Semiautomated analytical image correlation

Machine vision refers to computer programs consisting of a collection of pattern recognition and digital image processing algorithms (Fabel, G. Motion Control 2000, 53-54). A version of machine vision has been applied to correlating digital images generated by optical microscopy and secondary ion mass spectrometry (SIMS). By suitable application of image processing algorithms, semiautomated correlation between optical and secondary ion images is possible. For correlation of minor constituents evident in secondary ion images but invisible in optical images, correlation is performed by reference to the relative position of minor to major constituents. Precise coordinates of features apparent in one analytical image can be translated into the corresponding coordinates of an analytical image obtained by a different method. In principle, this capability yields a semiautomated system to combine complementary features of disparate imaging methods, such as secondary ion and optical microscopy.     

Poisson and multinomial mixture models for multivariate SIMS image segmentation

Multivariate statistical methods have been advocated for analysis of spectral images, such as those obtained with imaging time-of-flight secondary ion mass spectrometry (TOF-SIMS). TOF-SIMS images using total secondary ion counts or secondary ion counts at individual masses often fail to reveal all salient chemical patterns on the surface. Multivariate methods simultaneously analyze peak intensities at all masses. We propose multivariate methods based on Poisson and multinomial mixture models to segment SIMS images into chemically homogeneous regions. The Poisson mixture model is derived from the assumption that secondary ion counts at any mass in a chemically homogeneous region vary according to the Poisson distribution. The multinomial model is derived as a standardized Poisson mixture model, which is analogous to standardizing the data by dividing by total secondary ion counts. The methods are adapted for contextual image segmentation, allowing for spatial correlation of neighboring pixels. The methods are applied to 52 mass units of a SIMS image with known chemical components. The spectral profile and relative prevalence for each chemical phase are obtained from estimates of model parameters.     

Imaging of membrane lipids in single cells by imprint-imaging time-of-flight secondary ion mass spectrometry

A new method for identification and localization of organic molecules in biological samples is described. The method involves making an imprint of a biological sample on a silver (Ag) surface and subsequent analysis of the imprint by imaging time-of-flight secondary ion mass spectrometry (TOF-SIMS). Using this method, detection of unfragmented, Ag cationized molecules at a spatial resolution of <0.5 microm is possible. We have used the method to study the spatial distribution of phosphatidylcholine and cholesterol in blood cells adhering to a glass surface. The TOF-SIMS images show that cholesterol is preferentially located in the plasma membrane, whereas the phosphocholine shows highest concentration in the nuclear membrane. Scanning electron microscopy and fluorescence microscopy images show that the amount of transferred material during the imprinting process can be controlled by varying the imprinting pressure and pretreatment of the cell substrate prior to imprinting.     

Investigation into the nature of metal threads in a Renaissance tapestry and the cleaning of tarnished silver by UV/Ozone (UVO) treatment

Precious metal threads from a XVIth century tapestry were visually classified using optical microscopy and scanning electron microscopy (SEM). The nature of the surface metals and the nature of the corrosion products were studied by Energy dispersive X-ray analysis (EDX) and dynamic secondary ion mass spectrometry (SIMS).The cleaning of metal artefacts by UV/Ozone treatment was investigated and the surface properties before and after UVO treatment were determined by SIMS.     

Study of Zn diffusion in n-type GaSb by cathodoluminescence and scanning tunneling spectroscopy

We report here the studies carried out in zinc diffused n-type GaSb by cathodoluminescence (CL) microscopy and by scanning tunneling spectroscopy. Samples with different diffusion profiles measured by secondary ion mass spectrometry (SIMS) were obtained. CL plan-view observations show high homogeneity in the diffused layers. Cross-sectional measurements of the Zn diffused layers were performed by current imaging tunneling spectroscopy (CITS). The junction border was revealed clearly in the CITS images and conductance spectra recorded at differents points of the layers provided information on the local surface band gaps and the conductive behaviour. The results were related to the diffusion profiles and were found to agree with diffusion models suggested previously.     

Molecular depth profiling by wedged crater beveling

Time-of-flight secondary ion mass spectrometry and atomic force microscopy are employed to characterize a wedge-shaped crater eroded by a 40-keV C(60)(+) cluster ion beam on an organic film of Irganox 1010 doped with Irganox 3114 delta layers. From an examination of the resulting surface, the information about depth resolution, topography, and erosion rate can be obtained as a function of crater depth for every depth in a single experiment. It is shown that when measurements are performed at liquid nitrogen temperature, a constant erosion rate and reduced bombardment induced surface roughness is observed. At room temperature, however, the erosion rate drops by ～(1)/(3) during the removal of the 400 nm Irganox film and the roughness gradually increased to from 1 nm to ～4 nm. From SIMS lateral images of the beveled crater and AFM topography results, depth resolution was further improved by employing glancing angles of incidence and lower primary ion beam energy. Sub-10 nm depth resolution was observed under the optimized conditions on a routine basis. In general, we show that the wedge-crater beveling is an important tool for elucidating the factors that are important for molecular depth profiling experiments.     

Applicability of imaging time-of flight secondary ion MS to the characterization of solid-phase synthesized combinatorial libraries

We employ imaging time-of-flight secondary ion mass spectrometry to perform high-throughput analysis of solid-phase synthesized combinatorial libraries by acquiring mass spectra from arrays of polymer resin particles. To generalize this procedure to various types of resins and their associated chemical linkers, it is necessary to understand the dynamics associated with the analyte molecules during chemical pretreatment steps. Using stearic acid as a model compound, we examine the influence of three classes of linkers-acid or base labile linkers, a thermally labile linker, and a photochemically cleavable linker- all of which are used to anchor one end of the analyte to the polymer resin. With data obtained using secondary ion mass spectrometry, scanning electron microscopy, and X-ray photoelectron spectroscopy, we conclude that an effective treatment of the resin needs to include cleaving the linker and extracting the unbound analyte to the resin surface. We also demonstrate that the hydrophilicity of the polymeric constituents of a resin particle affects the experiments by changing the location of the analyte molecules during resin treatment. With this information, it is possible to utilize imaging TOF-SIMS to assay a range of material supports with assurance that high-quality spectra can be acquired.     

Protocols for three-dimensional molecular imaging using mass spectrometry

A protocol for three-dimensional molecular thin-film analysis is described that utilizes imaging time-of-flight secondary ion mass spectrometry and large-area atomic force microscopy. As a test study, a 300-nm trehalose film deposited on a Si substrate was structured by bombardment with a focused 15-keV Ga+ ion beam and analyzed using a 40-keV C60+ cluster ion beam. A three-dimensional sputter depth profile was acquired as a series of high-resolution lateral SIMS images with intermittent erosion cycles. As the most important result of this study, we find that the structured film exhibits a highly nonuniform erosion rate, thus preventing a simple conversion of primary ion fluence into eroded depth. Instead, the depth scale calibration must be performed individually on each pixel of the imaged area. The resulting laterally resolved depth profiles are discussed in terms of the chemical damage induced by the Ga+ bombardment along with the physics of the C60+ induced erosion process.     

Gold-enhanced biomolecular surface imaging of cells and tissue by SIMS and MALDI mass spectrometry

Surface metallization by plasma coating enhances desorption/ionization of membrane components such as lipids and sterols in imaging time-of-flight secondary ion mass spectrometry (TOF-SIMS) of tissues and cells. High-resolution images of cholesterol and other membrane components were obtained for neuroblastoma cells and revealed subcellular details (resolving power 1.5 mum). Alternatively, in matrix-enhanced SIMS, 2,5-dihydroxybenzoic acid electrosprayed on neuroblastoma cells allowed intact molecular ion imaging of phosphatidylcholine and sphingomyelin at the cellular level. Gold deposition on top of matrix-coated rat brain tissue sections strongly enhanced image quality and signal intensity in stigmatic matrix-assisted laser desorption/ionization imaging mass spectrometry. High-quality total ion count images were acquired, and the neuropeptide vasopressin was localized in the rat brain tissue section at the hypothalamic area around the third ventricle. Although the mechanism of signal enhancement by gold deposition is under debate, the results we have obtained for cells and tissue sections illustrate the potential of this sample preparation technique for biomolecular surface imaging by mass spectrometry.     

Comparative investigation of the surface properties of commercial titanium dental implants. Part I: chemical composition

The surfaces of five commercially available titanium implants (Brånemark Nobel Biocare, 3i ICE, 3i OSSEOTITE, ITI-TPS, and ITI-SLA) were compared by scanning electron microscopy, X-ray photoelectron spectroscopy, time-of-flight secondary ion mass spectroscopy. All five implant types were screw-shaped and fabricated from commercially pure (cp) titanium, but their surface properties differed both as regards surface morphology and surface chemical composition. The macro- and microstructure of the implant surfaces were investigated by scanning electron microscopy. The surfaces chemical composition was determined using the surface-sensitive analytical techniques of X-ray photoelectron spectroscopy and time-of-flight secondary ion spectrometry. Surface topographies were found to reflect the type of mechanical/chemical fabrication procedures applied by the manufacturers. The titanium oxide (passive) layer thickness was similar (5–6 nm) and typical for oxide films grown at or near room temperature. A variety of elements and chemical compounds not related to the metal composition were found on some implant types. They ranged from inorganic material such as sodium chloride to specific organic compounds believed to be due to contamination during fabrication or storage. The experimental findings are believed to make a contribution to a better understanding of the interplay between industrial fabrication procedure and physico-chemical implant surface properties.     

A combined ion probe/spark source analysis system

Spark source mass spectrometry and secondary ion mass spectrometry have complementary advantages in the elemental analysis of solids. The AEI IM20 ion probe and MS702R mass spectrometer form a composite instrument incorporating both techniques; the design and different operating modes are described. A primary ion beam diameter as low as 2 μm can be obtained under favourable circumstances; the minimum detectable concentration of a typical element is derived as a function of this diameter. The mass resolution is greater than 5000 (10% valley definition) with electrical detection and 10,000 (50% density definition) with photographic detection; this resolution is much higher than that obtained with other secondary ion mass spectrometers. Two typical applications illustrate that such high mass resolution is a necessary prerequisite to quantitative interpretation in secondary ion mass spectrometry: precise isotopic analysis of titanium is demonstrated and the concentrations of impurity transition elements derived from ion probe and spark source analyses of a geological olivine are compared.     

Integrating histology and imaging mass spectrometry

MALDI (matrix-assisted laser desorption/ionization) imaging mass spectrometry (IMS) is a new technology that generates molecular profiles and two-dimensional ion density maps of peptide and protein signals directly from the surface of thin tissue sections. This allows specific information to be obtained on the relative abundance and spatial distribution of proteins. One important aspect of this is the opportunity to correlate these specific ion images with histological features observed by optical microscopy. To facilitate this, we have developed protocols that allow MALDI mass spectrometry imaging and optical microscopy to be performed on the same section. Key components of these protocols involve the use of conductive glass slides as sample support for the tissue sections and MS-friendly tissue staining protocols. We show the effectiveness of these with protein standards and with several types of tissue sections. Although stain-specific intensity variations occur, the overall protein pattern and spectrum quality remain consistent between stained and control tissue samples. Furthermore, imaging mass spectrometry experiments performed on stained sections showed good image quality with minimal delocalization of proteins resulting from the staining protocols.     

Localization and quantitative analysis of antigen-antibody binding on 2D substrate using imaging NanoSIMS

Iron containing-antigen bound specifically to antibody immobilized on a surface is analyzed by nanoscale secondary ion mass spectrometry (NanoSIMS). This technique is well adapted compared with X-ray photoelectron spectroscopy and energy dispersive spectroscopy, which do not allow the detection of iron. The obtained Fe(+) map gives a good representation of the antigen repartition on the surface. NanoSIMS analysis of competition experiments performed with albumin and iron-free antigen are in good accordance with results obtained by a classical fluorescence microscopy approach. These results underline the interest of imaging NanoSIMS as a label-free method, allowing the localization and quantitative analysis of antigen-antibody binding with better spatial resolution than imaging ellipsometry and SPR.     

从飞行时间二次离子质谱像解析航天器污染的信息

飞行时间二次离子表面质谱能对航天材料上肉眼可见微量污染物实现包括元素、同位素和各种化合物在内的指纹鉴别,特别是样品量有限航天器污染成份分析的理想手段.本文重点讨论如何从带有金一次离子源的飞行时间二次离子像中提取有关航天器污染的信息.由于飞行时间二次离子质谱独具的并行质量登录能力,二次离子表面像上每一像素都储存着完整的质谱,任意质量的二次离子像都可重构.与四极和磁二次离子质谱相比,飞行时间二次离子质谱更适于复杂航天器污染物的成像分析.     

Synthesis and Characterization of(Na0.5K0.5)NbO3（NKN） Thin Films Formed by a Diol-based Sol-gel Process

Lead-free(Na_(0.5)K_(0.5))NbO_3(NKN) thin films were fabricated by spin coating on Pt/Ti/SiO2/Si substrates by a diol-based sol-gel process.Na-acetate,K-acetate,Nb-pentaethoxide and 1,3 propanediol were used to prepare the NKN precursor solution.Thermal analysis showed two characteristic temperatures of 360 and 600 ℃.Based on these temperatures,a heat treatment program with pyrolysis at 360 ℃ and calcination at 600 ℃ after every layer was used.To avoid inhomogeneities and secondary phases,an excess of sodium and potassium was necessary.To evaluate the proper excess amount of sodium and potassium secondary ion mass spectrometry(SIMS) lateral element maps and X-ray diffraction(XRD) patterns were recorded.An excess amount of 20% led to homogeneous distribution of the elements and to single phase perovskite NKN films with random crystal orientation.Scanning electron microscopy(SEM) images showed a pore free surface with 100 nm grains.The leakage current measurements showed a current of 1×10~(-3) A/cm~2 at 150 kV/cm.     

TOF-SIMS 3D biomolecular imaging of Xenopus laevis oocytes using buckminsterfullerene (C60) primary ions

Time-of-flight secondary ion mass spectrometry (TOF-SIMS) using buckminsterfullerene (C60) as the primary ion source has the ability to generate chemical images of surfaces with high sensitivities and minimal chemical damage. We studied the application of C60+ to depth profile a biological cell surface in a controlled manner and to subsequently image the revealed subsurfaces, in order to generate three-dimensional molecular images of the biological system. Such an analytical tool not only enables the surface localization of molecular species to be mapped but also enables the biomolecular distribution as a function of depth to be investigated with minimal sample preparation/intervention. Here we demonstrate the technique with a freeze-dried Xenopus laevis oocyte, which is a single cell. A C60+ ion beam was used with computer-controlled analyses and etch cycles. Mass spectra derived from the surface revealed peaks corresponding to cholesterol (m/z 369) and other lipids at m/z 540-570 and 800-1000, in the positive ion mode, and lipid fatty acid side chains (e.g., m/z 255) in the negative ion mode. To our knowledge, this is the first demonstration of the 3D biomolecular imaging within an actual biological system using TOF-SIMS.     

MALDI-FTICR imaging mass spectrometry of drugs and metabolites in tissue

A new approach is described for imaging mass spectrometry analysis of drugs and metabolites in tissue using matrix-assisted laser desorption ionization-Fourier transform ion cyclotron resonance (MALDI-FTICR). The technique utilizes the high resolving power to produce images from thousands of ions measured during a single mass spectrometry (MS)-mode experiment. Accurate mass measurement provides molecular specificity for the ion images on the basis of elemental composition. Final structural confirmation of the targeted compound is made from accurate mass fragment ions generated in an external quadrupole-collision cell. The ability to image many small molecules in a single measurement with high specificity is a significant improvement over existing MS/MS based technologies. Example images are shown for olanzapine in kidney and liver and imatinib in glioma.     

Identification of Mineral Phases on Basalt Surfaces by Imaging SIMS

A method for the identification of mineral phases on basalt surfaces utilizing secondary ion mass spectrometry (SIMS) with imaging capability is described. The goal of this work is to establish the use of imaging SIMS for characterization of the surface of basalt. The basalt surfaces were examined by interrogating the intact basalt (heterogeneous mix of mineral phases) as well as mineral phases that have been separated from the basalt samples. Mineral separates from the basalt were used to establish reference spectra for the specific mineral phases. Electron microprobe and X-ray photoelectron spectroscopy were used as supplemental techniques for providing additional characterization of the basalt. Mineral phases that make up the composition of the basalt were identified from single-ion images which were statistically grouped. The statistical grouping is performed by utilizing a program that employs a generalized learning vector quantization technique. Identification of the mineral phases on the basalt surface is achieved by comparing the mass spectra from the statistically grouped regions of the basalt to the mass spectral results from the mineral separates. The results of this work illustrate the potential for using imaging SIMS to study adsorption chemistry at the top surface of heterogeneous mineral samples.     

The Effects of Radicals on the Formation of Fullerene Ions in Laser Desorption Studies of C60 Adducts

We have studied the fragmentation and aggregation of C₆₀ and its radical adducts R_nC₆₀ by laser-desorption TOF mass spectrometry in the positive and negative ion channels. The mechanism of the formation of daughter fullerenes in the negative ion channel and the enhancement of fullerene aggregation products have been discussed. We consider that the electron transfer process between neutral cage-like clusters and the radicals is responsible for the appearance of strong mass peaks of daughter fullerene anions in the case of C₆₀ radical adducts. The effects of the radicals on the fullerene aggregation process have been discussed.     

Morphological effects and the diffusion of iodine in CdTe

The morphology of undiffused and iodine-diffused CdTe slices and its effect on iodine concentration profiles is discussed. Such CdTe slices were analysed using defect etching, scanning electron microscopy (SEM), infrared microscopy and secondary ion mass spectrometry (SIMS). The results suggest that during diffusion a layer of an iodine compound forms in the surface region of the CdTe while, over a limited range further into the material, clusters of either this or a similar compound form at defect sites causing a high degree of crystal distortion.