ToF-SIMS Imaging Research Articles

Automated Quantification of Tumor Viability in a Rabbit Liver Tumor Model after Chemoembolization Using Infrared Imaging

The rabbit VX2 tumor is a fast-growing carcinoma model commonly used to study new therapeutic devices, such as catheter-based therapies for patients with inoperable hepatocellular carcinoma. The evaluation of tumor viability after such locoregional therapies is essential to directing hepatocellular carcinoma management. We used infrared microspectroscopy for the automatic characterization and quantification of the VX2 liver tumor viability after drug-eluting beads transarterial chemoembolization (DEB-TACE). The protocol consisted of K-means clustering followed by principal component analysis (PCA) and linear discriminant analysis (LDA). The K-means clustering was used to classify the spectra from the infrared images of control or treated tumors and to build a database of many tissue spectra. On the basis of this reference library, the PCA-LDA analysis was used to build a predictive model to identify and quantify automatically tumor viability on unknown tissue sections. For the DEB group, the LDA model determined that the surface of tumor necrosis represented 91.6% ± 8.9% (control group: 33.1% ± 19.6%; Mann-Whitney P = 0.0004) and the viable tumor 2.6% ± 4% (control group: 62.2% ± 15.2%; Mann-Whitney P = 0.0004). Tissue quantification measurements correlated well with tumor necrosis (r = 0.827, P < 0.0001) and viable tumor (r = 0.840, P < 0.0001). Infrared imaging and PCA-LDA analysis could be helpful for easily assessing tumor viability.

Optical properties and chemical composition analyses of mixed rare earth oxyorthosilicate (R2SiO5, R=La, Gd and Y) doped Dy3+ phosphors prepared by urea-assisted solution combustion method

Dysprosium (Dy3+) doped lanthanum gadolinium oxyorthosilicate (LaGdSiO5), lanthanum yttrium oxyorthosilicate (LaYSiO5) and gadolinium yttrium oxyorthosilicate (GdYSiO5) phosphors (in powder form) were synthesized by urea-assisted combustion method. The X-ray diffractometer analysis confirmed that the LaGdSiO5, LaYSiO5 and GdYSiO5 crystalized in monoclinic phases. The chemical composition of the phosphors was analyzed by measuring the atomic and molecular ionic species using the time of flight secondary ion mass spectroscopy (ToF SIMS). In addition, ToF SIMS imaging technique was used to determine the distribution of the Dy3+ dopant ions on the surface on the phosphors. The average crystallite sizes and lattice strains of the phosphor were increased by Dy3+ doping. The field emission scanning electron microscope images showed that the powders were made up of an agglomeration of particles with no regular shape. The photoluminescence data showed narrow line emission peaks at the wavelengths of 485nm (minor emission) and 573nm (major emission) associated with the f→f transitions of Dy3+. The photoluminescence (PL) measurements showed that the emission peak of LaGdSiO5:Dy3+ was ~3× more intense than those of LaYSiO5:Dy3+ and GdYSiO5:Dy3+ when excited using monochromatic xenon lamp with a wavelength of 241nm. However, when the powders were excited using a 325nm He–Cd laser, the highest PL emission intensity was observed from GdYSiO5:Dy3+.

Surface characterization of dialyzer polymer membranes by imaging ToF-SIMS and quantitative XPS line scans.

The surfaces of polymeric dialyzer membranes consisting of polysulfone and polyvinylpyrrolidone were investigated regarding the lateral distribution and quantitative surface composition using time-of-flight secondary-ion-mass-spectrometry and x-ray photoelectron spectroscopy. Knowledge of the distribution and composition on the outer surface region is of utmost importance for understanding the biocompatibility of such dialyzer membranes. Both flat membranes and hollow fiber membranes were studied.

3D ToF-SIMS imaging of polymer multilayer films using argon cluster sputter depth profiling.

ToF-SIMS imaging with argon cluster sputter depth profiling has provided detailed insight into the three-dimensional (3D) chemical composition of a series of polymer multilayer structures. Depths of more than 15 μm were profiled in these samples while maintaining uniform sputter rates. The 3D chemical images provide information regarding the structure of the multilayer systems that could be used to inform future systems manufacturing and development. This also includes measuring the layer homogeneity, thickness, and interface widths. The systems analyzed were spin-cast multilayers comprising alternating polystyrene (PS) and polyvinylpyrrolidone (PVP) layers. These included samples where the PVP and PS layer thickness values were kept constant throughout and samples where the layer thickness was varied as a function of depth in the multilayer. The depth profile data obtained was observed to be superior to that obtained for the same materials using alternative ion sources such as C60(n+). The data closely reflected the "as manufactured" sample specification, exhibiting good agreement with ellipsometry measurements of layer thickness, while also maintaining secondary ion intensities throughout the profiling regime. The unprecedented quality of the data allowed a detailed analysis of the chemical structure of these systems, revealing some minor imperfections within the polymer layers and demonstrating the enhanced capabilities of the argon cluster depth profiling technique.

Identifying sources of Pb pollution in urban soils by means of MC-ICP-MS and TOF-SIMS.

Lead pollution was evaluated in 17 urban soils from parks and gardens in the city of Vigo (NW Spain). The Pb isotope ratios ((207)Pb/(206)Pb, (208)Pb/(204)Pb, (206)Pb/(204)Pb and (208)Pb/(206)Pb) were determined after being measured by MC-ICP-MS. The association of the isotopes ((204)Pb, (206)Pb, (207)Pb and (208)Pb) with the different components of the soil was studied using TOF-SIMS. The isotopic ranges obtained for the samples were between 1.116 and 1.203 ((206)Pb/(207)Pb), 2.044-2.143 ((208)Pb/(206)Pb), 37.206-38.608 ((208)Pb/(204)Pb), 15.5482-15.6569 ((207)Pb/(204)Pb) and 17.357-18.826 ((206)Pb/(204)Pb). The application of the three-end-member model indicates that the Pb derived from petrol is the main source of Pb in the soils (43.51% on average), followed by natural or geogenic Pb (39.12%) and industrial emissions (17.37%). The emissions derived from coal combustion do not appear to influence the content of Pb in the soil. TOF-SIMS images show that the Pb mainly interacts with organic matter. This technique contributes to the understanding of the association of anthropogenic Pb with the components of the soil, as well as the particle size of these associations, thus allowing the possible sources of Pb to be identified.

Biomedical studies by TOF-SIMS imaging.

Imaging by secondary ion mass spectrometry coupled to time-of-flight mass analysis (TOF-SIMS) is a method of which the applications have greatly increased since 10 years. Taking advantage of the development of cluster ion sources, TOF-SIMS offers images of molecular ions at a micrometer lateral resolution or slightly below and does not require complex sample preparation. Although TOF-SIMS has been primarily dedicated to surface analysis of inorganic or polymeric samples, several groups have successfully demonstrated that TOF-SIMS imaging is also perfectly suited for mapping organic compounds, such as drugs or lipids, directly on tissue sections from animals or from human biopsies. This minireview will enlighten some of these developments in the field of biomedical applications.

An efficient TOF-SIMS image analysis with spatial correlation and alternating non-negativity-constrained least squares.

Advances in analytical instrumentation towards acquiring high-resolution images of mass spectrometry constantly demand efficient approaches for data analysis. This is particularly true of time-of-flight secondary ion mass spectrometry imaging where recent advances enable acquisition of high-resolution data in multiple dimensions. In many applications, the distribution of different species from a sampled surface is spatially continuous in nature and a model that incorporates the spatial correlation across the surface would be preferable to estimations at discrete spatial locations. A key challenge here is the capability to analyse the high-resolution multidimensional data to extract relevant information reliably and efficiently. We propose a framework based on alternating non-negativity-constrained least squares which accounts for the spatial correlation across the sample surface. The proposed method also decouples the computational complexity of the estimation procedure from the image resolution, which significantly reduces the processing time. We evaluate the performance of the algorithm with biochemical image datasets generated from mixture of metabolites.

TOF-SIMS imaging of lipids on rat brain sections.

Since several decades, secondary ion mass spectrometry (SIMS) coupled to time of flight (TOF) is used for atomic or small inorganic/organic fragments imaging on different materials. With the advent of polyatomic ion sources leading to a significant increase of sensitivity in combination with a reasonable spatial resolution (1-10 μm), TOF-SIMS is becoming a more and more popular analytical platform for MS imaging. Even if this technique is limited to small molecules (typically below 1,000 Da), it offers enough sensitivity to detect and locate various classes of lipids directly on the surface of tissue sections. This chapter is thus dedicated to the TOF-SIMS analysis of lipids in positive and negative ion modes on rat brain tissue sections using a bismuth cluster ion source.

Probing the lipid chemistry of neurotoxin-induced hippocampal lesions using multimodal imaging mass spectrometry.

The environmental toxin β-N-methylamino-L-alanine (BMAA) has been causatively linked to neurodegenerative disease pathology. In a rat model, neonatal BMAA exposure resulted in selective uptake in the hippocampal formation and caused learning and memory impairments in adult animals. Moreover, high dose neonatal BMAA exposure resulted in formation of protein inclusions in the CA1 region of the adult hippocampus. However the mechanism underlying BMAA induced neuropathology remains elusive. Imaging mass spectrometry is a powerful method for spatial interrogation of biochemical distribution in biological tissue with high chemical specificity. The aim of this study was to therefore characterize the lipid microenvironment of BMAA-induced hippocampal lesions in adult rats using matrix-assisted laser desorption/ionization (MALDI) and time-of-flight SIMS (ToF-SIMS imaging). Multimodal imaging was carried out by ToF-SIMS scans of the hippocampal formation followed by whole tissue scans using MALDI imaging. Multivariate analysis was performed on the SIMS data in order to delineate the spatial biochemistry surrounding the lesions. The data show lesion-specific localization of phosphatidylcholine fragments, suggesting neuroinflammatory glial cell activation. Complementary MALDI imaging data showed increased levels of phosphoethanolamines colocalizing with the proteopathic lesions pointing to macroautophagic mechanisms associated with neurotoxin-induced protein accumulation. Multimodal IMS by means of ToF-SIMS and MALDI mass spectrometry proved to be a powerful technique for neurotoxicological research.

Localized lipidomics in cystic fibrosis: TOF-SIMS imaging of lungs from Pseudomonas aeruginosa-infected mice

A consistent body of research has linked cystic fibrosis (CF) with variations in the tissue and fluid content in a number of lipid molecules. However, little is known about the spatial localization of those variations. We have recently applied TOF-SIMS mass spectrometry imaging to detect differential lipid signatures at the colon epithelium between normal and cftr−/− mice. In the present work we have used this technology to investigate potential differences in the spatial distribution of lipids due to Pseudomonas aeruginosa (P.a.) infection in mouse lung expressing or not cftr. Wild-type and exon 10 cftr knockout mice were subjected to intranasal infection with a clinical strain of P.a. Lung cryosections from infected and non-infected mice were subjected to cluster TOF-SIMS analysis in the negative ion mode. We observed a highly specific localization of a phosphoinositol fragment ion at m/z 299.1 in bronchial epithelium. Using this ion to delineate a region of interest, we studied the relative abundance of ions below m/z 1500. We found a significant increase in m/z 465.4 (identified as cholesteryl sulfate) in cftr−/− epithelium and in response to bacterial infection, as well as a decrease in most carboxylic ions. In conclusion, the m/z 299.1 ion can be used as a marker of bronchial epithelium, where P.a. infection leads to increased presence of cholesteryl sulfate in this tissue. TOF-SIMS imaging reveals as a valuable tool for the study of respiratory epithelium.This article is part of a Directed Issue entitled: Cystic Fibrosis: From o-mics to cell biology, physiology, and therapeutic advances.

Resolving the internal structure of individual atmospheric aerosol particle by the combination of Atomic Force Microscopy, ESEM–EDX, Raman and ToF–SIMS imaging

In this study, internal structures of individual aerosol particles were resolved by using micro-analytical techniques in combination. We demonstrated the practical applicability of the combined use of Atomic Force Microscopy (AFM), Environmental Scanning Electron Microscopy coupled with Energy-Dispersive X-ray analysis (ESEM–EDX), Raman Microspectrometry (RMS) and Time-of-Flight Secondary Ion Mass Spectrometry (ToF–SIMS) to provide morphological, elemental, molecular and outer surface imaging of the same individual airborne particles for the first time. The characterization of single particles collected in the industrial atmosphere influenced by marine air masses demonstrated the physicochemical evolution of the particles in a short time period. The marine-derived particles were mainly encountered as genuine sea salts internally mixed with reacted sea salts such as NaNO3 and liquid NO3− which are covered by an organic thin layer. The particles collected downwind the industrial area were solid particles composed of an internal mixture of iron oxides and of marine-derived particles coated with an organic layer. The formation of these particles is a result of coalescence, agglomeration and drying processes occurring in the atmosphere during the transport of particles in a short time period (~15min). It is demonstrated that the combined use of the different types of spectral and imaging data from the same individual particles in atmospheric aerosol sample provides richer information on their physicochemical characteristics than when those techniques were used alone or when two techniques in combination.

Complimentary Use of MALDI FTICR-MS and TOF-SIMS Imaging Approaches in an Invertebrate

Extended abstract of a paper presented at Microscopy and Microanalysis 2013 in Indianapolis, Indiana, USA, August 4 – August 8, 2013.

Surface grafted glycopolymer brushes to enhance selective adhesion of HepG2 cells

This work demonstrates the application of carbohydrate based methacrylate polymer brush, poly(2-lactobionamidoethyl methacrylate), for the purpose of cell adhesion studies. The first part of the work illustrates the effects of the structure of the aminosilane based ATRP initiator layer on the polymerization kinetics of 2-lactobionamidoethyl methacrylate) (LAMA) monomer on thermally oxidized silicon wafer. Both monolayer and multilayered aminosilane precursor layers have been prepared followed by reaction with 2-bromoisobutyrylbromide to form the ATRP initiator layer. It is inferred from the kinetic studies that the rate of termination is low on a multilayered initiator layer compared to a disordered monolayer structure. However both initiator types results in similar graft densities. Furthermore, it is shown that thick comb-like poly(LAMA) brushes can be constructed by initiating a second ATRP process on a previously formed poly(LAMA) brushes. The morphology of human hepatocellular carcinoma cancer cells (HepG2) on the comb-like poly(LAMA) brush layer has been studied. The fluorescent images of the HepG2 cells on the glycopolymer brush surface display distinct protrusions that extend outside of the cell periphery. On the other hand the cells on bare glass substrate display spheroid morphology. Further analysis using ToF-SIMS imaging shows that the HepG2 cells on glycopolymer surfaces is enriched with protein fragment along the cell periphery which is absent in the case of cells on bare glass substrate. It is suggested that the interaction of the galactose units of the polymer brush with the asialoglycoprotein receptor (ASGPR) of HepG2 cells has resulted in the protein enrichment along the cell periphery.

Time-of-Flight Secondary Ion Mass Spectrometry with Principal Component Analysis of Titania–Blood Plasma Interfaces

Treatment of osseoimplant surfaces with autologous platelet-rich plasma prepared according to the plasma rich in growth factors (PRGF-Endoret) protocol prior to implantation yields promising results in the clinic. Our objective is to understand the organization of complex interfaces between blood plasma preparations of various compositions and model titania surfaces. Here we present the results of the morphological and chemical characterization of TiO(2) surfaces incubated with four types of blood plasma preparations devoid of leukocytes and red blood cells: either enriched in platelets (PRGF-Endoret) or platelet-depleted, and either activated with CaCl(2) to induce clotting, or not. Chemical characterization was done by time-of-flight secondary ion mass spectrometry with principal component analysis (ToF-SIMS/PCA). The interface morphology was studied with scanning electron and atomic force microscopy. Immunofluorescence microscopy was used to identify platelets and infer their activation state. We observe clear differences among the four types of interfaces by ToF-SIMS/PCA. Some of these could be straightforwardly related to the differences in the sample morphology and known effects of platelet activation, but others are more subtle. Strikingly, it was possible to differentiate between these samples by ToF-SIMS/PCA of the protein species alone. This clearly indicates that the composition, orientation, and/or conformation of the proteins in these specimens depend both on the platelets' presence and on their activation. The ToF-SIMS imaging functionality furthermore provides unique insight into the distribution of phospholipid species in these samples.

TOF-SIMS imaging of adsorbed proteins on topographically complex surfaces with Bi3+ primary ions

Although previous studies have demonstrated that TOF-SIMS is a powerful method for the characterization of adsorbed proteins due to its specificity and surface sensitivity, it was unclear from earlier work whether the differences between proteins observed on uniform flat surfaces were large enough to facilitate clear image contrast between similar proteins in small areas on topographically complex samples that are more typical of biological tissues. The goal of this study was to determine whether Bi(3) (+) could provide sufficiently high sensitivity to provide clear identification of the different proteins in an image. In this study, 10 μm polystyrene microspheres were adsorbed with one of three different proteins, human serum albumin (HSA), bovine serum albumin (BSA), and hemoglobin. Spheres coated with HSA were then mixed with spheres coated with either BSA (a very similar protein) or hemoglobin (a dramatically different protein), and deposited on silicon substrates. Fluorescent labeling was used to verify the SIMS results. With maximum autocorrelation factors (MAF) processing, images showed clear contrast between both the very different proteins (HSA and hemoglobin) and the very similar proteins (HSA and BSA). Similar results were obtained with and without the fluorescent labels. MAF images were calculated using both the full spectrum and only characteristic amino acid fragments. Although better image contrast was obtained using the full spectrum, differences between the spheres were still evident when only the amino acid fragments were included in the analysis, suggesting that we are truly observing differences between the proteins themselves. These results demonstrate that TOF-SIMS, with a Bi(3) (+) primary ion, is a powerful technique for characterizing interfacial proteins not only on large uniform surfaces, but also with high spatial resolution on the topographically complex samples typical in biological analysis.

Surface modification and chemical surface analysis of biomaterials

The chemical composition of the surface layers of synthetic biomaterials used for human medical devices and in biotechnology plays a key role in determining interfacial interactions between biological media (such as protein solutions, cells, tissue) and the synthetic material. Accordingly, considerable research efforts focus on improving the 'biocompatibility' of biomaterials by applying various surface modification and thin film coating approaches. Here we focus on the patterning of surface chemistries, often designed to exercise spatial control over events such as cell attachment and spreading. Secondly, we review recent developments in chemical characterisation of biomaterials surfaces, which is essential both for verifying the success of intended surface modification strategies and for reliable interpretation of observed biological responses. Biomaterials surface analysis by imaging ToF-SIMS and XPS and compositional depth profiling are discussed, as is the emerging complementary technique of Metastable Induced Electron Spectroscopy.

EEG effects of scopolamine in healthy subjects: A standardized low resolution brain electromagnetic tomography (sLORETA) study

As, astrocytes and microglia. Results: In some cases, Ch accumulations could be observed in the vicinity of As deposits. Figure 1a shows a region of a 3xTgAD brain hippocampus stained with DAPI and p-FTAA. The delineated area enriched with As is magnified in 1b. An area containing several deposits (area 1 in 1b) was imaged with ToF-SIMS, as shown in 1c. Ch signal (yellowish) is increased in the vicinity of As (purple). ToF-SIMS images can be quantified using mathematical tools. Figure 2 shows a histogram describing the Ch distribution shown in 1c. Several Ch subpopulations arise from the general distribution, suggesting the presence of different Ch accumulation patterns. In 3xTgAD, Ch seems to be distributed in a rather inhomogeneous pattern, whereas being more homogeneously distributed in wild-type brain sections. Figure 3 shows several deposits stained with pFTAA in a 3xTgAD hippocampus. According to our preliminary understanding of the p-FTAA fluorescence-emission profiles, 3xTgAD brains present two types of deposits; greenish-like accumulations with reddish diffuse surrounding, and reddish-like deposits likely to be associated with hyperphosphorylated tau. Conclusions: The combination of CLSM and ToFSIMS is a suitable approach to study Ch/lipid profile around As deposits. A complete study of the spatial colocalization between glial cells, Ch andAs is currently being completed. New results are expected by May 2011.

Metabolomics: Where seeing is believing

Metabolomics: Where seeing is believing

Retrospective sputter depth profiling using 3D mass spectral imaging.

A molecular multilayer stack composed of alternating Langmuir-Blodgett films was analyzed by ToF-SIMS imaging in combination with intermediate sputter erosion using a focused C60+ cluster ion beam. From the resulting dataset, depth profiles of any desired lateral portion of the analyzed field-of-view can be extracted in retrospect, allowing the influence of the gating area on the apparent depth resolution to be assessed. In a similar way, the observed degradation of depth resolution with increasing depth of the analyzed interface can be analyzed in order to determine the 'intrinsic' depth resolution of the method.

ToF-SIMS Imaging of Intracellular &lt;sup&gt;39&lt;/sup&gt;K/&lt;sup&gt;40&lt;/sup&gt;Ca Changes induced by ZnO-containing Nanomaterials

Pei-Ling Lee, Sin-Yu Shen, Yu-Sheng Yin, Shiu-Ling Lei, Cian-Ling Jhang , Woan-Ruoh Lee 4 and Yong-Chien Ling 1,2* Department of Chemistry, National Tsing Hua University, Hsinchu 30013, Taiwan Biomedical Mass Imaging Research Center, Taipei Medical University, Taipei 11031, Taiwan Graduate Institute of Medical Science, Taipei Medical University, Taipei 11031, Taiwan Department of Dermatology, Taipei Medical University, Taipei 11031, Taiwan *ycling@mx.nthu.edu.tw