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.     

Quantitative secondary ion mass spectrometry imaging of self-assembled monolayer films for electron beam dose mapping in the environmental scanning electron microscope

Fluorinated alkanethiol self-assembled monolayers (SAM) films immobilized on gold substrates have been used as electron-sensitive resists to map quantitatively the spatial distribution of the primary electronbeam scattering in an environmental scanning electron microscope (ESEM). In this procedure, a series of electron dose standards are prepared by exposing a SAM film to electron bombardment in well-defined regions at different levels of electron dose. Microbeam secondary ion mass spectrometry (SIMS) using Cs⁺ bombardment is then used to image the F^- secondary ion signal from these areas. From the reduction in F^- intensity as a function of increasing electron dose, a calibration curve is generated that allows conversion of secondary ion signal to electron dose on a pixel-by-pixel basis. Using this calibration, electron dose images can be prepared that quantitatively map the electron scattering distribution in the ESEM with micrometer spatial resolution. The SIMS imaging technique may also be used to explore other aspects of electron-surface interactions in the ESEM.     

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.     

Evaluation of fracture planes and cell morphology in complementary fractures of cultured cells in the frozen-hydrated state by field-emission secondary electron microscopy: feasibility for ion localization and fluorescence imaging studies

We have employed field-emission secondary electron microscopy (FESEM) for morphological evaluation of freeze-fractured frozen-hydrated renal epithelial LLC-PK₁ cells prepared with our simple cryogenic sandwich-fracture method that does not require any high-vacuum freeze-fracture instrumentation (Chandra et al. (1986) J. Microsc. 144 , 15–37). The cells fractured on the substrate side of the sandwich were matched one-to-one with their corresponding complementary fractured faces on the other side of the sandwich. The FESEM analysis of the frozen-hydrated cells revealed three types of fracture: (i) apical membrane fracture that produces groups of cells together on the substrate fractured at the ectoplasmic face of the plasma membrane; (ii) basal membrane fracture that produces basal plasma membrane-halves on the substrate; and (iii) cross-fracture that passes randomly through the cells. The ectoplasmic face (E-face) and protoplasmic face (P-face) of the membrane were recognized based on the density of intramembranous particles. Feasibility of fractured cells was shown for intracellular ion localization with ion microscopy, and fluorescence imaging with laser scanning confocal microscopy. Ion microscopy imaging of freeze-dried cells fractured at the apical membrane revealed well-preserved intracellular ionic composition of even the most diffusible ions (total concentrations of K⁺, Na⁺ and Ca⁺). Structurally damaged cells revealed lower K⁺ and higher Na⁺ and Ca⁺ contents than in well-preserved cells. Frozen-freeze-dried cells also allowed imaging of fluorescently labelled mitochondria with a laser scanning confocal microscope. Since these cells are prepared without washing away the nutrient medium or using any chemical pretreatment to affect their native chemical and structural makeup, the characterization of fracture faces introduces ideal sample types for chemical and morphological studies with ion and electron microscopes and other techniques such as laser scanning confocal microscopy, atomic force microscopy and near-field scanning optical microscopy.     

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Analysis of reactive oxygen species,Ca2+, and Hsp70 in the gill and mantle of clams Ruditapes philippinarum exposed in cadmium

In this study, the probes 2′,7′‐dichlorofluorescein diacetate (H₂DCF‐DA) and Fluo‐3 AM were used to investigate the instantaneous change of reactive oxygen species (ROS) and Ca²⁺ in the gill and mantle of clams Ruditapes philippinarum exposed in 0.05 mg L^?1 Cd²⁺ with the laser‐scanning confocal microscopy. The results indicated that Ca²⁺ level was declined in the gill and slightly increased in the mantle. The level of ROS was declined in the gill, while the oscillation of ROS level was observed in the mantle. These data revealed that Ca²⁺ could stimulate mitochondrial activity and enhance the respiratory chain in the gill and mantle. In addition, the expression of Hsp70 was increased in the gill and mantle of clams exposed in 0.05 mg L^?1 Cd²⁺. The change of Ca²⁺ and ROS level affected the expression of Hsp70 in the gill and mantle. An appropriate method was established to analyze the effects of Cd²⁺ on ROS, Ca²⁺, and Hsp70 in the gill and mantle of clams with confocal microscopy. Both confocal microscopy and chemical fluorescent are valuable tools for measurement of time‐dependent intracellular ROS and Ca²⁺ signals. Microsc. Res. Tech. 76:1297–1303, 2013. © 2013 Wiley Periodicals, Inc.     

Porous TiO2 nanowire microsphere constructed by spray drying and its electrochemical lithium storage properties

Porous TiO₂ nanowire microspheres with greatly decreasing agglomeration were successfully prepared by spray drying of hydrothermal reaction suspension, followed by calcination at 350°C. The as‐obtained nanowire microspheres with TiO₂‐B structure reach an initial discharge capacity 210 mAh g^?1 with an irreversible capacity 25 mAh g^?1 at a current density of 20 mA g^?1. For the 450°C‐calcined one with anatase TiO₂ crystal structure, the initial discharge capacity is 245 mAh g^?1 but with a much higher irreversible capacity of 80 mAh g^?1. The hierarchical porous structure in the 350°C‐calcined TiO₂ nanowire microspheres collapsed at 450°C, annihilating the main benefit of nanostructuring. Microsc. Res. Tech. 77:170–175, 2014. © 2013 Wiley Periodicals, Inc.     

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.     

Evaluation of the effective cross‐sections for recombination and trapping in the case of pure spinel

In this paper, we have investigated the evolution of the secondary electron emission in the case of pure spinel during electron irradiation, achieved in a scanning electron microscope at room temperature, which is derived from the measurement of the induced and the secondary electron currents. It was observed from the experimental results, that there are two regimes during the charging process: a plateau followed by a linear variation, which are better identified by plotting the logarithm of the secondary electron emission yield lnσ as function of the total surface density of trapped charges in the material Q_T. For positive charging, E₀ = 1.1 and 5 keV, the slope of the linear part, whose value is of about 10^?10 cm² charge^?1, is independent of the primary electron energy. It is interpreted as a microscopic cross section for electron–hole recombination. For negative charging of pure spinel, E₀ = 15 and 30 keV, the slope is associated with an electron trapping cross section close to 10^?14 cm² charge^?1, which can be assigned to the microscopic cross section for electron trapping. This trapping cross section is four orders of magnitude lower than the recombination one.     

Rapid and Sensitive Determination of Nucleic Acids by Enhanced Resonance Light Scattering Spectroscopy of Tetraphenyl Porphyrin Cobalt(II)Chloride

Abstract

A novel probe, tetraphenyl porphyrin Cobalt(II)chloride (CoTPPCl), was first developed for the determination of nucleic acids at a nanogram level by a resonance light scattering (RLS) technique. Under optimum conditions, the weak RLS signal of CoTPPCl was enhanced greatly by nucleic acids at 444.0 nm; the enhanced RLS intensity is proportional to the concentration of nucleic acids in the range of 0.05–3.5 mg L^?1 for calf thymus DNA and 0.03–4.2 mg L^?1 for fish sperm DNA. The detection limits (3δ) are 3.5 ng mL^?1 for calf thymus DNA and 4.5 ng mL^?1 for fish sperm DNA, respectively. The results show that determination of nucleic acids with CoTPPCl as a probe is much more sensitive than with α, β, γ, δ‐ tetrakis[4‐(trimethylammoniumyl)phenyl]porphine (TAPP). Synthetic samples and plasmid DNA extracted from K‐12‐HB101 colt were determined with satisfactory results.     

FT-ICR mass spectrometry: Superconducting magnet,external ion source,ion–molecule reactions,and ion–ion traps

The world of Fourier-transform ion cyclotron resonance (FT-ICR) mass spectrometry has witnessed, especially in the last 30 years significant advances in many fields of science, such as electronics, magnets, new ICR cell designs, developed ICR event sequences, modern external ionization sources, and linear ion beam guides, as well as modern vacuum technology. In this review, a brief account is given focusing especially on the studies performed in Wanczek's group and ICR research laboratory at the University of Bremen. An FT-ICR mass spectrometer has been developed with a high magnetic field superconducting magnet, operating at 4.7 T. At this magnetic field, a trapping time of 13.5 h was obtained with 30% efficiency. For the tetrachloromethane molecular ion, m/z 166, a mass-resolving power m/Δm = 1.5 × 10⁶ was measured at a pressure of 2 × 10⁻⁸ Torr. The transition from magnet sweep to frequency sweep and the application of Fourier-transform has greatly enhanced the ICR technology. External ion sources were invented and differential pumping schemes were developed for enabling ultrahigh vacuum condition for ICR detection, while guiding ions at relatively higher pressures, during their flight to the ICR cell. With the external ion source, a time-of-flight ICR tandem instrument is built. A method to measure the ion flight time and to trap the ions in the ICR cell is described. Many ICR cell characteristics such as z-axis ion ejection and coupling of radial and axial ion motions in a superposed homogeneous magnetic and inhomogeneous trapping electric field were extensively studied. Gas-phase ion–molecule reactions of several reactive inorganic compounds with a focus on phosphorous and sulfur as well as silicon chemistry were also studied in great detail. The gas-phase ion chemistry of several trifluoromethyl-reagents such as trifluoromethyltrimethylsilane and tris(trifluoromethyl)phosphine were also investigated in ICR. Dual polarities multisegmented ICR cells were invented and deeply characterized. Sophisticated ICR pulse event programs were developed to enable long-range ion–ion interactions between simultaneously trapped positive and negative ions.     

Scanning ion microprobe analysis of composite materials

Applications of scanning ion imaging with high lateral resolution in the microchemical investigation of metal – and ceramic-matrix composites are described. The technique, which combines a scanning ion microprobe with secondary ion mass spectrometry (SIMS), is ideally suited to the study of complex, multicomponent composite structures. Most elements can be detected with good sensitivity, enabling the determination of spatial distributions for major and minor elements. Analytical images obtained with this technique reveal unprecedented chemical information about interfacial segregation and interdiffusion phenomena. As examples, the characterization of both ceramic–matrix (Al borate–SiC) and metal–matrix (Ni alloy–Al₂O₃) composite materials is described.     

Radiation damage to lyophilized mineral solutions during electron probe analysis: quantitative study of chlorine loss as a function of beam current-density and sample mass-thickness

The quantitative effects of beam current-density and sample mass-thickness on the loss of chlorine which occurs from lyophilized solutes of micro-droplets of mineral salt solutions irradiated in an electron probe analyser were studied. Results are reported for chlorine loss from lyophilized deposits with mass-thickness varying between 5 and 50 mg mm^?2 for NaCl salts and 5 and 80 mg mm^?2 for KCl salts. Electron accelerating voltage was kept constant at 15 kV. The range of beam current-density (I/S, current/sample surface area) was from 0.1 to 1.5 A mm^?2. Samples were irradiated for 1200 s. The results show that under some conditions there is a period of stable chlorine signal before chlorine loss occurs. This is observed between 0.1 and 1 A mm^?2, for a period which can last several hundred seconds depending on beam current-density and sample mass-thickness. For each value of I/S, however, no stable chlorine signal can be observed for samples whose mass-thickness exceeds a value negatively correlated with I/S. The curves of decrease of characteristic chlorine X-ray signal (expressed as per cent of count rate in the initial counting interval) versus irradiation time can be fitted by the sum of two exponentials with half lives T₁ and T₂. In NaCl, T₁ and T₂ values are highly correlated with I/S but not with mass-thickness. In KCl, T₁ is correlated only with mass-thickness and T₂ only with I/S. Mixing plasma with mineral solutions prevents chlorine loss.     

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.     

Embossed‐carving processing of cytoskeletons of cultured cells by using focused ion beam technology

The focused ion beam (FIB) technology has drawn considerable attention in diverse research fields. FIB can be used to mill samples at the nanometer scale by using an ion beam derived from electrically charged liquid gallium (Ga). This powerful technology with accuracy at the nanometer scale is now being applied to life science research. In this study, we show the potential of FIB as a new tool to investigate the internal structures of cells. We sputtered Ga⁺ onto the surface or the cross section of animal cells to emboss the internal structures of the cell. Ga⁺ sputtering can erode the cell surface or the cross section and thus emboss the cytoskeletons quasi‐3 dimensionally. We also identified the embossed structures by comparing them with fluorescent images obtained via confocal laser microscopy because the secondary ion micrographs did not directly provide qualitative information directly. Furthermore, we considered artifacts during the FIB cross sectioning of cells and propose a way to prevent undesirable artifacts. We demonstrate the usefulness of FIB to observe the internal structures of cells. Microsc. Res. Tech. 76:290–295, 2013. © 2013 Wiley Periodicals, Inc.     

Fluorescence in situ hybridization on human metaphase chromosomes detected by near-field scanning optical microscopy

Fluorescence in situ hybridization on human metaphase chromosomes is detected by near-field scanning optical microscopy. This combination of cytochemical and scanning probe techniques enables the localization and identification of several fluorescently labelled genomic DNA fragments on a single chromosome with an unprecedented resolution. Three nucleic acid probes are used: pUC1. 77. p1–79 and the plasmid probe α-spectrin. The hybridization signals are very well resolved in the near-field fluorescence images, while the exact location of the probes can be correlated accurately with the chromosome topography as afforded by the shear force image.     

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.     

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.     

DESIGN OF A SENSITIVE MEMBRANE OPTODE FOR IRON DETERMINATION

An optical chemical sensor has been developed for the sensitive determination of Fe (III) ions by spectrophotometry. The optical membrane was constructed by immobilization of methyltrioctylammonium chloride on triacetylcellulose polymer. The exchange of thiocyanate as counter ion in the membrane sensitized this film to Fe (III). The sensing membrane is capable of determining Fe(III) reversibly over a dynamic range of 7.11 × 10^?7?8.88 × 10^?5 mol L^?1 with a limit of detection of 6.02 × 10^?7 mol L^?1 and a response time of 5 min. This optode can easily be regenerated by 0.1 mol L^?1 of sodium fluoride solution. The relative standard deviation for eight replicate measurements of 7.11 × 10^?6 and 5.33 × 10^?5 mol L^?1 of Fe (III) was 4.2 and 3.7%, respectively. The sensor was successfully applied for the determination of iron in tablet and water samples.     

Influence of ion bombardment on tribological properties of UHMWPE

Medical grade UHMWPE-s: GUR 1020 and 1050, were subjected to ion bombardment with H₂⁺, He⁺, Ar⁺ or Ag⁺ of different energy and various doses. The work presents changes to micromechanical profiles and surface morphology (AFM, SEM) of polyethylenes due to the treatment. Mechanisms behind the modification have been proposed owing to structural and chemical analyses by s-SIMS, FTIR-IRS, confocal Raman microscopy and “nuclear depth profiling”. Studies revealed that hardness of the surface layer increases due to radiolysis, probably leading to cross-polymerization of polyethylene. The depth of modification is limited to the penetration of ion beam, being dependent on a dose and a kind of ions applied. The treatment results additionally in oxidation, which together with a development of the surface geometry is responsible for its hydrofilization and, in some cases bacteriostatic character. Friction and wear of polyethylenes under a high load, adequate to extreme conditions of exploitation of hip joints, can be reduced even by three times, due to a proper ion beam treatment.