Nanotome cluster bombardment to recover spatial chemistry after preparation of biological samples for SIMS imaging

A C₆₀⁺ cluster ion projectile is employed for sputter cleaning biological surfaces to reveal spatio-chemical information obscured by contamination overlayers. This protocol is used as a supplemental sample preparation method for time of flight secondary ion mass spectrometry (ToF-SIMS) imaging of frozen and freeze-dried biological materials. Following the removal of nanometers of material from the surface using sputter cleaning, a frozen-patterned cholesterol film and a freeze-dried tissue sample were analyzed using ToF-SIMS imaging. In both experiments, the chemical information was maintained after the sputter dose, due to the minimal chemical damage caused by C₆₀⁺ bombardment. The damage to the surface produced by freeze-drying the tissue sample was found to have a greater effect on the loss of cholesterol signal than the sputter-induced damage. In addition to maintaining the chemical information, sputtering is not found to alter the spatial distribution of molecules on the surface. This approach removes artifacts that might obscure the surface chemistry of the sample and are common to many biological sample preparation schemes for ToF-SIMS imaging.     

A new particle mounting method for surface analysis

The chemical analysis of microparticles is challenging due to the need to mount the particles on a substrate for analysis; double-sided adhesive tape is often used (sometimes conductive), however that is usually coated with poly (dimethyl siloxane) (PDMS) that is often used as a release agent. PDMS is a common surface contamination that can mask surface chemistries and hinder material performance where it is dependent on this contaminated interface. It is known that PDMS contains a very mobile oligomeric fraction that readily diffuses across surfaces resulting in the contamination of mounted particulate samples before and during surface chemistry analysis. This makes it impossible to determine whether the PDMS has arisen from the analysis procedure or from the sample itself. A new sample preparation method is proposed where polymer microparticles are mounted on a poly (hydroxyethyl methacrylate) (pHEMA) polymer solution, which we compare with particles that have been mounted on adhesive discs using time-of-flight secondary ion mass spectrometry (ToF-SIMS) and 3D OrbiSIMS analysis. Particles mounted on the pHEMA substrate results in a reduction of PDMS signal by 99.8% compared with microparticles mounted on adhesive discs. This illustrates how a simple, quick and inexpensive polymer solution can be used to adhere particles for analysis by ToF-SIMS, or other surface chemical analysis techniques such as X-ray photoelectron spectroscopy (XPS), without introduction of large amounts of silicone contaminant.     

Investigations of matrix-assisted laser desorption/ionization sample preparation by time-of-flight secondary ion mass spectrometry

Matrix-assisted laser desorption/ionization (MALDI) and time-of-flight secondary ion mass spectrometry (ToF-SIMS) analyses are compared to gain insight into some of the details of sample preparation for MALDI analysis of synthetic polymers. ToF-SIMS imaging of MALDI samples shows segregation of the cationization agent from the matrix crystals. The amount of observed segregation can be controlled by the sample preparation technique. Electrospray sample deposition minimizes segregation. Comparing ToF-SIMS and MALDI mass spectra from the same samples confirms that ToF-SIMS is significantly more surface sensitive than MALDI. This comparison shows that segregation of the oligomers of a polymer sample can occur during MALDI sample preparation. Our data indicate that MALDI is not as sensitive to those species dominating the sample surface as to species better incorporated into the matrix crystals. Finally, we show that matrix-enhanced SIMS can be an effective tool to analyze synthetic polymers, although the sample preparation conditions may be different than those optimized for MALDI.     

Chemical and structural characterisation of DGEBA-based epoxies by time of flight secondary ion mass spectrometry (ToF-SIMS) as a preliminary to polymer interphase characterisation

Time-of-flight secondary ion mass spectrometry (ToF-SIMS) has become a powerful tool in the field of surface analysis since it provides information about the top few monolayers of a sample, i.e. on the chemical composition of the sample surface. Thus, the general question arises whether a surface-sensitive technique like ToF-SIMS would be appropriate to detect systematic chemical and/or structural changes in organic bulk polymers caused by varying a chemical content of the initial components or by tracking, e.g. curing processes in such materials. It is shown that careful sample preparation and the use of multivariate methods permit the quantitative acquisition of chemical and structural information about bulk polymers from the secondary ion signals. The hardener concentration and a cross-linking coefficient in diglycidyl ether of bisphenol A based epoxies were determined by ToF-SIMS measurements on samples with different resin to hardener ratio and varying curing time. In future work, we will use the developed method to investigate the local composition of adhesively bonded joints. In particular, the mapping of the chemical and structural properties in the so-called interphase will then be of interest.     

ToF-SIMS measurements with topographic information in combined images

In 2D and 3D time-of-flight secondary ion mass spectrometric (ToF-SIMS) analysis, accentuated structures on the sample surface induce distorted element distributions in the measurement. The origin of this effect is the 45° incidence angle of the analysis beam, recording planar images with distortion of the sample surface. For the generation of correct element distributions, these artifacts associated with the sample surface need to be eliminated by measuring the sample surface topography and applying suitable algorithms. For this purpose, the next generation of ToF-SIMS instruments will feature a scanning probe microscope directly implemented in the sample chamber which allows the performance of topography measurements in situ. This work presents the combination of 2D and 3D ToF-SIMS analysis with topographic measurements by ex situ techniques such as atomic force microscopy (AFM), confocal microscopy (CM), and digital holographic microscopy (DHM). The concept of the combination of topographic and ToF-SIMS measurements in a single representation was applied to organic and inorganic samples featuring surface structures in the nanometer and micrometer ranges. The correct representation of planar and distorted ToF-SIMS images was achieved by the combination of topographic data with images of 2D as well as 3D ToF-SIMS measurements, using either AFM, CM, or DHM for the recording of topographic data.     

Methods for generating protein molecular ions in ToF-SIMS

One of the greatest challenges in mass spectrometry lies in the generation and detection of molecular ions that can be used to directly identify the protein from the molecular weight of the molecular ion. Typically, proteins are large (MW > 1000), nonvolatile, and/or thermally labile, but the vaporization process produced by many mass spectrometry techniques including time-of-flight secondary ion mass spectrometry (ToF-SIMS) is inherently limited to generating ions from smaller compounds or fragments of the parent molecule, making the identification of proteins complex. The application of specific molecules to aid in the generation of high molecular weight ions in ToF-SIMS has been recognized for some time. In this study we have developed a matrix-SAM substrate preparation technique based on the self-assembly of a matrix-like molecule, mercaptonicotinic acid (MNA), on gold. We then compare this substrate with two existing ToF-SIMS sample preparation techniques, cationized alkane thiol and matrix-enhanced SIMS (MESIMS). The results of this study illustrate that while there is a range of methods that can be used to improve the molecular ion yield of proteins in ToF-SIMS, their efficacy and reproducibility vary considerably and crucially are linked to the sample preparation and/or protein application methods used. Critically, the MNA modified substrate was able to simultaneously induce molecular ions for each protein present in a multicomponent solution, suggesting that this sample preparation technique may have future application in proteomics and DNA analysis.     

Probing the orientation of surface-immobilized immunoglobulin G by time-of-flight secondary ion mass spectrometry

Static time-of-flight secondary ion mass spectrometry (ToF-SIMS) is a powerful surface analysis technique for the characterization of protein films because of its chemical selectivity and surface sensitivity. In this study, static ToF-SIMS and principal component analysis (PCA), a multivariate data analysis method, were combined to probe the orientation of surface-immobilized immunoglobulin G (IgG). IgG orientation can enhance its ability to detect its antigen in immunoassay techniques. The IgG used in this work is the mouse monoclonal anti-human chorionic gonadotropin (anti-hCG). Anti-hCG films on different well-defined substrates have been studied using its F(ab')2 and Fc fragments as references. Atomic force microscopy was used to characterize these protein films before static ToF-SIMS analysis. The results from PCA of ToF-SIMS spectra were related to the antibody primary amino acid composition and its three-dimensional structure.     

ToF-SIMS analysis of poly(l-lysine)-graft-poly(2-methyl-2-oxazoline) ultrathin adlayers

Understanding of the interfacial chemistry of ultrathin polymeric adlayers is fundamentally important in the context of establishing quantitative design rules for the fabrication of nonfouling surfaces in various applications such as biomaterials and medical devices. In this study, seven poly(l-lysine)-graft-poly(2-methyl-2-oxazoline) (PLL–PMOXA) copolymers with grafting density (number of PMOXA chains per lysine residue) 0.09, 0.14, 0.19, 0.33, 0.43, 0.56, and 0.77, respectively, were synthesized and characterized by means of nuclear magnetic resonance spectroscopy (NMR). The copolymers were then adsorbed on Nb₂O₅ surfaces. Optical waveguide lightmode spectroscopy method was used to monitor the surface adsorption in situ of these copolymers and provide information on adlayer masses that were then converted into PLL and PMOXA surface densities. To investigate the relationship between copolymer bulk architecture (as shown by NMR data) and surface coverage as well as surface architecture, time-of-flight secondary ion mass spectrometry (ToF-SIMS) analysis was performed. Furthermore, ToF-SIMS method combined with principal component analysis (PCA) was used to verify the protein resistant properties of PLL–PMOXA adlayers, by thorough characterization before and after adlayer exposure to human serum. ToF-SIMS analysis revealed that the chemical composition as well as the architecture of the different PLL–PMOXA adlayers indeed reflects the copolymer bulk composition. ToF-SIMS results also indicated a heterogeneous surface coverage of PLL–PMOXA adlayers with high grafting densities higher than 0.33. In the case of protein resistant surface, PCA results showed clear differences between protein resistant and nonprotein-resistant surfaces. Therefore, ToF-SIMS results combined with PCA confirmed that the PLL–PMOXA adlayer with brush architecture resists protein adsorption. However, low increases of some amino acid signals in ToF-SIMS spectra were detected after the adlayer has been exposed to human serum.

Mapping Dicorynia guianensis Amsh. wood constituents by submicron resolution cluster‐TOF‐SIMS imaging

The preparation of tropical wood surface sections for time‐of‐flight secondary ion mass spectrometry imaging is described, and the use of delayed extraction of secondary ions and its interest for the analysis of vegetal surface are shown. The method has been applied to the study by time‐of‐flight secondary ion mass spectrometry imaging with a resolution of less than one micron of a tropical wood species, Dicorynia guianensis, which is one of the most exploited wood in French Guiana for its durable heartwood. The heartwood of this species exhibits an economical importance, but its production is not controlled in forestry. Results show an increase of tryptamine from the transition zone and a concomitant decrease of inorganic ions and starch fragment ions. These experiments lead to a better understanding of the heartwood formation and the origin of the natural durability of D. guianensis. Copyright © 2016 John Wiley & Sons, Ltd.     

XPS characterization of wood chemical composition after heat‐treatment

XPS was used to characterize the chemical changes occurring after drying or applying a heat‐treatment to beech wood samples. Our results indicate that the surface of this air‐exposed material could be strongly affected either by the ambient atmosphere during storage or by the complex atmosphere in the oven during drying or heat‐treatment. However, the O/C ratio measured after removal of a thin slice of a few millimetres of an untreated sample is in reasonable agreement with that calculated from the well‐established chemical composition of beech. Through this methodology (equivalent to scraping for hard materials) it is expected to get a realistic characterization of the wood. The reliability and repeatability of the XPS measurements have been checked and the method applied to the study of the chemical changes of the beech samples subjected to heat‐treatment. Heating at 240 °C induces a significant decrease of the O/C ratio from 0.55 before to 0.44 after the treatment. Heat‐treatment induces also a decrease of the C₂ carbon contribution (carbon atom bound to a single non‐carbonyl oxygen) associated with an increase of the C₁ carbon contribution (carbon atoms bound only to carbon or hydrogen atoms), in agreement with chemical modifications reported previously in the literature. Thanks to the small analysed area of the equipment used in this study, different spots were analysed to demonstrate the presence or absence of a gradient of chemical composition due to thermal degradation or migration of extractives from within the wood structure to its surface. At the scale of our observations, the different wood samples investigated (dried or heat treated) appear to be homogeneous. Copyright © 2006 John Wiley & Sons, Ltd.     

Preparation techniques alter the mineral and organic fractions of fish otoliths: insights using Raman micro-spectrometry

The high spatial resolution analysis of the mineral and organic composition of otoliths using Raman micro-spectrometry involves rigorous protocols for sample preparation previously established for microchemistry and trace elements analyses. These protocols often include otolith embedding in chemically neutral resin (i.e., resins which do not contain, in detectable concentration, elements usually sought in the otoliths). Such embedding may however induce organic contamination. In this paper, Raman micro-spectrometry reveals the presence of organic contamination onto the surface obtained from the use of epoxy resin, specifically Araldite. This contamination level varies depending on otolith structures. Core and checks, known as structural discontinuities, exhibit the most important level of contaminations. Our results suggest that otolith embedding with resin affects the organic matrix of the otolith, probably through an infiltration of the resin in the crystalline structure. The interpretation of chemical otolith signatures, especially Raman otolith signatures, and stable isotope analyses should then be revised in light of these results. In this respect, we propose a method for the correction of Raman otolith signatures for contamination effects.     

Efficient and sample-specific interpretation of ToF-SIMS data by additional postprocessing of principal component analysis results

Time-of-flight secondary ion mass spectrometry (ToF-SIMS) is a powerful tool for surface analysis, but fragmentation of molecular species during the SIMS process may lead to complex mass spectra. While the fragmentation pattern is typically characteristic for each compound, industrial samples are engineered materials, and, thus, may contain a mixture of many compounds, which may result in a variety of overlapping peak patterns in ToF-SIMS spectra. Consequently, the process of data evaluation is challenging and time-consuming. Principal component analysis (PCA) can be used to simplify data analysis for complex sample systems. Especially, correlation loadings were observed as an ideal tool to identify relevant signals in PCA results, which induce the separation of different sample groups. This is because correlation loadings show the relevance of signals independent from their intensity in the raw data. In correlation loadings, however, fragmentation patterns are no longer observed and the identification of peaks' sum formulas is challenging. In this study, a new approach is presented, which simplifies peak identification and assignment in ToF-SIMS spectra after PCA is performed. The approach uses a mathematical transformation that projects PCA results, in particular loadings and correlation loadings, in the direction of specific sample groups. The approach does not change PCA results but rather presents them in a new way. This method allows to visualize characteristic spectra for specific sample groups that contain only relevant signals and, additionally, visualize fragmentation patterns. Data analysis is simplified and helps the user to focus on data interpretation rather than processing.     

A methodology to investigate heterogeneous oxidation of thermally aged cross-linked polyethylene by ToF-SIMS

Artificial ageing of polymeric insulation jackets is routinely performed in order to assess end-of-life material characteristics. Practical constraints including high temperatures/short times ageing treatments lead to strong influence of diffusion-limited oxidation (DLO) resulting in unreliable life-time predictions. This study proposes a new experimental approach to the investigation of cable insulation ageing, exploiting analytical techniques capable of resolving chemistry at the length scale relevant for DLO (nano-microscale). When studying the potential effects of DLO using time of flight secondary ion mass spectrometry (ToF-SIMS) sample preparation becomes crucial. This paper presents the development of a methodology to generate suitable specimens to investigate the DLO effect using ToF-SIMS. A reference polymeric material has been thermally aged in various DLO conditions. Cross sections of aged samples were generated using three different methods. In order to assess the most suitable approach for this study, cross-section topography were scanned using a profilometer and the surface chemistry was investigated using ToF-SIMS together with multivariate analysis methods.     

Microbeam analysis applied to adhesion, surfaces and interfaces

Typical applications of microbeam surface analysis techniques of XPS and ToF-SIMS in adhesion research are being described. Three areas of endeavour are identified as being important in the use of surface analysis in adhesion research; the assessment of surface cleanliness prior to the adhesion process; the forensic analysis of interfacial failure surfaces; probing the interfacial chemistry of adhesion. Examples, chosen from work in the author’s laboratory, are used to illustrate the levels of information that may be attainable from both model systems and from fully formulated, commercial, systems using these techniques. It is concluded that both XPS and ToF-SIMS have important roles to play in adhesion research and for a complete picture of interfacial chemistry and subsequent failure both must be employed.     

Analysis of lignin from archaeological waterlogged wood by direct exposure mass spectrometry (DE-MS) and PCA evaluation of mass spectral data

The chemical characterisation of waterlogged archaeological wood is of fundamental importance to understand the degradation processes undergone by wooden objects and consequently to develop suitable consolidation and conservation procedures. Lignin extracted from archaeological waterlogged wood samples was characterized using direct exposure electron ionisation mass spectrometry (DE-MS). DE-MS achieves a mass spectral fingerprint of the sample in a few minutes, avoiding any chemical pre-treatment and requiring only few micrograms of material.Mass spectral data were put in relation to the chemical composition of lignin and evaluated by means of principal component analysis (PCA). The preliminary results, presented in this study, demonstrate the feasibility and the potential of DE-MS as a reproducible and rapid screening method for archaeological waterlogged wood samples.     

Surface characterization of untreated and hydro‐thermally pre‐treated Turkey oak woods after UV‐C irradiation

The aim of this study was to determine the effect of UV‐C irradiation on the Turkey oak wood surface (Quercus cerris L.). In order to compare the effect of irradiation, both untreated wood samples and those treated with steam and heat were analyzed. The steam treatments were carried out in an autoclave at 130 °C; samples were then heated in an oven for 2 h at 180 °C. The physical and chemical changes brought about in the untreated and treated wood samples by the UV‐C light were monitored by colorimetry (color changes), X‐ray photoelectron spectroscopy (XPS), Fourier transform infrared spectroscopy (FTIR) (chemical composition) and scanning electron microscopy (SEM) (microstructure and morphology). A detailed analysis of the results indicates that the UV‐C treatment caused irreversible changes in both the chemical composition and morphology of the wood samples via photooxidation and photodegradation processes. Depending on the type of pre‐treatment used, these processes affected the wood samples differently. Copyright © 2014 John Wiley & Sons, Ltd.     

ToF-SIMS characterization of microplastics in soils

Microplastics pollution is becoming one of the most serious threats to the surface ecosystem of the earth; it is widespread in oceans, rivers, sediments, soils, and organisms. It is a growing concern as an environmental pollutant, which currently has no clear detection standard. Detection methods still need to be constantly supplemented and improved. This study explored a novel method called time-of-flight secondary ion mass spectrometry (ToF-SIMS) in this field. Four types of microplastics in farmland soils, namely, polypropylene, polyvinyl chloride, polyethylene terephthalate, and polyamide 6, were successfully identified in terms of particle size and abundance by combining the high molecular specificity with ion imaging capability of ToF-SIMS. The procedure based on ToF-SIMS analysis also provides a methodological reference and basic data for the investigation and research of microplastics in soil, coastal beaches, and sediment.     

ToF-SIMS and XPS investigations of fibers, coatings and biomedical materials

The use of ToF-SIMS and XPS in industrial research is demonstrated by a number of applications of surface analysis within Akzo Nobel: surface treatment of carbon fibers, adhesion activation of aramid fibers, weathering and protection of wood, surfactant adsorption on pigments, grafting of polypropylene with acrylic monomers and treatment of a perfluorinated membrane with an amphiphile. The examples illustrate the broad use of these techniques on non-conducting organic and polymeric materials.     

Characterization of insulin adsorption in the presence of albumin by time-of-flight secondary ion mass spectrometry and X-ray photoelectron spectroscopy

With a view to develop an encapsulation membrane for a bioartificial pancreas, we have studied the adsorption of insulin and human serum albumin (HSA) on it. The aim of this study was to determine the possibility of insulin detection on a polycarbonate membrane surface in the presence of HSA, an abundant blood protein. The first step of the work consisted in the identification of time-of-flight secondary ion mass spectrometry (ToF-SIMS) and X-ray photoelectron spectroscopy (XPS) specific signals for insulin and albumin. For this purpose, adsorption isotherms in physiological conditions (pH = 7.2, T = 37 degrees ) were established for the two proteins by looking at the SIMS intensity variations of the characteristic protein and substrate fragments when increasing the protein concentration in the solution. The CHS+ ToF-SIMS fragment and the S2p XPS peak were identified as representative insulin signals. The second step of the work consisted in performing simultaneous adsorption of the two proteins with increasing insulin concentration. We observed an increase of the insulin signal in ToF-SIMS and XPS for insulin concentration beyond 5 microg/mL. Principal component analysis (PCA) of the ToF-SIMS results permits us to obtain information about the protein layer composition. The results show that at low relative insulin concentration in solution, the mixed adsorbed layers are enriched in insulin compared to the solution.     

Oxygen diffusion in grain boundaries: a ToF-SIMS investigation on hot-rolled steel sheets

A study of grain boundary diffusion of oxygen in hot-rolled steel sheets is carried out by means of time-of-flight secondary-ion-mass-spectrometry (ToF-SIMS). This involves polishing of the sample surface prior to the oxygen exposure. A nickel layer deposited after exposure ensures a homogeneous extraction field for ToF-SIMS measurements at the Ni–steel interface. The sample is bevelled at an angle of 11.5° to spread up the diffusion pathway by a factor of 5. The oxygen distribution is then acquired via ToF-SIMS in imaging mode from which diffusion parameters are calculated according to the Whipple–Le Claire’s approach.