Quantification of silver nanoparticle uptake and distribution within individual human macrophages by FIB/SEM slice and view

Background: Quantification of nanoparticle (NP) uptake in cells or tissues is very important for safety assessment. Often, electron microscopy based approaches are used for this purpose, which allow imaging at very high resolution. However, precise quantification of NP numbers in cells and tissues remains challenging. The aim of this study was to present a novel approach, that combines precise quantification of NPs in individual cells together with high resolution imaging of their intracellular distribution based on focused ion beam/ scanning electron microscopy (FIB/SEM) slice and view approaches. Results: We quantified cellular uptake of 75 nm diameter citrate stabilized silver NPs (Ag 75 Cit) into an individual human macrophage derived from monocytic THP-1 cells using a FIB/SEM slice and view approach. Cells were treated with 10 μg/ml for 24 h. We investigated a single cell and found in total 3138 ± 722 silver NPs inside this cell. Most of the silver NPs were located in large agglomerates, only a few were found in clusters of fewer than five NPs. Furthermore, we cross-checked our results by using inductively coupled plasma mass spectrometry and could confirm the FIB/SEM results. Conclusions: Our approach based on FIB/SEM slice and view is currently the only one that allows the quantification of the absolute dose of silver NPs in individual cells and at the same time to assess their intracellular distribution at high resolution. We therefore propose to use FIB/SEM slice and view to systematically analyse the cellular uptake of various NPs as a function of size, concentration and incubation time

Quantification of silver nanoparticle uptake and distribution within individual human macrophages by FIB/SEM slice and view

Background Quantification of nanoparticle (NP) uptake in cells or tissues is very important for safety assessment. Often, electron microscopy based approaches are used for this purpose, which allow imaging at very high resolution. However, precise quantification of NP numbers in cells and tissues remains challenging. The aim of this study was to present a novel approach, that combines precise quantification of NPs in individual cells together with high resolution imaging of their intracellular distribution based on focused ion beam/ scanning electron microscopy (FIB/SEM) slice and view approaches. Results We quantified cellular uptake of 75 nm diameter citrate stabilized silver NPs (Ag 75 Cit) into an individual human macrophage derived from monocytic THP-1 cells using a FIB/SEM slice and view approach. Cells were treated with 10 μg/ml for 24 h. We investigated a single cell and found in total 3138 ± 722 silver NPs inside this cell. Most of the silver NPs were located in large agglomerates, only a few were found in clusters of fewer than five NPs. Furthermore, we cross-checked our results by using inductively coupled plasma mass spectrometry and could confirm the FIB/SEM results. Conclusions Our approach based on FIB/SEM slice and view is currently the only one that allows the quantification of the absolute dose of silver NPs in individual cells and at the same time to assess their intracellular distribution at high resolution. We therefore propose to use FIB/SEM slice and view to systematically analyse the cellular uptake of various NPs as a function of size, concentration and incubation time.TU Berlin, Open-Access-Mittel - 201

Complementary 2D/3D Imaging of Functional Materials using X-ray & Electron Microscopy

Catalysts and other functional materials are generally hierarchically structured materials. Hence, the detailed characterization at different length scales, and especially under reaction conditions, are necessary to unravel their mechanisms and to improve their performance and catalytic activities. Besides, a combination of several techniques is required to acquire complementary information owing to the lack of a single technique able to cover all the length scales. With respect to length, the best way to investigate is by microscopy either in 2D or more preferably in 3D. The work began with an exploration of three different 3D imaging techniques, i.e. ptychographic X-ray computed tomography, electron tomography, and focused ion beam slice-and view. Using nanoporous gold as the model, this study aimed to exhibit the versatility of 3D microscopy as a method beyond imaging as well as to confirm the necessity of complementary nature between them, where electron offers better spatial resolution and X-ray provides larger field of view. The study then continued by utilizing ptychographic X-ray computed tomography for quasi in situ thermal treatment of the same materials under atmospheric pressure. This study highlighted its ease of use of implementing in situ studies, complemented by electron tomography to prove its powerful ability to resolve what ptychographic tomography cannot. The resulting 3D volumes were then used for air permeability and CO2 diffusion simulations, along with material’s electrical and thermal conductivity simulations in order to further expose another excellent benefit from 3D microscopy. Ultimately, the work proceeded into developing two cells in order to perform full in situ investigations under controlled temperatures and atmospheres, where one cell was built for 2D only (X-ray) ptychography experiments with simultaneous X-ray fluorescence mapping, and the other was constructed with an additional capability for 3D limited-angle ptychographic tomography experiments. The feasibility tests were conducted using several functional materials, i.e. nanoporous gold, zeolite, and cobalt-manganese-oxides hollow sphere, as the models for thermal annealing process under specific atmospheres. This work eventually attests the importance of in situ studies in precisely determining the onset annealing temperatures under particular environments, to visualize the morphology online either in 2D or 3D, and to simultaneously map elemental distributions live. Moreover, a complementary technique via transmission electron microscopy was also demonstrated on the same sample, adding up another advantage in using the cells. Despite the preliminary results from in situ limited-angle ptychographic tomography experiments for limitation in data reconstruction, a new tomographic reconstruction technique was recently developed as a solution to acquire 3D images with shortened acquisition times. In conclusions, the work here converges into the ideal case of performing all-around in situ 3D imaging of functional materials for quantitative analysis and simulation

Occurrence and fate of micro- and nanoplastic in the terrestrial environment

The worldwide production of plastic has grown exponentially since the 1950’s and revolutionized our daily life. Simultaneously, plastic pollution in the environment has become a global issue and micro- (MP) and nanoplastics (NP) have now been detected even in the most remote ecosystems. There is currently a data gap due to a lack of analytical methods on the occurrence and characterisation of two highly relevant categories of plastic in the soil environment: tire wear particles (TWP), which concentration in the environment is expected to be high and carry toxic additives, and NP, which toxicity has been demonstrated on soil organisms and is characterized by its ability to cross cell membranes. The effects of micro- and nanoplastic (MNP) on their surrounding environment are determined by their size, morphology, surface characteristics and chemical composition, which can be affected by soil residence time. As the soil is often considered as sink for MNP, it is crucial to investigate and understand the different weathering factors which might affect the MNP properties. To address these knowledge gaps, three main objectives were identified in the scope of this study: develop an extraction and single particle identification method for the quantification and characterisation of (i) TWP and (ii) NP in soil samples and (iii) characterise the physico-chemical properties at the surface of plastic debris occurring in the soil environment, as well as assess the effect of soil and UV weathering as single ageing factors. In order to realise the first objective, a method of extraction and identification of TWP in soil samples based on their black colour was developed using optical microscopy. Cryo-grinded TWP down to a size of 35 μm could be detected with a >85% but the tests conducted with environmental TWP showed that the density used in this study was not efficient to separate the whole range of TWP occurring in different densities. Yet, TWP concentration in highway adjacent soil samples ranged between 8084 ±1059 and 2562 ± 1160 TWP kg-1 dry soil and showed similar trends and magnitude order than previously reported concentrations. Thus, the developed protocol was estimated sufficiently accurate for TWP monitoring in soil samples. Regarding the second objective, an extraction and identification method for NP in soil samples was developed using X-ray spectro-microscopy (STX-NEXAFS). The results demonstrated the suitability of the technique for the imaging and chemical characterisation of individual NP with a minimum dimension of ≈100 nm and its application to the analysis of pure NP and for NP present in environmental and food matrices. However, it was not possible to obtain quantitative data on the NP present in the samples, as the method was too time consuming to allow the measurement of a high number of particles. For the last objective, STXM-NEXAFS was applied to the characterisation of the surface alterations of natural-soil weathered, soil-incubated and UV exposed polymers. A surface alteration on a depth varying between 150 and 1000 nm on could be observed and the analysis of the replicate’s measurement acquired on the same plastic debris highlighted the heterogeneity of the processes affecting polymers surface. The comparison of UV weathered and natural-soil weathered samples showed that the two treatments led to different surface alterations and the absence of surface alteration after one-year soil incubation indicated slow aging of polymers in this medium. Moreover, the very first step of surface fragmentation was observed on a PS fragment, providing an insight on the factors and processes leading to the release of MP and NP in soils. Overall, the present research contributed significantly to the development of innovative methods to characterise MNP in the soil environment. The results obtained helped to provide ground information on the characteristic of environmental MP and NP, which is of high importance to design ecotoxicological test using environmentally relevant material as well as validate predictive models to better understand the potential risk that MP and NP represent for the ecosystems

Experimental and Data-driven Workflows for Microstructure-based Damage Prediction

Materialermüdung ist die häufigste Ursache für mechanisches Versagen. Die Degradationsmechanismen, welche die Lebensdauer von Bauteilen bei vergleichsweise ausgeprägten zyklischen Belastungen bestimmen, sind gut bekannt. Bei Belastungen im makroskopisch elastischen Bereich hingegen, der (sehr) hochzyklischen Ermüdung, bestimmen die innere Struktur eines Werkstoffs und die Wechselwirkung kristallografischer Defekte die Lebensdauer. Unter diesen Umständen sind die inneren Degradationsphänomene auf der mikroskopischen Skala weitgehend reversibel und führen nicht zur Bildung kritischer Schädigungen, die kontinuierlich wachsen können. Allerdings sind einige Kornensembles in polykristallinen Metallen, je nach den lokalen mikrostrukturellen Gegebenheiten, anfällig für Schädigungsinitiierung, Rissbildung und -wachstum und wirken daher als Schwachstellen. Daher weisen Bauteile, die solchen Belastungen ausgesetzt sind, oft eine ausgeprägte Lebensdauerstreuung auf. Die Tatsache, dass ein umfassendes mechanistisches Verständnis für diese Degradationsprozesse in verschiedenen Werkstoffen nicht vorliegt, hat zur Folge, dass die derzeitigen Modellierungsbemühungen die mittlere Lebensdauer und ihre Varianz in der Regel nur mit unbefriedigender Genauigkeit vorhersagen. Dies wiederum erschwert die Bauteilauslegung und macht die Nutzung von Sicherheitsfaktoren während des Dimensionierungsprozesses erforderlich. Abhilfe kann geschaffen werden, indem umfangreiche Daten zu Einflussfaktoren und deren Wirkung auf die Bildung initialer Ermüdungsschädigungen erhoben werden. Die Datenknappheit wirkt sich nach wie vor negativ auf Datenwissenschaftler und Modellierungsexperten aus, die versuchen, trotz geringer Stichprobengröße und unvollständigen Merkmalsräumen, mikrostrukturelle Abhängigkeiten abzuleiten, datengetriebene Vorhersagemodelle zu trainieren oder physikalische, regelbasierte Modelle zu parametrisieren. Die Tatsache, dass nur wenige kritische Schädigungen bezogen auf das gesamte Probenvolumen auftreten und die hochzyklische Ermüdung eine Vielzahl unterschiedlicher Abhängigkeiten aufweist, impliziert einige Anforderungen an die Datenerfassung und -verarbeitung. Am wichtigsten ist, dass die Messtechniken so empfindlich sind, dass nuancierte Schwankungen im Probenzustand erfasst werden können, dass die gesamte Routine effizient ist und dass die korrelative Mikroskopie räumliche Informationen aus verschiedenen Messungen miteinander verbindet. Das Hauptziel dieser Arbeit besteht darin, einen Workflow zu etablieren, der den Datenmangel behebt, so dass die zukünftige virtuelle Auslegung von Komponenten effizienter, zuverlässiger und nachhaltiger gestaltet werden kann. Zu diesem Zweck wird in dieser Arbeit ein kombinierter experimenteller und datenverarbeitender Workflow vorgeschlagen, um multimodale Datensätze zu Ermüdungsschädigungen zu erzeugen. Der Schwerpunkt liegt dabei auf dem Auftreten von lokalen Gleitbändern, der Rissinitiierung und dem Wachstum mikrostrukturell kurzer Risse. Der Workflow vereint die Ermüdungsprüfung von mesoskaligen Proben, um die Empfindlichkeit der Schädigungsdetektion zu erhöhen, die ergänzende Charakterisierung, die multimodale Registrierung und Datenfusion der heterogenen Daten, sowie die bildverarbeitungsbasierte Schädigungslokalisierung und -bewertung. Mesoskalige Biegeresonanzprüfung ermöglicht das Erreichen des hochzyklischen Ermüdungszustands in vergleichsweise kurzen Zeitspannen bei gleichzeitig verbessertem Auflösungsvermögen der Schädigungsentwicklung. Je nach Komplexität der einzelnen Bildverarbeitungsaufgaben und Datenverfügbarkeit werden entweder regelbasierte Bildverarbeitungsverfahren oder Repräsentationslernen gezielt eingesetzt. So sorgt beispielsweise die semantische Segmentierung von Schädigungsstellen dafür, dass wichtige Ermüdungsmerkmale aus mikroskopischen Abbildungen extrahiert werden können. Entlang des Workflows wird auf einen hohen Automatisierungsgrad Wert gelegt. Wann immer möglich, wurde die Generalisierbarkeit einzelner Workflow-Elemente untersucht. Dieser Workflow wird auf einen ferritischen Stahl (EN 1.4003) angewendet. Der resultierende Datensatz verknüpft unter anderem große verzerrungskorrigierte Mikrostrukturdaten mit der Schädigungslokalisierung und deren zyklischer Entwicklung. Im Zuge der Arbeit wird der Datensatz wird im Hinblick auf seinen Informationsgehalt untersucht, indem detaillierte, analytische Studien zur einzelnen Schädigungsbildung durchgeführt werden. Auf diese Weise konnten unter anderem neuartige, quantitative Erkenntnisse über mikrostrukturinduzierte plastische Verformungs- und Rissstopmechanismen gewonnen werden. Darüber hinaus werden aus dem Datensatz abgeleitete kornweise Merkmalsvektoren und binäre Schädigungskategorien verwendet, um einen Random-Forest-Klassifikator zu trainieren und dessen Vorhersagegüte zu bewerten. Der vorgeschlagene Workflow hat das Potenzial, die Grundlage für künftiges Data Mining und datengetriebene Modellierung mikrostrukturempfindlicher Ermüdung zu legen. Er erlaubt die effiziente Erhebung statistisch repräsentativer Datensätze mit gleichzeitig hohem Informationsgehalt und kann auf eine Vielzahl von Werkstoffen ausgeweitet werden

Methods, microstructure and mudrocks : towards an improved understanding of deep-water mudrocks

Microstructure controls the petrophysical properties of mudrocks. Knowledge about mudrock microstructure in general is maturing and there is an on-going research field aimed at developing accurate, reliable and fast methods to characterise them. The focus of this research is on mudrocks from the deep-water setting, which are deposited by three principal processes operating in the deep-water: downslope turbidity currents (turbidites), along-slope bottom currents (contourites) and vertical fall-out from surface suspension (hemipelagites). Distinguishing between these respective deposits is challenging but very important in understanding the deep-water environment in terms of their petroleum systems and reservoir characteristics. Hence, the present research entails methodology development and documenting the microstructure of the three principal sedimentary facies in the deep-water. Scanning electron microscopy is a common technique for studying mudrock microstructure. An efficient and effective method for analysing grain size of mudrocks was developed, which gives closely comparable results to laser diffraction granulometry. A fast and reliable approach for characterising detailed mudrock microstructure using automated large-area, high-resolution scanning electron microscopy and image processing is also presented. The method is automated, free of human subjectivity and provides robust information on mudrock microstructure. Interestingly, the developed method gives comparable results with a synchrotron x-ray diffraction technique. Furthermore, the research presents exciting new insights on microstructure of deep-water fine grained sediments. A microfabric model is presented for the deep-water fine grained sediments. Turbidites have pronounced preferred bedding parallel fabric, produced by turbulence and high sedimentation rate, with little or no bioturbation effect. Contourites possess mixed fabric (random - semi random and parallel to the bedding). The mixed fabric is suggested to be developed by weak turbulence (bottom currents) and distortion of the fabric by bioturbation. Hemipelagites are characterised by random and oblique preferred microfabrics, which are produced by absence of current and pervasive bioturbation. The oblique preferred microfabric is suggested to be a product of extensive burrowing, in which grains are aligned along the length of the burrows. Additional important findings are that, depositional processes and sedimentation rate as well as burial depth are the most important controlling factors of microstructure development within the deep-water sediments. Mudrocks are prevalent is all sedimentary environments. The need for a cleaner source of energy for the 21st century has revolutionized interest in mudrocks, as they are recognised as potential unconventional reservoirs. They are also important in terms of carbon storage, as repositories for nuclear waste and as records of environmental change. The contributions presented on mudrock microstructure in this research are relevant to studying the generality of mudrocks without recourse to their environment and not just restricted to deep-water mudrocks

Microscopy Conference 2017 (MC 2017) - Proceedings

Das Dokument enthält die Kurzfassungen der Beiträge aller Teilnehmer an der Mikroskopiekonferenz "MC 2017", die vom 21. bis 25.08.2017, in Lausanne stattfand

Multi-scale 3D Imaging for Characterization of Microstructural Properties of Gas Shales

In recent years, gas shale has attracted renewed attention as an unconventional energy resource, with massive, fast-growing, and largely untapped reserves. Shale is a fine-grained sedimentary rock containing a high content of organic matter (kerogen) from which gas can be extracted. The identification of the pore structure and quantification of the geometry, sizes, volume, connectivity, and distribution of extremely fine-grain pores, kerogen, and minerals are all extremely significant for the characterization of gas shale reservoirs. These features determine fluid flow and ultimate hydrocarbon recovery, however, they are also highly challenging to determine accurately. X-ray micro and nano-computed tomography (μ-CT and Nano-CT) combined with 3D focused ion beam scanning electron microscopy (FIB-SEM) are used in this thesis to address this challenge and to provide more information for understanding the complex microstructures in 3D from multiple scales within shale samples. In this thesis, state-of-the-art multi-scale imaging with multi-dimensional potential was applied to the image and quantified the microstructures' properties of gas shale. Samples were first imaged with X-ray micro-and nano-tomography (μ-CT and Nano-CT), and then with Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) measurements. Results of image analysis using SEM (2D), μ-CT (3D), ultrahigh-resolution Nan-CT (3D), and FIBSEM (3D) under backscattered electron (BSE) images reveal a complex fine-grained structure at specified phases such as pores, kerogen, and minerals within samples. The results show low connectivity of pores and high connectivity of kerogen which suggests that porous gas flow through samples used in this study cannot be the main transport through the pores. This implies that the gas transport through the pores is unlikely to be important, but cannot be ignored, as it is very important and constitutes the basis for understanding permeability in the rock. However, the high connectivity of kerogen provides the potential pathways for gas flow throughout the whole sample. The combination of multiscale 3D X-ray CT techniques (micro-nano) with 3D FIB-SEM provides a powerful combination of tools for quantifying microstructural information including pore volume, size, pore aspect ratio and surface area to volume distributions, porosity, permeability in shales, and also allowing the visualization of pores, kerogen and minerals phases over a range of scales. Other methods, such as physical measurements Gas Research Institute (GRI), mercury injection (MIP), and nitrogen adsorption (N2) are also presented in this study. The combination of these data sets has allowed the examination of the microstructure of the shale in unprecedented depth across a wide range of scales (from about 20 nm to 0.5 mm). Overall, the shale samples from Bowland shale formation shows a porosity of 0.10 ± 0.01%, 0.52 ± 0.05%, and 0.94 ± 0.09% from three FIB-SEM measurements, 0.67 ± 0.009% from the nano-CT data and 0.06 ± 0.008% from one μ-CT measurement, which compare with 0.0235 ± 0.003% from nitrogen adsorption, and 0.60 ± 0.07% from MIP. The porosity was also observed to be 0.43± 0.009% and 0.7% ± 0.007% for FIB-SEM and Nano-CT methods, respectively in different shale reservoirs from a Sweden formation. The data vary due to the different scales at which each technique interrogates the rock and whether the pores are openly accessible (especially in the case of nitrogen adsorption). The measured kerogen fraction is 32.4 ± 1.45% from nano-CT compared with 34.8 ± 1.74%, 38.2 ± 1.91%, 41.4 ± 2.07%, and 44.5 ± 2.22% for three FIB-SEM and one μ-CT measurement. The pore size imaged by nano-CT ranged between 100 and 5000 nm, while the corresponding ranges were between 3 and 2000 nm for MIP analysis and between 2 and 90 nm for N2 adsorption. The distribution of pore aspect ratio and scale-invariant pore surface area to volume ratio (σ) as well as the calculated permeability shows the shale sample in this study to have a high shale gas potential. Aspect ratios indicate that most of the pores that contribute significantly to pore volume are oblate, which is confirmed by the range of σ (3−30). Oblate pores have greater potential for interacting with other pores compared to needle-shaped prolate pores as well as optimizing surface area for the gas to desorb from the kerogen into the pores. Permeability has also been calculated and values of 2.61 ± 0.42 nD were obtained from the nano-CT data, 2.65 ± 0.45 nD from MIP, 13.85 ± 3.45 nD, 4.16 ± 1.04 nD, and 150 ± 37.5 nD from three FIB-SEM measurements and 2.98 ± 0.75 nD from one μ-CT measurement, which are consistent with expectations for generic gas shales (i.e., tens of nD). The quantitative results of 2D and 3D imaging datasets across nm-μm-mm length scales provided a view of understanding the heterogeneous rock types, as well as great value to better understand, predict and model the pore structure, hydrocarbon transport, and production from gas shale reservoirs