Micro-PIXE investigations on some Transylvanian chondritic meteorite fragments and on small Moon soil pieces from the LUNA-16 mission were performed at the Legnaro and Rossendorf proton microprobes. The most exciting finding of the study was the presence of Pt grains in the Moci meteorite. The results are compared to previously published data.

A novel approach to the identification and quantitative elemental analysis of amyloid deposits—Insights into the pathology of Alzheimer’s disease

There is considerable interest in the role of metals such as iron, copper, and zinc in amyloid plaque formation in Alzheimer’s disease. However to convincingly establish their presence in plaques in vivo, a sensitive technique is required that is both quantitatively accurate and avoids isolation of plaques or staining/fixing brain tissue, since these processes introduce contaminants and redistribute elements within the tissue. Combining the three ion beam techniques of scanning transmission ion microscopy, Rutherford back scattering spectrometry and particle induced X-ray emission in conjunction with a high energy (MeV) proton microprobe we have imaged plaques in freeze-dried unstained brain sections from CRND-8 mice, and simultaneously quantified iron, copper, and zinc. Our results show increased metal concentrations within the amyloid plaques compared with the surrounding tissue: iron (85 ppm compared with 42 ppm), copper (16 ppm compared to 6 ppm), and zinc (87 ppm compared to 34 ppm).

Parallel Mappings as a Key for Understanding the Bioinorganic Materials

Important bioinorganic objects, both living and fossilized are as a rule characterized by a complex microscopic structure. For biological samples, the cell-like and laminar as well as growth ring structures are among most significant. Moreover, these objects belong to a now widely studied category of biominerals with composite, inorganic-organic structure. Such materials are composed of a limited number of inorganic compounds and several natural organic polymers. This apparently simple composition leads to an abnormal variety of constructions significant from the medical (repairs and implants), natural (ecological effectiveness) and material science (biomimetic synthesis) point of view. The analysis of an image obtained in an optical microscope, optionally in a scanning electron microscope is a topographical reference for further investigations. For the characterization of the distribution of chemical elements and compounds in a material, techniques such as X-ray, electronor proton microprobes are applied. Essentially, elemental mappings are collected in this stage. The need for the application of an X-ray diffraction microprobe is obvious and our experience indicates on the necessity of using the synchrotron-based devices due to their better spatial resolution and good X-ray intensity. To examine the presence of the organic compounds, the Raman microprobe measurements are good options. They deliver information about the spatial distribution of functional groups and oscillating fragments of molecules. For the comprehensive investigation of bioinorganic material structural and chemical features, we propose the following sequence of methods: optical imaging, elemental mapping, crystallographic mapping, organic mapping and micromechanical mapping. The examples of such an approach are given for: petrified wood, human teeth, and an ammonite shell.

Three-dimensional imaging of aerosol particles with scanning proton microprobe in a confocal arrangement

We have reconstructed three-dimensional (3D) element-specific distributions of aerosol microparticles captured in a thick quartz filter. The characteristic x rays were induced by a proton microprobe and detected by a Si(Li) spectrometer equipped with a polycapillary lens. Combining a fine microbeam scanning in the lateral plane with the sample movement parallel to the beam axis, the aerosol particles were driven across the sensitive microvolume and for Fe, Ca, S, and Si a sequence of two-dimensional lateral x-ray images was recorded. This is the first example of an x-ray element selective 3D imaging of a few micrometer sized objects with a proton beam.

Scanning proton microprobe analysis of mercury and other trace elements in Fe-sulfides from a Kentucky coal

Abstract Scanning proton microprobe analysis of a series of Fe-sulfides (pyrite and marcasite) from the Manchester coal bed, Clay County, eastern Kentucky, demonstrated that Hg could be detected in some grains. The low concentration of Hg in coal, and even in the sulfides where it is generally concentrated, makes the detection difficult. Other elements, notably Ni, Cu, Zn, As, Se, Tl, and Pb, occur in greater concentrations and could be detected in more samples than Hg. All of the Fe-sulfides analyzed As substitution, and most were even more complex. Overall, there was a large variation in the amount of substitution in the sulfides. Arsenic, as an example, ranges in concentration from 11 μg/g to 12,000 μg/g in Fe-sulfides from the same coal sample. Much of the trace element concentration in this coal is in the coarse sulfides or, possibly, in other mineral phases included in the pyrite or marcasite, favorable for the pre-combustion removal of the trace elements.

EPMA and PIXE dating of monazite in granulites from Stary Gierałtów, NE Bohemian Massif, Poland

Chemical Th–U–total Pb (CHIME) dating of monazite by electron probe microanalyzer (EPMA) and proton microprobe (PIXE) was carried out on felsic granulites from Stary Gierałtów, Poland, which represent part of the Orlica-Śnieżnik Dome in the NE Bohemian Massif. Analyzed monazite is characterized by mosaic zoning rather than simple core-to-rim growth, and strontium contents of up to 750ppm. An isochron age of 347 ± 13Ma represents timing of amphibolite-facies metamorphism, in agreement with previously published estimates.

Analysis of ancient embroideries by IBA techniques

Ion beam analysis (IBA) techniques are often used to study works of art, mainly for their non-destructive character. In these techniques, products of interactions between beams and targets are exploited to determine the elemental composition of the sample surface down to depths from few micrometres to hundreds of micrometres, depending on incident particles, their energy and target material.For samples with surface inhomogeneities, information about elemental distribution can be essential to discriminate contributions from different areas. At the authors’ laboratory in Florence (LABEC), the external scanning proton microprobe facility — installed on a beam line of the 3 MV Tandem Accelerator — allows samples to be analysed in atmosphere, with spatial resolution down to around micrometres, and to extract elemental maps from the investigated areas.Recently, there has been the opportunity of analysing a ‘gold lace embroidery’ (using silk threads, spiral wrapped by a sort of miniature golden tape) after a cartoon by Raffaellino del Garbo (the fifteenth–sixteenth centuries).

Proton microprobe for chemical dating of monazite

Although quantitative chemical analysis by proton microprobe has become an established technique, it has been rarely applied to problems in the earth sciences. The method, having lower detection limit (better than 10 ppm for U, Th and Pb) and higher spatial resolution than electron microprobe (typically 1 μm vs 3 μm), can be successfully used in geology. Here, we present a procedure for the chemical dating of monazite, (REE)PO 4, by proton microprobe. The procedure is compared with electron probe microanalysis technique (EPMA).

Exploring Ocean Biogeochemistry by Single‐Cell Microprobe Analysis of Protist Elemental Composition1

The biogeochemical cycles of many elements in the ocean are linked by their simultaneous incorporation into protists. In order to understand these elemental interactions and their implications for global biogeochemical cycles, accurate measures of cellular element stoichiometries are needed. Bulk analysis of size-fractionated particulate material obscures the unique biogeochemical roles of different functional groups such as diatoms, calcifying protists, and diazotrophs. Elemental analysis of individual protist cells can be performed using electron, proton, and synchrotron X-ray microprobes. Here we review the capabilities and limitations of each approach and the application of these advanced techniques to cells collected from natural communities. Particular attention is paid to recent studies of plankton biogeochemistry in low-iron waters of the Southern Ocean. Single-cell analyses have revealed significant inter-taxa differences in phosphorus, iron, and nickel quotas. Differences in the response of autotrophs and heterotrophs to iron fertilization were also observed. Two-dimensional sub-cellular mapping indicates the importance of iron to photosynthetic machinery and of zinc to nuclear organelles. Observed changes in diatom silicification and cytoplasm content following iron fertilization modify our understanding of the relationship between iron availability and silicification. These examples demonstrate the advantages of studying ocean biogeochemistry at the level of individual cells.

Novel pattern of foliar metal distribution in a manganese hyperaccumulator.

The primary sequestration of foliar manganese (Mn) in Mn-hyperaccumulating plants can occur in either their photosynthetic or non-photosynthetic tissues, depending on the species. To date, only non-photosynthetic tissues have been found to be the major sinks in other hyperaccumulators. Here, electron (SEM) and proton (PIXE) microprobes were used to generate qualitative energy dispersive (EDS) X-ray maps of leaf cross sections. Two Mn hyperaccumulators, Garcinia amplexicaulis Vieill. (Clusiaceae) and Maytenus fournieri (Panch. and Sebert) Loesn. (Celastraceae), and the Mn accumulator Grevillea exul Lindley (Proteaceae) were studied. PIXE/EDS data obtained here for M. fournieri were in agreement with existing SEM/EDS data showing that the highest localised foliar Mn concentrations were in the epidermal tissues. However, this is the first in situ microprobe investigation of G. amplexicaulis and G. exul. The Mn X-ray maps of G. amplexicaulis revealed a previously undescribed third spatial distribution pattern among Mn-hyperaccumulating species. Manganese was relatively evenly distributed throughout the leaf photosynthetic and non-photosynthetic tissues, while in G. exul it was most highly concentrated in the epidermal cells.

Early Calcifications in Human Coronary Arteries As Determined with a Proton Microprobe

The current paradigm reads that calcifications characterize the advanced and complex lesions in the atherosclerotic process. To explore the possibility that coronary artery wall calcifications already commence at an early stage of atherosclerosis, a combination of proton beam techniques with a (sub-) micrometer resolution, i.e., micro-proton induced X-ray emission, backward and forward scattering spectroscopy, was applied on human coronary arteries with lesions preceding overt atheromas. The detection limits of phosphorus and calcium in each separate pixel, 0.88*0.88 microm2 in size, were approximately 150 and 80 microg/g dry weight, respectively. Calcium distributions of entire coronary artery cross section were obtained, and calcifications were demonstrated at a preatheroma stage of the atherosclerotic process. The size of the microcalcifications varied between 1 and 10 microm. The composition of the microcalcifications was deduced from the calcium-to-phosphorus ratio. In order to quantify this ratio, the thickness of the specific X-ray absorber used for PIXE had to be accurately determined. Also, thick target PIXE calculations were performed and the method was validated. The calcium-to-phosphorus ratios of the microcalcifications were assessed with good accuracy and varied from 1.62 to 2.79, which corresponds with amorphous calcium phosphate.

Microchemical imaging of iodine distribution in the brown alga Laminaria digitata suggests a new mechanism for its accumulation.

Brown algal kelp species are the most efficient iodine accumulators among all living systems, with an average content of 1.0% of dry weight in Laminaria digitata. The iodine distributions in stipe and blade sections from L. digitata were investigated at tissue and subcellular levels. The quantitative tissue mapping of iodine and other trace elements (Cl, K, Ca, Fe, Zn, As and Br) was provided by the proton microprobe with spatial resolutions down to 2 mum. Chemical imaging at a subcellular resolution (below 100 nm) was performed using the secondary ion mass spectrometry microprobe. Sets of samples were prepared by both chemical fixation and cryofixation procedures. The latter prevented the diffusion and the leaching of labile inorganic iodine species, which were estimated at around 95% of the total content by neutron activation analysis. The distribution of iodine clearly shows a huge, decreasing gradient from the meristoderm to the medulla. The contents of iodine reach very high levels in the more external cell layers, up to 191 +/- 5 mg g(-1) of dry weight in stipe sections. The peripheral tissue is consequently the main storage compartment of iodine. At the subcellular level, iodine is mainly stored in the apoplasm and not in an intracellular compartment as previously proposed. This unexpected distribution may provide an abundant and accessible source of labile iodine species which can be easily remobilized for potential chemical defense and antioxidative activities. According to these imaging data, we proposed new hypotheses for the mechanism of iodine storage in L. digitata tissues.