Signal standardization of the secondary ion mass spectrometry (SIMS) microscope for quantification of halogens and calcium in biological applications

The secondary ion mass spectrometry (SIMS) microscope is able to map chemical elements in tissue sections. Although absolute quantification of an element remains difficult, a relative quantitative approach is possible for soft tissue by using carbon (¹²C) as an internal reference present at large homogeneous and constant concentration in specimen and embedding resin. In this study, this approach is used to standardize the signal of an SIMS microscope for the quantification of halogens (¹⁹F^—, ³⁵Cl^— and ⁷⁹Br^—) and calcium (⁴⁰Ca⁺). Standard preparation was determined based on homogeneity and stability criteria by molecular incorporation (halogens) or mixing (calcium) in methacrylate resin. Standard measurements were performed by depth analysis on areas of 8 μm (halogens) and 150 μm (calcium) in diameter for 10–30 min, under Cs⁺ (halogens) or O₂⁺ (calcium) bombardment. Results obtained from 100–120 measurements for each standard dilution show that the relationship between the signal intensity measured and the elemental concentration (μg/mg of wet tissue or mm ) is linear in the range of biological concentrations. This quantitative approach was applied firstly to bromine of the 5-bromo-2′-deoxyuridine (BrdU) used as nuclear marker of rat hepatocytes in proliferation. The second model concerns depletion of calcium concentration in cortical compartment in Paramecium tetraurelia during exocytosis. Then signal standardization in SIMS microscopy allows us to correlate quantitative results with those obtained from other methods.     

Using the scanning electron microprobe and secondary ion mass spectrometry to locate 14C and 13C labelled plant residues within soil aggregates

Increasing concentrations of CO2 in the atmosphere are placing emphasis on the necessity for sequestering carbon (C) into soil organic matter (SOM). By studying the interior parts of soil aggregates, a better understanding of the incorporation and sequestration of plant residue materials within these aggregates could be obtained. The location of newly added plant residues within soil aggregates may also assist in the investigation of the impact of these newly added plant materials on soil aggregation. This study investigated two different techniques for determining the location of newly added plant residues within soil aggregates by using plant materials labelled with 14C and 13C isotopes incorporated into two different soil types, Black Earth (Pellic Vertisol) and Red Clay (Chromic Vertisol). Both autoradiography combined with scanning electron microprobe analysis (14C) and secondary ion mass spectrometry (SIMS) (13C) were successfully used for detecting the presence and location of the newly added plant residues fragments within soil aggregates of both soil types. The use of labelled plant materials is essential for the study of the location of newly added plant materials within soil aggregates, and this has proven to be a useful tool for studying the impact of residue additions on soil aggregate formation. Furthermore, these methods have been shown to be useful for determining the incorporation and sequestration of C materials within soil aggregates. The development of the 13C SIMS technique could alleviate the necessity for the use of the radioactive isotope 14C in soil studies.     

Using the scanning electron microprobe and secondary ion mass spectrometry to locate 14C- and 13C-labelled plant residues within soil aggregates

Increasing concentrations of CO2 in the atmosphere are placing emphasis on the necessity for sequestering carbon (C) into soil organic matter (SOM). By studying the interior parts of soil aggregates, a better understanding of the incorporation and sequestration of plant residue materials within these aggregates could be obtained. The location of newly added plant residues within soil aggregates may also assist in the investigation of the impact of these newly added plant materials on soil aggregation. This study investigated two different techniques for determining the location of newly added plant residues within soil aggregates by using plant materials labelled with 14C and 13C isotopes incorporated into two different soil types, Black Earth (Pellic Vertisol) and Red Clay (Chromic Vertisol). Both autoradiography combined with scanning electron microprobe analysis (14C) and secondary ion mass spectrometry (SIMS) (13C) were successfully used for detecting the presence and location of the newly added plant residues fragments within soil aggregates of both soil types. The use of labelled plant materials is essential for the study of the location of newly added plant materials within soil aggregates, and this has proven to be a useful tool for studying the impact of residue additions on soil aggregate formation. Furthermore, these methods have been shown to be useful for determining the incorporation and sequestration of C materials within soil aggregates. The development of the 13C SIMS technique could alleviate the necessity for the use of the radioactive isotope 14C in soil studies.     

Appraisal of SIMS applicability to boron studies in plants

In the search for a new methodological approach applicable to the determination of the still poorly known primary role of boron in plant physiology, we have undertaken to appraise the potential of the SIMS method for the analytical imaging of the boron isotopes, (10)B and (11)B, at physiological concentrations in plants. With our own, CAMECA IMS4F SIMS ion analyser, and using O(2)(+) as primary ions for the detection of B(+) (plus (12)C(+) and (40)Ca(+)) secondary ions, we have been able to map quantitatively the two boron isotopes in control and boron-enriched plants, to evaluate boron concentrations at the level of individual cells and to determine boron isotopic ratios. This provides the opportunity to carry out the simultaneous labeling and imaging of boron, using enrichment with the stable isotopes, (10)B and (11)B. The method has also the potential for the simultaneous, quantitative detection of the boron isotopes and of the borate-binding sites in plant cells.     

The preparation of cryosections from plant tissue: An alternative method appropriate for secondary ion mass spectrometry studies of nutrient tracers and trace metals

A method involving cryostat sectioning (10 μm thickness) and freeze-drying is presented for the preparation of plant tissue for microanalytical studies. The method is well suited for semi-quantitative imaging by secondary ion mass spectrometry (SIMS) and offers significant advantages over bulk freeze-dried or freeze-substitution preparations. Segments of corn or soybean root (5 mm) are quench-frozen, embedded externally, sectioned in a cryostat (10 μm), pressed onto ultrapure Si and slowly freeze-dried. Images of these sections with secondary electron microscopy and SIMS indicated good morphological preservation. It was possible to section tissues of a wide developmental range, as well as roots varying sixfold in diameter. SIMS images are presented which demonstrate the ability to detect and localize nutrient tracers, such as Rb⁺, following brief exposures (10 min) to the intact plant. Likewise, a toxic metal (Al) was localized in root tissue after brief exposure (<1 day) of the intact plant root to micromolar external concentrations. Elemental redistribution during processing was minimal, as demonstrated most explicitly by the lack of movement of loosely bound Ca from the outer cell walls into the adjacent embedding material. Preservation of compositional differences between cellular content and cell wall was supported by a semi-quantitative treatment of SIMS images.     

Some proposed improvements in the scanning ion microprobe

Three improvements, which we have investigated, promise to lead to a scanning ion microprobe with a space resolution of about 15 nm. The improvements are: (1) use of a field-evaporation (EHD) ion source with liquid gallium to give a brightness exceeding 10¹⁰ A m^?2 sr¹ at 21 keV, (2) a high efficiency (10%) collecting system for secondary ions, and (3) enhancement of the secondary-ion yield by cesium deposition. With this instrument, tracing with stable isotopes would offer a number of advantages over autoradiography.     

Analysis of boron-10 in soft tissue by dynamic secondary ion mass spectrometry

We report here a preliminary study in which dynamic secondary ion mass spectrometry (SIMS) has provided images of boron‐10 (¹⁰B) in biological tissue as used in research into boron neutron capture therapy. Cultured tumour cells incubated in media containing known concentrations of a ¹⁰B‐containing compound, p‐boronophenylalanine (BPA), and intracranial tumour tissue from animals previously injected with BPA were analysed by an in‐house constructed SIMS. Investigations were conducted in positive secondary ion detection mode using a 25‐keV, 5‐nA gallium primary ion source. For calibration purposes, tissue standards were also analysed and their boron‐to‐carbon signal ratios correlated to bulk boron concentrations measured by inductively coupled plasma atomic emission spectroscopy (ICP‐AES). Ion maps of ¹⁰B, ¹²C, ²³Na and ³⁹K showing gross tissue and cell features were acquired. SIMS and ICP‐AES standard measurements were in good agreement. Tissue regions with high or low ¹⁰B concentrations were identified along with ¹⁰B hotspots in normal brain areas. Cultured cells revealed the intracellular localization of ¹⁰B. SIMS is capable of producing images showing the distribution of ¹⁰B at p.p.m. levels in cells and in normal and tumour‐bearing brain tissue.     

The use of secondary ion mass spectrometry coupled with image analysis to identify and locate chemical elements in soil minerals: The example of phosphorus

The mobility andbioavailability of elements in soils and sediments largely depends on their distribution on the diverse inorganic and organic constituents. This work addresses the example of phosphorus (P) associated to goethite and calcite, that is, to the major minerals involved in the retention of P in soils and sediments in calcareous environments. Synthetic goethite (FeOOH) and calcite (CaCO₃) were reacted with P prior to being analysed by dynamic secondary ion mass spectrometry (SIMS). Powdery samples were embedded in resin, cut in thin sections, and imaged with a Cameca IMS 4F ion microscope used in scanning mode with a primary ion beam of caesium that produced negatively charged secondary ions (?) (Cameca, Cedex, France). Carbon, O, P, and calcium (Ca) were directly imaged at m/z 12, 16, 31, and 40, respectively, while Fe was imaged via the polyatomic ion FeO^? ion at m/z 72. The SIMS data were treated by image analysis procedures. The visual comparison of images and the scatterplot method showed that P strongly interacted with goethite, probably following an adsorption process, and was thus evenly distributed at its surface. Conversely, P was not evenly distributed at the surface of calcite which rather suggests a precipitation process, and the scatterplot method confirmed a poor relationship between P and Ca. For the goethite‐calcite mixture, visual examination suggested that P occurred as clusters which were largely associated with calcite, whereas a statistical analysis of the various images showed that the distribution of P was largely related to that of goethite particles. This work confirms the potential contribution of iron oxides in the retention of P in calcareous environments and shows that coupling image analysis to sensitive analytical techniques such as SIMS is a powerful approach for providing quantitative information on the location of elements at low bulk concentrations.     

Anthropogenic Plutonium in Soils of the Ural Region

The Pu content and distribution in soils of certain territories of the Ural region located in zones of nuclear accidents and regular operation of power reactor facilities are studied. The maximum content of ^239;240Pu isotopes (up to 5000 Bc/m²) was revealed in soils of the central axis of the East Ural radioactive contamination at a distance of 30 km from the accident point. Regular gas-aerosol releases from the PO “Mayak” plants appreciably contribute to contamination of the neighboring territories. It is shown that almost 10¹² Bc of ^239;240Pu was released in the environment as a result of two nuclear accidents in 1957 and 1967. The nonuniform terrestrial plutonium contamination (from 200 to 5000 Bc/m²) is noted within the expected central axis of the Totsk radioactive trail. The contribution of the Beloyarsk nuclear power plant to the soil contamination in the fall-out zone of the aerosol emission plume is estimated on the basis of an analysis of the ²³⁸Pu/^239;240Pu isotope ratio.     

Quasicrystalline Materials

SIMS matrix effects (mass interferences, sputter yield variations and practical ion yield variations) were evaluated in freeze-fractured, freeze-dried cultured cells at the ～0.5 μm spatial resolution of the Cameca IMS-3f ion microscope. Cell lines studied include normal rat kidney (NRK), 3T3 mouse fibroblast, L6 rat myoblast, chinese hamster ovary (CHO) and rat kangaroo kidney (PtK2) cells. High mass resolution studies indicated that the secondary ion signals of H^—, C^—, O^—, Na⁺, Mg⁺, CN^—, P^—, S^—, Cl^—, K⁺ and Ca⁺ were free from major mass interferences. However, a large mass interference was observed for nitrogen at mass 14. No significant sputtering yield difference between the nuclear and cytoplasmic compartments of the cells studied was observed. The subcellular distributions of the major (H, C, N and O) and minor (P, S, K, Cl, Na, Mg and Ca) matrix elements were found to be largely homogeneous with the exception of Ca, which was observed mainly in the cell cytoplasm. Practical ion yield variations were compared by three different approaches: (i) by the use of cells doped with known electrolyte concentrations, (ii) by quantitative ion implantation, and (iii) by analysis of the same cell with both electron probe and ion microscope. Each approach indicated an absence of significant practical ion yield differences between the nuclear and cytoplasmic regions of these specimens. These observations indicate that secondary ion signals in this type of sample are not significantly affected by local matrix effect variations. Hence, qualitative imaging of such specimens provides a true representation of subcellular elemental distribtions. These observations should allow the development of quantitative ion imaging methodologies and enhance the applicability of ion microscopy to biomedical problems.     

Localization of chemical elements and isotopes in the leaf of soybean (Glycine max) by secondary ion mass spectrometry microscopy: critical choice of sample preparation procedure

Secondary ion mass spectrometry (SIMS) is one of the few microscopical methods that potentially can detect and in situ localize the various isotopes of virtually all elements. Recent work with SIMS has demonstrated the possibility of imaging the distribution of various elements in plant cell and tissues. However, in these studies, the elements were incorporated in cell macromolecules or associated with structural polymers, precipitated or immobilized in dry seeds. The localization of mineral ions is of particular significance for the physiology of higher plants owing to their quantitative importance and the impact of their cellular distribution on metabolic regulation. Here we analyse the possibility of mapping different elements (K, Ca, Mg, P, S, ¹⁵N and ¹⁴N) present as soluble and/or bound forms in highly vacuolated leaf cells. Cryoprocedures to prepare samples for SIMS detection are described and discussed. The quality of the results is assessed at each step of the sample preparation and analysis. Various methodologies are used, including photonic and electronic microscopies, and the agreement of the observed ion distribution with current knowledge of ion compartmentalization in plant cells. The K/Ca emission ratio is proposed as an index of the degree of preservation of the natural ion distribution to critically evaluate the results and identify where artefacts are likely to occur.     

Ion microscopy imaging of various pyrimidic nucleotides and analogs in the nuclei of cultured tumor cells

New applications for ion microscopy are presented. This method has been used primarily to detect mineral elements (K⁺, Na⁺, AL⁺). It also can be used to detect organic molecules containing halogen atoms and radioactive isotopes. ¹⁴C-nucleotides and halogenated pyrimidic nucleotides and analogs were employed in this study. The various images obtained were correlated with the mechanism of action of these compounds, thus opening new lines of research.     

Historical and contemporary stable isotope tracer approaches to studying mammalian protein metabolism

Over a century ago, Frederick Soddy provided the first evidence for the existence of isotopes; elements that occupy the same position in the periodic table are essentially chemically identical but differ in mass due to a different number of neutrons within the atomic nucleus. Allied to the discovery of isotopes was the development of some of the first forms of mass spectrometers, driven forward by the Nobel laureates JJ Thomson and FW Aston, enabling the accurate separation, identification, and quantification of the relative abundance of these isotopes. As a result, within a few years, the number of known isotopes both stable and radioactive had greatly increased and there are now over 300 stable or radioisotopes presently known. Unknown at the time, however, was the potential utility of these isotopes within biological disciplines, it was soon discovered that these stable isotopes, particularly those of carbon (¹³C), nitrogen (¹⁵N), oxygen (¹⁸O), and hydrogen (²H) could be chemically introduced into organic compounds, such as fatty acids, amino acids, and sugars, and used to “trace” the metabolic fate of these compounds within biological systems. From this important breakthrough, the age of the isotope tracer was born. Over the following 80 yrs, stable isotopes would become a vital tool in not only the biological sciences, but also areas as diverse as forensics, geology, and art. This progress has been almost exclusively driven through the development of new and innovative mass spectrometry equipment from IRMS to GC‐MS to LC‐MS, which has allowed for the accurate quantitation of isotopic abundance within samples of complex matrices. This historical review details the development of stable isotope tracers as metabolic tools, with particular reference to their use in monitoring protein metabolism, highlighting the unique array of tools that are now available for the investigation of protein metabolism in vivo at a whole body down to a single protein level. Importantly, it will detail how this development has been closely aligned to the technological development within the area of mass spectrometry. Without the dedicated development provided by these mass spectrometrists over the past century, the use of stable isotope tracers within the field of protein metabolism would not be as widely applied as it is today, this relationship will no doubt continue to flourish in the future and stable isotope tracers will maintain their importance as a tool within the biological sciences for many years to come. © 2016 The Authors. Mass Spectrometry Reviews Published by Wiley Periodicals, Inc. Mass Spec Rev     

Chemical and morphological changes in human dentin after Er:YAGlaser irradiation: EDS and SEM analysis

Sixty samples of human dentin were divided into six groups (n = 10) and were irradiated with Er:YAG laser at 100 mJ–19.9 J/cm², 150 mJ–29.8 J/cm², 100 mJ–35.3 J/cm², 150 mJ–53.0 J/cm², 200 mJ–70.7 J/cm², and 250 mJ–88.5 J/cm², respectively, at 7 Hz under a water spray. The atomic percentages of carbon, oxygen, magnesium, calcium, and phosphorus and the Ca‐to‐P molar ratio on the dentin were determined by energy dispersive X‐ray spectroscopy. The morphological changes were observed using scanning electron microscopy. A paired t‐test was used in statistical analysis before and after irradiation, and a one‐way ANOVA was performed (P ≤ 0.05). The atomic percent of C tended to decrease in all of the groups after irradiation with statistically significant differences, O and Mg increased with significant differences in all of the groups, and the Ca‐to‐P molar ratio increased in groups IV, V, and VI, with statistically significant differences between groups II and VI. All the irradiated samples showed morphological changes. Major changes in the chemical composition of dentin were observed in trace elements. A significant increase in the Ca‐to‐P ratio was observed in the higher energy density groups. Morphological changes included loss of smear layer with exposed dentinal tubules. The changes produced by the different energy densities employed could have clinical implications, additional studies are required to clarify them. Microsc. Res. Tech. 78:1019–1025, 2015. © 2015 Wiley Periodicals, Inc.     

Subcellular localization of boron in cultured melanoma cells by electron energy-loss spectroscopy of freeze-dried cryosections

Boron neutron capture therapy (BNCT) is based on the ability of the non‐radioactive isotope ¹⁰B to capture thermal neutrons and to disintegrate instantaneously. This reaction opens a way to selectively destroy tumour cells after specific uptake of ¹⁰B. In this paper, a method based on electron energy‐loss spectroscopy is presented for detecting and quantifying boron in freeze‐dried cryosections of human melanoma cells. A practical detection limit of around 6 mmol kg^?1 in 0.1‐µm² areas is estimated using specimens prepared from standard boron solutions. Preliminary results of boron mapping in the spectrum‐imaging acquisition mode reveal boron penetration and probably spot‐like accumulation within melanoma cells when exposed to culture medium containing sodium borocaptate.     

A Method for Increasing the Sensitivity of Mass-Spectrometric Measurements

A method for increasing the sensitivity of mass-spectrometric measurements in the analysis of small amounts of gases, which are insufficient for organizing a stationary flow through an ion source, is considered. Measurements are carried out upon a single injection of a portion of a gas. The use of this method for increasing the sensitivity in isotope helium analyses on MI9303 magnetic-resonance mass spectrometers allowed the sensitivity of the spectrometer to be increased to ~10⁵ atoms in a sample, i.e., it allowed measurements for a helium volume in a sample of up to ~10^?14 cm³. The experience of carrying out isotope analyses in samples of inert gases of various origins using different methods for evacuating chambers of mass analyzers was generalized.     

Morphological and elemental integrity of freeze-fractured,freeze-dried cultured cells during ion microscopic analysis

The effects of progressive ion beam bombardment on freeze-fractured, freeze-dried cultured cells during ion microscopic (SIMS) analysis were studied with scanning electron microscopy (SEM) and ion microscopy. The freeze-fracture, freeze-dry sample preparation method was generally found to preserve cell morphology to a level far exceeding the spatial resolution of the ion microscope, with splitting at the nuclear envelope being the most commonly observed artefact. SEM monitoring of surface topography of an NRK-49F fibroblast after various ion bombardment doses showed relatively uniform erosion of cellular material, with some apparent selective retention of small cytoplasmic granules. Prolonged bombardment produced no detectable lateral elemental translocation. ⁴¹K+/²⁴Mg+ signal ratios from Swiss 3T3 fibroblasts and RBL rat basophilic leukaemia cells were shown to vary generally by less than 10% during the course of extended ion bombardment. GM0415 human skin fibroblasts containing engorged lysosomes characteristic of Hurler's Syndrome were used to evaluate the effects of ion bombardment during a typical analysis session, where ion images of ³⁹K+, ²³Na+, ⁴⁰Ca+ and ²⁴Mg+ are sequentially recorded. This cell line was chosen as a worst-case system, because these cells are often thinly spread and possess extreme surface topography. Thin cell edges were shown sometimes to sputter away during analysis, giving misleadingly low ion signals from these regions in some ²⁴Mg+ micrographs. Various nonuniform sputtering phenomena occurring in the submicrometre spatial domain had little or no measurable impact on local intensities in ion micrographs, indicating that freeze-dried, freeze-fractured cells are sampled in a sufficiently uniform fashion that quantitative ion microscopic evaluations of intracellular elemental levels in the general cytoplasmic or nuclear regions are feasible.     

Production of <Superscript>10</Superscript>Be isotope from boron carbide irradiated with fast neutrons

A method for producing ¹⁰Be isotope from spent rods of the emergency protection and compensation systems of a fast neutron reactor is described. This isotope has found application in targets and radioactive beams for experiments at modern heavy-ion accelerators. The mass of ¹⁰Be obtained using this technique is 2.7 mg, which is five times the mass of this isotope produced throughout the world to date. A method for determining the ¹⁰Be concentration in the source material and intermediate fractions of its processing has been developed. This method is based on analysis of γ and β spectra of samples, measured by HPGe and Si(Li) detectors. A prototype of the ¹⁰Be oxide target has been manufactured. The parameters of the target prototype have been determined, and its vacuum testing has been performed.     

Calcium hypochlorite as a dentin deproteinization agent: Microleakage,scanning electron microscopy and elemental analysis

Purpose: This study investigated the influence of collagen removal with calcium hypochlorite on the surface morphology of acid‐etched dentin and on the microleakage of composite restorations. In addition, the elemental composition (EC) of dentin after removal of the collagen fibrils was analyzed. Materials and Methods: Forty third molars received two cavities and were divided into four groups according to dentin treatment: CTRL—no pre‐treatment; Na10—10% NaOCl for 30 s; Ca10—10% CaOCl for 30 s, and Ca15—15% CaOCl for 30 s. The cavities were filled using an acetone‐based adhesive system and a resin composite; they were then subjected to thermal cycling for 5,000 cycles, immersed in methylene blue for 4 h and sectioned into 1‐mm thick slabs. Two examiners evaluated two slices per tooth using a stereomicroscope and assigned the degree of infiltration (scores 0–3). The data were analyzed using the Kruskal–Wallis (α = 0.05). Four teeth received surface treatment according to the groups and were submitted to SEM and EDS to carry at the EC. Results: There was no significant difference between the experimental groups (P = 0.533). CaOCl alters the morphology and surface composition of the dentin, resulting in an increase in the amount of calcium in the interface. Conclusions: When used prior to an acetone‐based adhesive system, CaOCl did not produce any differences in microleakage when compared to the CTRL group or to the Na10 group. Microsc. Res. Tech. 78:676–681, 2015. © 2015 Wiley Periodicals, Inc.