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.     

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.     

High spatial resolution secondary ion imaging and secondary ion mass spectrometry of aluminium-lithium alloys

Samples of aluminium-lithium alloys have been observed by scanning ion microscopy and analysed by secondary ion mass spectrometry. The high signal-to-noise ratio of the positive secondary lithium ion opens up the possibility of both high resolution imaging and microanalysis of lithium distributions in aluminium and other materials. Some of the problems encountered due to sample preparation are discussed and ion images of both the artefacts and the true lithium distribution are shown.     

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.     

Comparison in spatial spreads of secondary electron information between scanning ion and scanning electron microscopy

Monte Carlo simulations have been carried out to compare the spatial spreads of secondary electron (SE) information in scanning ion microscopy (SIM) with scanning electron microscopy (SEM). Under Ga ion impacts, the SEs are excited by three kinds of collision-partners, that is, projectile ion, recoiled target atom, and target electron. The latter two partners dominantly contribute to the total SE yield gamma for the materials of low atomic number Z2. For the materials of high Z2, on the other hand, the projectile ions dominantly contribute to gamma. These Z2 dependencies generally cause the gamma yield to decrease with an increasing Z2, in contrast with the SE yield delta under electron impacts. Most of the SEs are produced in the surface layer of about 5lambda in depth (lambda: the mean free path of SEs), as they are independent of the incident probe. Under 30 keV Ga ion impacts, the spatial spread of SE information is roughly as small as 10 nm, decreasing with an increasing Z2. Under 10 keV electron impacts, the SEI excited by the primary electrons has a small spatial spread of about 5lambda, but the SEII excited by the backscattered electrons has a large one of several 10 to several 100 nanometers, decreasing with an increasing Z2. The main cause of a small spread of SE information at ion impact is the short ranges of the projectile ions returning to the surface to escape as backscattered ions, the recoiled target atoms, and the target electrons in collision cascade. The 30 keV Ga-SIM imaging is better than the 10 keV SEM imaging in spatial resolution for the structure/material measurements. Here, zero-size probes are assumed.     

Nucleotide and protein distribution in BrdU-labelled polytene chromosomes revealed by ion probe mass spectrometry

Detailed chemical maps of BrdU-labelled polytene chromosomes of Drosophila melanogaster, obtained by imaging secondary ion mass spectrometry, reveal separately the distribution of DNA and proteins in the chromosomes. The thymidine-analogue BrdU within the chromosomal DNA is localized by detecting the Br^? secondary ion signal, while both nucleic acid and protein content are mapped through the abundantly emitted CN^? signal. This novel approach supercedes, and helps explain the origin of, the banding patterns that are observed by conventional staining techniques. The high spatial resolution and chemical and isotopic sensitivity of this technique should enhance the localization of specific genes by in situ hybridization in mitotic chromosomes.     

Elemental and molecular imaging of human hair using secondary ion mass spectrometry.

Secondary ion mass spectrometry (SIMS) is used to image the spatial distribution of elemental and molecular species on the surface and in cross sections of doped human hair using a magnetic sector SIMS instrument operated as an ion microprobe. Analysis of electrically insulating, non-planar hair samples requires one of two different methods of charge compensation to be used depending on the polarity of the sputtered secondary ions. For detection of positive secondary ions, the hair is imaged using a approximately 0.5 micron diameter, 19.5 keV impact energy, O- microbeam with no auxiliary electron bombardment. For detection of negative secondary ions, a approximately 0.2 micron diameter, 14.5 keV impact energy Cs+ microbeam is used in conjunction with normal incidence, low-energy electron bombardment. Both of these methods allow submicrometer spatial resolution elemental and molecular secondary ion images to be obtained from hair samples without metallic coating of the sample surface prior to analysis. Several examples are presented that reflect potential application areas for these analytical methods.     

Tools and procedures for quantitative microbeam isotope ratio imaging by secondary ion mass spectrometry

In this work we demonstrate the use of secondary ion mass spectrometry (SIMS) combined with the Lispix image processing program (Bright 1995) to generate quantitative isotope ratio images from a test sample of a calcium-aluminum rich inclusion from the Allende meteorite that is known to contain discrete mineral grains with perturbed Mg isotopic ratios. Using 19.5 keV impact O- primary ion bombardment and detection of positive secondary ions, microbeam imaging SIMS has allowed us to identify, from the isotope ratio images, enrichments in the 26Mg/24Mg isotope ratio of approximately 5-15% in selected mineral grains. Using custom image processing software, each isotopic ratio image is corrected on an individual pixel basis for a number of factors including detector dead-time, mass bias effects, and isobaric interferences. We have developed procedures for correlating the isotopic images with polarized optical microscopy so that targeted mineral grains could be identified for further SIMS analysis. Finally, additional image processing tools have been developed to allow for pixel-by-pixel evaluation of the influence of detector dead-time and count rate errors on the isotopic ratio images and for correlation of the isotopic images with elemental distribution maps.     

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.     

An ion microprobe study of the microchemistry of Ni-base superalloy-Al2O3 metal-matrix composites

The chemical microstructure of Ni-base superalloy/Al₂O₃ metal-matrix composites (MMCs) has been studied by scanning ion microprobe microanalysis, using the secondary ion mass spectrometry (SIMS) technique. The MMCs were fabricated using the transient-liquid-phase bonding (TLP) process, with B-doped superalloy powder as an interlayer. Boron was found to diffuse rapidly throughout the matrix to form boride phases, mostly at the grain boundaries in the matrix. These borides contain excess Cr (also Mo, Si, W) in comparison with the Ni alloy-matrix, but are depleted in Ni (also in Al and Co). Carbides form at the grain boundaries as thin platelets and inside the grains as fine particles. Chemical reaction occurs between the sapphire fibre and the matrix; formation of NiAl₂O₄ spinel at the interface is suggested. This interface reaction layer is friable and parts of it peel off during consolidation to become inclusions in the matrix near the fibre/matrix interface.     

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.     

Development of environmental scanning electron microscopy electron beam profile imaging with self-assembled monolayers and secondary ion mass spectroscopy

A method for demonstrating the scattering of the primary electron beam in the presence of a gas has been developed. A self-assembled decanethiol monolayer is damaged by primary beam electrons. The damaged portion of the mono-layer is exchanged with another thiol-containing molecule by immersion in solution. The resulting film is imaged using a secondary ion mass spectrometer. Three-dimensional reconstruction of the data yields a representation of scattered electrons in the gaseous environment of the environmental scanning electron microscope.     

Measurement technique for the incident electron current in secondary electron detectors and its application in scanning electron microscopes.

A measurement technique for incident electron current in secondary electron (SE) detectors, especially the Everhart-Thornley (ET) detector, based on signal-to-noise ratio (SNR), which uses the histogram of a digital scanning electron microscope (SEM) image, is described. In this technique, primary electrons are directly incident on the ET detector. This technique for measuring the correlation between incident electron current and SNR is applicable to the other SE detectors. This correlation was applied to estimate the efficiency of the ET detector itself, to evaluate SEM image quality, and to measure the geometric SE collection efficiency and the SE yield. It was found that the geometric SE collection efficiency at each of the upper and lower detectors of a Hitachi S-4500 SEM was greater than 0.78 at all working distances.     

High‐quality imaging in environmental scanning electron microscopy – optimizing the pressure limiting system and the secondary electron detection of a commercially available ESEM

In environmental scanning electron microscopy applications in the kPa regime are of increasing interest for the investigation of wet and biological samples, because neither sample preparation nor extensive cooling are necessary. Unfortunately, the applications are limited by poor image quality. In this work the image quality at high pressures of a FEI Quanta 600 (field emission gun) and a FEI Quanta 200 (thermionic gun) is greatly improved by optimizing the pressure limiting system and the secondary electron (SE) detection system. The scattering of the primary electron beam strongly increases with pressure and thus the image quality vanishes. The key to high‐image quality at high pressures is to reduce scattering as far as possible while maintaining ideal operation conditions for the SE‐detector. The amount of scattering is reduced by reducing both the additional stagnation gas thickness (aSGT) and the environmental distance (ED). A new aperture holder is presented that significantly reduces the aSGT while maintaining the same field‐of‐view (FOV) as the original design. With this aperture holder it is also possible to make the aSGT even smaller at the expense of a smaller FOV. A new blade‐shaped SE‐detector is presented yielding better image quality than usual flat SE‐detectors. The electrode of the new SE detector is positioned on the sample table, which allows the SE‐detector to operate at ideal conditions regardless of pressure and ED.     

傅立叶变换离子回旋共振质谱及其在药学领域的应用

傅立叶变换离子回旋共振质谱可以实现超高分辨率和质量精确度,在药物结构鉴定和元素分析中展现出强大的优势,并发展成为分析复杂混合物的强有力工具。本文简述了傅立叶变换离子回旋共振质谱的分析特性以及在药学领域的应用。     

Quantitative microlocalization of diffusible ions in normal and galactose cataractous rat lens by secondary ion mass spectrometry

Secondary ion mass spectrometry (SIMS) is a surface analytical technique with high sensitivity for elemental detection and microlocalization capabilities within the micrometre range. Quantitative analysis of epoxy resins and gelatin have been reported (Burns-Bellhorn & File, 1979).     

Accuracy and precision of quantitative energy-dispersive x-ray spectrometry in the environmental scanning electron microscope

The accuracy and precision of quantitative energy-dispersive x-ray spectrometry in the environmental scanning electron microscope have been estimated using a series of copper / gold alloys of known composition. The mean values (five to six replicate experiments) had relative errors within +/- 5%, and most were within +/- 3.5%. All relative standard deviations were < 5% and most were < 3%. Since the standard specimens were large (approximately 500 microm) in diameter, electron scattering in the 2 torr of water vapor above the specimen did not affect the results. This level of accuracy and precision was possible only by using a novel specimen surface charge neutralization scheme.     

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.