Focussed Ion Beam Milling and Scanning Electron Microscopy of Brain Tissue

This protocol describes how biological samples, like brain tissue, can be imaged in three dimensions using the focussed ion beam/scanning electron microscope (FIB/SEM). The samples are fixed with aldehydes, heavy metal stained using osmium tetroxide and uranyl acetate. They are then dehydrated with alcohol and infiltrated with resin, which is then hardened. Using a light microscope and ultramicrotome with glass knives, a small block containing the region interest close to the surface is made. The block is then placed inside the FIB/SEM, and the ion beam used to roughly mill a vertical face along one side of the block, close to this region. Using backscattered electrons to image the underlying structures, a smaller face is then milled with a finer ion beam and the surface scrutinised more closely to determine the exact area of the face to be imaged and milled. The parameters of the microscope are then set so that the face is repeatedly milled and imaged so that serial images are collected through a volume of the block. The image stack will typically contain isotropic voxels with dimenions as small a 4 nm in each direction. This image quality in any imaging plane enables the user to analyse cell ultrastructure at any viewing angle within the image stack

Transmission-electron-microscopy study of quasi-epitaxial tungsten-bronze (Sr2.5Ba2.5Nb10O30) thin film on perovskite (SrTiO3) single crystal

Strontium barium niobate (Sr2.5Ba2.5Nb10O30) thin films were deposited on (001) SrTiO3 single-crystalline substrates by pulsed laser deposition. The growth nature was investigated by transmission electron microscopy (TEM). Selected-area electron diffraction and high-resolution transmission electron microscopy revealed the existence of six types of grains. These grains grew on the substrate in a partially epitaxial fashion. Geometrical models were built, which were confirmed by TEM observations. Based on the TEM results and geometrical analysis, a crystallographic model was developed. The strain nature resulting from the growth columns is discussed in this repor

Advances in 3D focused ion beam tomography

This article summarizes recent technological improvements of focused ion beam tomography. New in-lens (in-column) detectors have a higher sensitivity for low energy electrons. In combination with energy filtering, this leads to better results for phase segmentation and quantitative analysis. The quality of the 3D reconstructions is also improved with a refined drift correction procedure. In addition, the new scanning strategies can increase the acquisition speed significantly. Furthermore, fast spectral and elemental mappings with silicon drift detectors open up new possibilities in chemical analysis. Examples of a porous superconductor and a solder with various precipitates are presented, which illustrate that combined analysis of two simultaneous detector signals (secondary and backscattered electrons) provides reliable segmentation results even for very complex 3D microstructures. In addition, high throughput elemental analysis is illustrated for a multi-phase Ni-Ti stainless steel. Overall, the improvements in resolution, contrast, stability, and throughput open new possibilities for 3D analysis of nanostructured materials

Advances in 3D focused ion beam tomography

This article summarizes recent technological improvements of focused ion beam tomography. New in-lens (in-column) detectors have a higher sensitivity for low energy electrons. In combination with energy filtering, this leads to better results for phase segmentation and quantitative analysis. The quality of the 3D reconstructions is also improved with a refined drift correction procedure. In addition, the new scanning strategies can increase the acquisition speed significantly. Furthermore, fast spectral and elemental mappings with silicon drift detectors open up new possibilities in chemical analysis. Examples of a porous superconductor and a solder with various precipitates are presented, which illustrate that combined analysis of two simultaneous detector signals (secondary and backscattered electrons) provides reliable segmentation results even for very complex 3D microstructures. In addition, high throughput elemental analysis is illustrated for a multi-phase Ni-Ti stainless steel. Overall, the improvements in resolution, contrast, stability, and throughput open new possibilities for 3D analysis of nanostructured material

An integrated human health risk assessment framework for alkylphenols due to drinking water and edible crop consumption

INTRODUCTION The scarcity of clean freshwater is becoming a major issue for present and future generations, especially in densely urbanised areas. This situation promotes the potential cross-contamination of different environmental compartments by contaminants of emerging concern (CECs) which, in fact, have already been detected worldwide in surface water, groundwater and soils. In particular, the CECs released by wastewater treatment plants (WWTPs) can end up both in the recipient surface water and groundwater, both of which are used as drinking water (DW) sources. Furthermore, if those water sources and reclaimed wastewater are used for irrigation, CECs can be directly absorbed by crops intended for human consumption or accumulate in soil and translocate to crops over time. Hence, both DW and edible crops are critical CEC exposure pathways for humans, the combined effect of which requires further investigation. This work is aimed at developing an integrated framework for a quantitative chemical risk assessment due to CECs in complex multiple-use scenarios, combining DW and edible crop consumption, as a decision-making support tool for optimising solutions to minimise risks and social costs. METHODOLOGY The developed procedure includes several steps. Firstly, the analysed system boundaries are defined, to evaluate all the phenomena affecting the fate of CECs from source to end user. Then, CEC migration (e.g. diffusion in surface water, infiltration in soil, uptake by food crops) and human exposure (via water and edible crop consumption) are modelled in an integrated framework as a function of boundary conditions, CECs and by-products characteristics, and proposed interventions. Exposure models are calibrated through literature data, field monitoring and lab tests where, for instance, the CECs’ fate and uptake by vegetables from contaminated soils have been investigated. In the hazard assessment step, a toxicological characterisation was performed to obtain single CEC adverse effect potencies, aimed at applying the Relative Potency Factors methodology for combining CECs that affect the same endpoint. Lastly, exposure and hazard assessment steps are combined to quantitatively estimate the risk to human health from a mixture of CECs, which includes uncertainty analyses to account for knowledge gaps and to provide decision-makers with the confidence level of the risk estimation. RESULTS The developed quantitative risk assessment procedure has been applied to a case study on the mixture of two alkylphenols, i.e. bisphenol-A (BPA) and nonylphenol (NP), used as reference CECs. Literature and field-monitoring data were used to feed the model, with an estimate of BPA and NP concentration in DW up to 0.1 and 0.35 μg/L, respectively, as a function of different system boundary conditions. As for their uptake in edible crops, lab tests with contaminated soil (BPA=75 μg/kg and NP=10 mg/kg, according to the range reported in literature for soil irrigated with reclaimed wastewater or amended with biosolids) demonstrated a significant transfer of NP from soil to vegetables, with concentrations of up to 230 μg/kg fresh weight (f.w.) in the edible parts. No BPA (<8 μg/kg f.w.) was found in vegetables, unlike its metabolite para-hydroxybenzoic acid (up to 56 μg/kg f.w). Those results highlight that both DW and edible crop consumption exposure pathways are critical for the risk to human health due to BPA, NP and their by-products. Several interventions in WWTPs or in DW treatment plants and distribution networks were simulated, demonstrating promising cumulative risk reduction. DISCUSSION Integrated modelling of the fate of CEC mixtures in complex multiple-use water systems, combined with quantitative risk assessment, has proven to be an effective tool to identify the main causes of risk for humans and to assign the various CEC source contributions. Lab tests proved to be useful to investigate the fate of CECs, including metabolites, in the soil system and potential transfer to food crops, corroborating the information from literature and monitoring data for model calibration. Integrated modelling also made it possible to explore several intervention strategies to be adopted at different points of the water system, identifying those that achieve the minimum overall mixture risk. Moreover, in addition to CEC toxicological characterisation, this procedure allows decision-makers to prioritise CECs to be regulated not only based on their exposure levels but looking at their contribution to the overall mixture risk. Lastly, uncertainty analysis made it possible to properly consider the availability and quality of CEC data, especially as regards their physical-chemical behaviour and toxicity, thereby providing the degree of confidence for the estimated risk, which is a key factor for taking informed decisions concerning CEC