Reliable Surface Analysis Data of Nanomaterials in Support of Risk Assessment Based on Minimum Information Requirements.

The minimum information requirements needed to guarantee high-quality surface analysis data of nanomaterials are described with the aim to provide reliable and traceable information about size, shape, elemental composition and surface chemistry for risk assessment approaches. The widespread surface analysis methods electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDS), X-ray photoelectron spectroscopy (XPS) and secondary ion mass spectrometry (SIMS) were considered. The complete analysis sequence from sample preparation, over measurements, to data analysis and data format for reporting and archiving is outlined. All selected methods are used in surface analysis since many years so that many aspects of the analysis (including (meta)data formats) are already standardized. As a practical analysis use case, two coated TiO2 reference nanoparticulate samples, which are available on the Joint Research Centre (JRC) repository, were selected. The added value of the complementary analysis is highlighted based on the minimum information requirements, which are well-defined for the analysis methods selected. The present paper is supposed to serve primarily as a source of understanding of the high standardization level already available for the high-quality data in surface analysis of nanomaterials as reliable input for the nanosafety community.

Evaluation of Time-of-Flight Secondary Ion Mass Spectrometry Spectra of Peptides by Random Forest with Amino Acid Labels: Results from a Versailles Project on Advanced Materials and Standards Interlaboratory Study.

We report the results of a VAMAS (Versailles Project on Advanced Materials and Standards) interlaboratory study on the identification of peptide sample TOF-SIMS spectra by machine learning. More than 1000 time-of-flight secondary ion mass spectrometry (TOF-SIMS) spectra of six peptide model samples (one of them was a test sample) were collected using 27 TOF-SIMS instruments from 25 institutes of six countries, the U. S., the U. K., Germany, China, South Korea, and Japan. Because peptides have systematic and simple chemical structures, they were selected as model samples. The intensity of peaks in every TOF-SIMS spectrum was extracted using the same peak list and normalized to the total ion count. The spectra of the test peptide sample were predicted by Random Forest with 20 amino acid labels. The accuracy of the prediction for the test spectra was 0.88. Although the prediction of an unknown peptide was not perfect, it was shown that all of the amino acids in an unknown peptide can be determined by Random Forest prediction and the TOF-SIMS spectra. Moreover, the prediction of peptides, which are included in the training spectra, was almost perfect. Random Forest also suggests specific fragment ions from an amino acid residue Q, whose fragment ions detected by TOF-SIMS have not been reported, in the important features. This study indicated that the analysis using Random Forest, which enables translation of the mathematical relationships to chemical relationships, and the multi labels representing monomer chemical structures, is useful to predict the TOF-SIMS spectra of an unknown peptide.

Distribution of Paramagnetic Fe2O3/SiO2⁻Core/Shell Nanoparticles in the Rat Lung Studied by Time-of-Flight Secondary Ion Mass Spectrometry: No Indication for Rapid Lipid Adsorption.

Amorphous silica nanoparticles comprise a class of widely used industrial nanomaterials, which may elicit acute inflammation in the lung. These materials have a large specific surface to which components of the pulmonary micro-milieu can bind. To conduct appropriate binding studies, paramagnetic Fe2O3/SiO2 core/shell nanoparticles (Fe-Si-NP) may be used as an easy-to-isolate silica surrogate, if several prerequisites are fulfilled. To this end, we investigated the distribution of Fe, Si, protein and phosphatidylcholine (PC) by Time-of-Flight secondary ion mass spectrometry (ToF-SIMS) in cryo-sections from the rat lungs to which Fe-Si-NP had been administered for 30 min. Regions-of-interest were identified and analyzed with incident light and enhanced dark-field microscopy (DFM). Fe-Si-NP particles (primary particle size by electron microscopy: 10⁻20 nm; aggregate size by tracking analysis: 190 ± 20 nm) and agglomerates thereof were mainly attached to alveolar walls and only marginally internalized by cells such as alveolar macrophages. The localization of Fe-Si-NP by DFM was confirmed by ToF-SIMS signals from both, Fe and Si ions. With respect to an optimized signal-to-noise ratio, Fe⁺, Si⁺, CH4N⁺ and the PC head group (C5H15NO4P⁺) were the most versatile ions to detect iron, silica, protein, and PC, respectively. Largely congruent Fe⁺ and Si⁺ signals demonstrated that the silica coating of Fe-Si-NP remained stable under the conditions of the lung. PC, as a major lipid of the pulmonary surfactant, was colocalized with the protein signal alongside alveolar septa, but was not detected on Fe-Si-NP, suggesting that silica nanoparticles do not adsorb lipids of the lung surfactant under native conditions. The study shows that ToF-SIMS is a valuable technique with adequate spatial resolution to analyze nanoparticles together with organic molecules in the lung. The paramagnetic Fe-Si-NP appear well suited to study the binding of proteins to silica nanomaterials in the lung.

Detection of ZrO2 Nanoparticles in Lung Tissue Sections by Time-of-Flight Secondary Ion Mass Spectrometry and Ion Beam Microscopy.

The increasing use of nanoparticles (NP) in commercial products requires elaborated techniques to detect NP in the tissue of exposed organisms. However, due to the low amount of material, the detection and exact localization of NP within tissue sections is demanding. In this respect, Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS) and Ion Beam Microscopy (IBM) are promising techniques, because they both offer sub-micron lateral resolutions along with high sensitivities. Here, we compare the performance of the non-material-consumptive IBM and material-consumptive ToF-SIMS for the detection of ZrO2 NP (primary size 9-10 nm) in rat lung tissue. Unfixed or methanol-fixed air-dried cryo-sections were subjected to IBM using proton beam scanning or to three-dimensional ToF-SIMS (3D ToF-SIMS) using either oxygen or argon gas cluster ion beams for complete sample sputtering. Some sample sites were analyzed first by IBM and subsequently by 3D ToF-SIMS, to compare results from exactly the same site. Both techniques revealed that ZrO2 NP particles occurred mostly agglomerated in phagocytic cells with only small quantities being associated to the lung epithelium, with Zr, S, and P colocalized within the same biological structures. However, while IBM provided quantitative information on element distribution, 3D ToF-SIMS delivered a higher lateral resolution and a lower limit of detection under these conditions. We, therefore, conclude that 3D ToF-SIMS, although not yet a quantitative technique, is a highly valuable tool for the detection of NP in biological tissue.

Detection of SiO2 nanoparticles in lung tissue by ToF-SIMS imaging and fluorescence microscopy.

The direct detection of nanoparticles in tissues at high spatial resolution is a current goal in nanotoxicology. Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS) is widely used for the direct detection of inorganic and organic substances with high spatial resolution but its capability to detect nanoparticles in tissue sections is still insufficiently explored. To estimate the applicability of this technique for nanotoxicological questions, comparative studies with established techniques on the detection of nanoparticles can offer additional insights. Here, we compare ToF-SIMS imaging data with sub-micrometer spatial resolution to fluorescence microscopy imaging data to explore the usefulness of ToF-SIMS for the detection of nanoparticles in tissues. SiO2 nanoparticles with a mean diameter of 25 nm, core-labelled with fluorescein isothiocyanate, were intratracheally instilled into rat lungs. Subsequently, imaging of lung cryosections was performed with ToF-SIMS and fluorescence microscopy. Nanoparticles were successfully detected with ToF-SIMS in 3D microanalysis mode based on the lateral distribution of SiO3- (m/z 75.96), which was co-localized with the distribution pattern that was obtained from nanoparticle fluorescence. In addition, the lateral distribution of protein (CN-, m/z 26.00) and phosphate based signals (PO3-, m/z 78.96) originating from the tissue material could be related to the SiO3- lateral distribution. In conclusion, ToF-SIMS is suitable to directly detect and laterally resolve SiO2 nanomaterials in biological tissue at sufficient intensity levels. At the same time, information about the chemical environment of the nanoparticles in the lung tissue sections is obtained.

Structural and photosensitizing features of phthalocyanine--zeolite hybrid nanomaterials.

In this work, we have quantified for the first time the fluorescence and singlet oxygen quantum yields of a silicon(IV) phthalocyanine bound to the surface of zeolite L nanocrystals. The photophysical properties were correlated with the absorption spectra and the morphology of the nanoparticles, and most importantly, with the fraction of photoactive chromophores. By comparison with the fluorescence and singlet oxygen quantum yields of the free phthalocyaninate in dilute solution (ΦF = 0.50 and Φ∆ = 0.50, respectively), we conclude that for the most efficient nanoparticles nearly 80% of chromophores are active as monomeric units on the surface, as indicated by the corresponding quantum yields (ΦF = 0.40 and Φ∆ = 0.40). We further functionalized and raised the ζ-potential of the best performing nanomaterial to improve its water dispersibility. The functionalization was monitored by thermogravimetric analysis and time-of-flight secondary-ion mass spectrometry, and its influence on the photophysical properties was assessed. The resulting nanomaterials are capable of establishing stable suspensions in water while retaining the ability to form reactive oxygen species upon irradiation with red light. This provides a basis for the rational design of photoactive nanomaterials for photodynamic therapy or water decontamination.

Bioimaging TOF-SIMS: High resolution 3D imaging of single cells.

The distribution of phosphocholine ions (m/z 184, m/z 86), sodium ions, and potassium ions in thyroid tumor cells was analyzed by imaging TOF-SIMS. Repeated sputtering with a C(60) (+) source and subsequent analysis with a Bi(3) (+) gun produced a series of 138 images that were stacked to make a 3D display of the chemistry of cells. Phosphocholine was seen in the plasma membrane (m/z 184) and intracellular membranes (m/z 86). The different fragmentation of the phospholipid probably reflects the chemical composition of membranes at these sites. High intensity of secondary ion signals of potassium was seen in membrane-encompassed cellular compartments. The data indicate that potassium ions are compartmentalized in thyroid tumor cells.

Localization of fatty acids with selective chain length by imaging time-of-flight secondary ion mass spectrometry.

Localization of fatty acids in biological tissues was made by using TOF-SIMS (time-of-flight secondary ion mass spectrometry). Two cell-types with a specific fatty acid distribution are shown. In rat cerebellum, different distribution patterns of stearic acid (C18:0), palmitic acid (C16:0), and oleic acid (C18:1) were found. Stearic acid signals were observed accumulated in Purkinje cells with high intensities inside the cell, but not in the nucleus region. The signals colocalized with high intensity signals of the phosphocholine head group, indicating origin from phosphatidylcholine or sphingomyelin. In mouse intestine, high palmitic acid signals were found in the secretory crypt cells together with high levels of phosphorylinositol colocalized in the crypt region. Palmitic acid was also seen in the intestinal lumen that contains high amounts of mucine, which is known to be produced in the crypt cells. Linoleic acid signals (C18:2) were low in the crypt region and high in the villus region. Oleic acid signals were seen in the villi and stearic acid signals were ubiquitous with no specific localization in the intestine. We conclude that the results obtained by using imaging TOF-SIMS are consistent with known brain and intestine biochemistry and that the localization of fatty acids is specific in differentiated cells.

Localization of lipids in the aortic wall with imaging TOF-SIMS.

Time-of-flight secondary-ion-mass-spectrometry (TOF-SIMS) was utilized to address the issue of localization of lipids and inorganic ions in healthy rat aorta and human atherosclerotic plaque. Pieces of rat aorta were high pressure frozen, freeze-fractured and freeze dried. The samples were analyzed by imaging TOF-SIMS equipped with a Bi(1-7)(+)-source. Reference lipid samples were analyzed and compared to data obtained by analysis of the rat aorta samples. Fatty acids, cholesterol, oxysterol and diacylglycerols were detected and localized. A heterogeneous lipid distribution could be shown in the aorta, where the lamellae of the aorta, distinguished by imaging of CN(-), appeared enriched in cholesterol, oxysterol and diacylglycerols, while the smooth muscle tissue, identified by imaging of PO(3), appeared enriched in phosphocholine. Palmitic/palmitoleic acid and stearic/oleic acid appeared to be heterogeneously distributed over the aorta with high concentration areas located especially in the tunica media region of the aorta. Human atherosclerotic plaque showed an irregular cholesterol distribution mainly located in spots in the intima region with elongated diacylglycerol regions located mainly in the media region.

Distribution of cholesterol and galactosylceramide in rat cerebellar white matter.

White matter and the inner granular layer of rat cerebellum was analysed by imaging time-of-flight secondary-ion mass spectrometry (TOF-SIMS) equipped with a Bi+ ion cluster gun. Samples were prepared by high pressure freezing, freeze-fracturing and freeze drying or by plunge freezing and cryostat sectioning. The identified and localized chemical species were: sodium, potassium, phosphocholine, cholesterol and galactosylceramide (GalC) with carbon chain lengths C18:0 (N-stearoyl-galactosylceramide) and C24:0 (N-lignoceroylgalactosylceramide) with CH24:0 (hydroxy-lignoceroylgalactosylceramide). We report new findings regarding the organization of myelin in white matter. One is cholesterol-rich, ribbon-shaped 10-20 microm areas excluding Na+ and K+. The second finding is the different distribution of GalC C18 and GalC C24 in relation to these areas, where GalC C18 was localized in cholesterol-rich areas and GalC C24 was localized in Na/K-enriched areas. The distribution of GalC was in small spots, homogeneous in size, of 0.8-1.5 microm. Sample preparation with high pressure freezing allowed separate localization of sodium and potassium in tissue samples.

Imaging TOF-SIMS of rat kidney prepared by high-pressure freezing.

Phosphocholine, potassium ions, and sodium ions were localized in rat kidney with imaging TOF-SIMS. Tissue preparation was performed with high-pressure freezing, freeze-fracturing and freeze-drying. The distribution of sodium ions was visualized by imaging the signal at m/z 23 of positively charged secondary ions, and the distribution of potassium ions was visualized by imaging the signal at m/z 39. Potassium was found localized within cells of the proximal tubulus epithelium and within cells of the glomeruli. High signals of sodium ions were seen in the interstitial tissue and also in epithelial cells of the collecting ducts and in glomeruli. The overlay image showed that the distribution of sodium ions and potassium ions were largely complementary with color mixing in glomeruli and in the interstitium surrounding proximal tubules. The ion distribution was further analyzed by correlation analysis. Phosphocholine-containing phospholipids were visualized by imaging the phosphocholine head group at m/z 184 of positively charged ions. The m/z 184 signal shows a ubiquitous distribution with a high intensity of phosphocholine in epithelial cells. Overlay image of m/z 184, m/z 39, and m/z 23 and multivariate analysis showed that the localization of high levels of phosphocholine colocalizes with high levels of potassium ions, as expected for an ion with intracellular localization.

Localization of cholesterol, phosphocholine and galactosylceramide in rat cerebellar cortex with imaging TOF-SIMS equipped with a bismuth cluster ion source.

Time-of-flight secondary-ion-mass-spectrometry (TOF-SIMS) was utilized to address the issue of co-localization of cholesterol, phosphocholine and galactosylceramide in rat cerebellar cortex. Rat cerebellum was fixed, freeze-protected by sucrose, frozen and sectioned by cryoultramicrotomy and dried at room temperature. The samples were analyzed in an imaging TOF-SIMS instrument equipped with a Bi(1-7)+-source. The cholesterol signal (m/z 369 and 385) was localized in Purkinje cells and in nuclei of granular layer cells. The phosphocholine headgroup of phosphatidylcholine and sphingomyelin was localized by imaging a specific fragment (m/z 86). This signal was localized in the molecular layer of cerebellar cortex, in Purkinje cells and in parts of the granular layer probably representing the synapse-rich glomeruli. The galactosylceramide was localized by imaging the quasi-molecular ions at m/z 835 and 851, showed a clear colocalization with cholesterol, but also a specific localization in dots (diameter

Tissue molecular ion imaging by gold cluster ion bombardment.

The use of gold cluster focused ion beams produced by a liquid metal ion gun in a TOF-SIMS mass spectrometer is shown to dramatically enhance secondary ion emission of phospholipids and peptides. The method has been successfully tested with cells grown onto plastic slips and with mouse brain slices, without any treatment of the samples. Very reliable time-of-flight mass spectra are acquired with a low primary ion dose of a few 10(7) ions, and high lateral resolution molecular ion images are obtained for heavy ions of great biological interest. This approach offers new opportunities in pharmacological and biological research fields by localizing compounds of interest such as drugs or metabolites in tissues.