The area of particle characterization is expansive; it contains many technologies and methods of analysis. Light spectroscopy techniques yield information on the joint property distribution of particles, comprising the chemical composition, size, shape, and orientation of the particles. The objective of this dissertation is to develop a hybrid scattering-absorption model incorporating Mie and Rayleigh-Debye-Gans theory to characterize submicron particles in suspension with multiwavelength spectroscopy.Rayleigh-Debye-Gans theory (RDG) was chosen as a model to relate the particles joint property distribution to the light scattering and absorption phenomena for submicron particles. A correction model to instrument parameters of relevance was implemented to Rayleigh-Debye-Gans theory for spheres. Behavior of nonspherical particles using RDG theory was compared with Mie theory (as a reference). A multiwavelength assessment of Rayleigh-Debye-Gans theory for spheres was conducted where strict adherence to the limits could not be followed. Reported corrections to the refractive indices were implemented to RDG to try and achieve Mies spectral prediction for spheres.The results of studies conducted for RDG concluded the following. The angle of acceptance plays an important role in being able to assess and interpret spectral differences. Multiwavelength transmission spectra contains qualitative information on shape and orientation of non-spherical particles, and it should be possible to extract this information from carefully measured spectra. There is disagreement between Rayleigh-Debye-Gans and Mie theory for transmission simulations with spherical scatterers of different sizes and refractive indices.

Septinas são GTPases consideradas como um novo componente do citoesqueleto. Essas proteínas interagem entre si para formar heterocomplexos filamentosos e estruturas de alta ordem que são importantes para a citocinese e uma variedade de outros processos celulares. Existem muitos aspectos mecânicos dessas proteínas que não são totalmente compreendidos, incluindo a forma como os heterocomplexos se agrupam corretamente. Em humanos, há 13 genes que codificam septinas, classificadas em quatro grupos quanto à similaridade em relação à estrutura primária. O complexo hexamérico SEPT7-SEPT6-SEPT2-SEPT2-SEPT6-SEPT7 foi o melhor caracterizado, com uma estrutura cristalina resolvida à 4 Å. Segundo às Regras de Kinoshita, as septinas desse complexo podem ser substituídas nesse arranjo por outras pertencentes ao mesmo grupo. Neste trabalho utilizamos a técnica de microscopia eletrônica de transmissão com análise de partícula única para estudar dois complexos de septinas. Um dos complexos estudados neste projeto é formado por septinas humanas, para as quais atualmente não há informações estruturais disponíveis. O complexo SEPT5-SEPT6-SEPT7 foi expresso heterólogamente em E. coli e purificado em alta concentração salina para evitar a polimerização. A análise de partículas únicas de imagens por contrastação negativa mostrou a presença de partículas alongadas de aproximadamente 25 nm de comprimento, compostos por seis monômeros, como esperado. Com o objetivo de localizar a posição da SEPT5 no complexo, foi realizada uma fusão com MBP (Maltose Binding Protein) e imunomarcação com anticorpo monoclonal anti-SEPT5, concluindo que a SEPT5 está localizada na extremidade do complexo hexamérico. Porém, a SEPT5 pertence ao mesmo grupo da SEPT2, que foi relatada estar localizada no centro do hexâmero. Este resultado possibilitou uma nova discussão sobre a maneira que as septinas formam os complexos e, como a sensibilidade à concentração salina está relacionada com a fragilidade da interface NC, análogo ao observado em complexos de Saccharomyces cerevisiae. Um complexo de Ciona intestinalis incluindo a SEPT2, SEPT6, SEPT7 e SEPT9, também expresso heterólogamente em E. coli, foi preparado por contrastação negativa. A análise de partícula única das imagens coletadas mostrou um heterocomplexo aparentemente hexamérico, embora fosse esperado um octâmero devido à presença das quatro septinas diferentes, sendo uma pertencente à cada um dos quatro grupos. Os resultados deste trabalho proporcionaram um avanço na compreensão da formação de heterocomplexos de septinas e como essas proteínas interagem umas com as outras nesta montagem. / Septins are GTPases that appear to be a novel component of the cytoskeleton. These proteins interact with each other to form filamentous heterocomplexes and high order structures which are important for cytokinesis and a variety of other cellular processes. There are many mechanistic aspects of these proteins that are not fully understood, including how the heterocomplexes correctly assemble. In humans, there are 13 genes encoding septins, classified in four groups based on primary structure. The SEPT7-SEPT6-SEPT2-SEPT2-SEPT6-SEPT7 hexameric complex was the best characterized, with a crystalline structure solved at 4 Å. According to Kinoshita´s Rules, the septins of this complex can be replaced in this arrangement by others belonging to the same group. In this work, we used transmission electron microscopy with single particle analysis to study two septin complexes. One of the complexes studied in this project is composed of three human septins, for which there is currently no structural information available. The SEPT5-SEPT6-SEPT7 complex was heterologously expressed in E. coli and purified at high salt concentration to avoid polymerization. Single particle analysis of negatively stained samples showed the presence of elongated particles of approximately 25 nm in length. To locate SEPT5 in the complex, a fusion with MBP (Maltose Binding Protein) and immunoblotting with anti-SEPT5 monoclonal antibody was performed, concluding that SEPT5 is located at the end of the hexameric complex. However, SEPT5 belongs to the same group as SEPT2, which was reported to be located in the center of the hexamer. This result allowed for a new discussion on the way that septins form heterocomplexes and also, on how the sensitivity of the NC interface in related to salt concentration, analogous to that observed in the heterocomplex of Saccharomyces cerevisiae. A Ciona intestinalis complex including SEPT2, SEPT6, SEPT7 and SEPT9, also expressed heterologously in E. coli, was prepared by negative staining. The single particle analysis of the collected images showed an apparently hexameric heterocomplex, although an octamer was expected due to the presence of the four different septins, one belonging to each of the four groups. The results of this work represent advances in the understanding of the formation of septin heterocomplexes and how these proteins interact with each other during assembly.

Semester project deals with the processing of data from TEM and their analysis (to- mography, single particle analysis). The main aim of this work is to determine the 3D structure of the studied enzyme. As a test sample with low symmetry is used restriction endonuclease EcoR124I.

Particle contamination control is a critical issue for the semiconductor industry. In the near future, this industry will be concerned with the chemical identities of contaminant particles as small as 0.01 pm in size. Therefore, analytical techniques with both high chemical sensitivity and spatial resolution are required. Transmission electron microscopy (TEM) provides excellent spatial resolution and yields structural and compositional information. It is rarely used, however, due to the difficulty of sample preparation. The goals of this research are to promote the use of TEM as an ultrafine particle analysis tool by developing new sample preparation methods, and to exploit the new TEM techniques for analysis of particles in semiconductor processing fluids. A TEM methodology for the analysis of particulate contaminants in fluids with an elemental detectability limit as low as 0.1 part per trillion (ppt), and a particle concentration detectability limit as low as 1 particle/ml for particles greater than 0.2 pm was developed and successfully applied to the analysis of particles in HF, H202, de-ionized (DI) water, and on the surface of an electronic device. HF samples from three manufacturers were examined. For HF (B), the maximum particle concentration was 8.3 x 103 particles/ml. Both a viscous material and lath-shaped particles were observed. The Sb concentration was less than 0.6 part per billion (ppb). HF (C) was the cleanest. CaF2 and TiO2 particles were identified in HF (D). For H2 02, iron and tin oxides and hydroxides were identified. The maximum particle concentration was 990 particles/ml. The Sn and Fe concentrations were less than 0.3 ppb. Spherical and dendritic particles were observed. For DI water, spherical and dendritic particles (<2 particles/ml), and particles containing Fe or Si with concentrations less than 0.1 ppt were observed. Contaminants on an electronic device surface were also analyzed. Clusters of small particles were determined to be a mixture of aluminum oxides and aluminum silicates.

The aim of this thesis is to investigate applicability of regulariza- tion by Krylov subspace methods to discrete inverse problems arising in single particle analysis (SPA). We start with a smooth model formulation and describe its discretization, yielding an ill-posed inverse problem Ax ≈ b, where A is a lin- ear operator and b represents the measured noisy data. We provide theoretical background and overview of selected methods for the solution of general linear inverse problems. Then we focus on specific properties of inverse problems from SPA, and provide experimental analysis based on synthetically generated SPA datasets (experiments are performed in the Matlab enviroment). Turning to the solution of our inverse problem, we investigate in particular an approach based on iterative Hybrid LSQR with inner Tikhonov regularization. A reliable stopping criterion for the iterative part as well as parameter-choice method for the inner regularization are discussed. Providing a complete implementation of the proposed solver (in Matlab and in C++), its performance is evaluated on various SPA model datasets, considering high levels of noise and realistic distri- bution of orientations of scanning angles. Comparison to other regularization methods, including the ART method traditionally used in SPA,...

Pseudomonas aeruginosa est une bactérie Gram-négative qui présente une grande résistance aux antibiotiques, lui permettant de sévir dans le milieu hospitalier en infectant plus particulièrement les patients immunodéprimés. Cette résistance est principalement due au système d’efflux membranaire MexAB-OprM, capable d’exporter les antibiotiques en dehors de la cellule. Cette pompe à efflux est composée de trois protéines, MexA, MexB et OprM, incorporées dans les membranes internes et externes de la paroi bactérienne. Les structures de MexA, OprM et AcrB -une protéine présente chez E. coli, homologue de MexB- ont été déterminées individuellement par cristallographie des rayons X. Cependant, la structure du complexe entier, regroupant les trois protéines en interaction, ainsi que le mécanisme de cette pompe font toujours défaut. Le renforcement de nos connaissances structurales et fonctionnelles est donc capital pour lutter plus efficacement contre ces bactéries, par de nouvelles stratégies médicamenteuses. Ce travail porte sur l’étude de la structure et de la stœchiométrie de l’assemblage des protéines OprM et MexA au sein d’une membrane lipidique. La caractérisation du complexe OprM/MexA a été réalisée à l’aide de nouvelles techniques de caractérisation physico-chimique des surfaces, telle que la Microbalance à Cristal de Quartz avec Mesure de Dissipation (QCM-D), et par des méthodes d’imagerie, telles que la Cryo-Microscopie Electronique en Transmission (CryoMET) et la Cryo-Tomographie Electronique (CryoTE). En QCM-D, les mesures d’interaction entre OprM et MexA ont été réalisées sur support solide en contrôlant l’orientation d’OprM placée dans un environnement lipidique. Après ajout de la protéine MexA, la formation de complexes OprM/MexA a été mise évidence. Pour comprendre l’organisation de ce complexe, nous avons procédé à une étude comparative de l’organisation des protéines OprM, MexA et du complexe OprM/MexA incorporés dans une membrane lipidique, par CryoMET. Trois types d’organisation, respectivement spécifiques d’OprM, de MexA et du complexe OprM/MexA, ont été mis en évidence. Une analyse structurale de ces trois différents assemblages, pris en sandwich entre deux membranes lipidiques, a été menée par CryoTE. La reconstitution de la protéine OprM conduit à la formation de protéoliposomes, dû à des interactions intervenant entre les protéines OprM au niveau de leurs hélices périplasmiques. La protéine MexA s’organise sous forme d’une structure annulaire de 13 nm de hauteur au sein des membranes lipidiques, et d’une structure plus complexe de 26 nm de hauteur, résultant de l’empilement tête-bêche de deux structures annulaires de 13 nm. Ce travail révèle les dimensions exactes de l’assemblage formé par MexA, et permet de localiser à proximité des membranes les domaines non résolus dans la structure cristallographique. La reconstitution du complexe OprM/MexA révèle une disposition régulière des deux protéines dans les membranes lipidiques. Au sein des complexes, les protéines OprM sont présentes sous forme de trimères. Dans la membrane opposée, à l’aplomb d’une molécule d’OprM, MexA ne forme pas une structure annulaire similaire à celle décrite précédemment, indiquant un état d’oligomérisation différent de celui observé dans les assemblages MexA. Les densités de MexA sont compatibles avec la présence de quelques molécules de MexA. Cependant des structures annulaires de MexA, positionnées à l’aplomb de trois trimères d’OprM sont visibles. Notre étude montre que MexA adopte des structures oligomériques spécifiques en fonction de ses interactions avec les membranes lipidiques ou avec son partenaire OprM. / The structure determination of membrane protein in lipid environment can be carried out using cryo electron microscopy combined with the recent development of data collection and image processing. We describe a protocol to study assemblies or stacks of membrane protein reconstitued into a lipid membrane using both cryo electron tomography and single particle analysis which is an alternative approach to electron crystallography for solving 3D structure. We show the organization of the successive layers of OprM molecules revealing the protein-protein interactions between OprM molecules of two successive lipid bilayers.

The transcription initiation is the first step in gene expression and an important regulation step in all living organisms. In bacteria, it has been proposed that DNA bending and its wrapping on the surface of E. coli RNAP might facilitate the opening of the transcription bubble, which is necessary for the initiation of gene transcription. In this work, it is shown the first structural study to evaluate a DNA wrapping model, including its length and the relative position in the bacterial transcription initiation complex (RP complex), assembled between RNA polymerase-&sigma;70 holoenzyme (RNAP) and a &lambda;PR promoter (-100 to +30 wild type). RP complex was prepared and negatively stained with 2% uranyl acetate on a thin-carbon coated grid and the data acquisition of 500 images was performed in a JEM-2100 (JEOL, Japan) microscope equipped with an F-416 CMOS camera (TVIPS, Germany). Single particle analysis of 16,015 particles, grouped in 666 class-averages, was conducted using IMAGIC 4D software (Image Science, Germany) to obtain a three-dimensional model of the RP complex at 20&Aring; resolution. After the rigid-body fitting of the RNAP crystallographic structure (PDB 4YG2) and the modeled DNA promoter, it was observed that the regions 1.2 and 4.2 of the &sigma;70 subunit interacts with the consensus zones, -10 and -35 hexamers of the promoter. Furthermore, it was possible to observe that &alpha;CTDs (C-terminal domain) in both alpha subunits would be oriented to facilitate the interaction with the first and second UP-elements regions, respectively (centered around &ndash;50 and -75 positions in the promoter). These was enabled by the presence of the characteristics motifs helix-hairpin-helix in these domains. In addition, the downstream DNA, from the transcription bubble, appears to be inside the protein main channel, oriented in a way to enable interactions with the RNAP clamp and jaws. Finally, it was observed that the DNA wrapping has ~32 nm of total length and involves a promoter bent of ~255&deg; around the RNAP surface. The 3D-model obtained in this study is the very first direct structural confirmation of the DNA promoter wrapping in a bacterial transcription initiation complex. / A iniciação da transcrição é o primeiro passo na expressão gênica e importante ponto de regulação em todos os organismos vivos. Em bactérias, foi proposto que o enovelamento do DNA na superfície da RNAP de E. coli pode facilitar a abertura da bolha de transcrição, necessária para o início da transcrição gênica. Neste trabalho, é apresentado o primeiro estudo estrutural direto para avaliar o comprimento do enovelamento do DNA e sua posição no complexo de iniciação da transcrição bacteriana (complexo RP), montado entre a holoenzima RNA polimerase-&sigma;70 (RNAP) e um promotor &lambda;PR (-100 para +30, tipo selvagem). Amostras do complexo RP foram preparadas e contrastadas negativamente com 2% de acetato de uranila em uma grade com filme fino de carbono e a aquisição de 500 imagens foi realizada em um microscópio JEM-2100 (Jeol, Japão) equipado com uma câmera CMOS F-416 (TVIPS, Alemanha). A análise de partículas isoladas de 16.015 partículas, agrupadas em 666 médias de classe, foi conduzida usando o software IMAGIC 4D (Image Science, Alemanha) para obter um modelo tridimensional do complexo RP, a 20&Aring; de resolução, estimado pelo critério de &frac12; bit. Após o ajuste de corpo rígido da estrutura cristalográfica da RNAP (PDB 4YG2) e do promotor de DNA modelado, observou-se que as regiões 1.2 e 4.2 da subunidade &sigma;70 interagem com as zonas de consenso, hexâmeros -10 e -35, do promotor. Além disso, foi possível observar que os &alpha;CTDs (domínio C-terminal) em ambas as subunidades alfa estariam orientados para facilitar uma possível interação com a primeira e segundas regiões dos elementos UP, respectivamente (centradas em torno das posições &ndash;50 e -75 do promotor). Estas seriam possíveis devido à presença de alguns motivos de características hélice-grampo-hélice nesses domínios. Além disso, a região do promotor, downstream da bolha de transcrição, parece estar dentro do canal principal da proteína, orientado de forma a possibilitar interações com o clamp e jaw da RNAP. Finalmente, foi observado que o comprimento total do enovelamento de DNA envolve cerca de 32 nm e 255&deg; de rotação do DNA ao redor da superfície da RNAP. Portanto, este modelo 3D é a primeira confirmação estrutural direta do enovelamento de DNA em um complexo bacteriano de iniciação da transcrição.

The overall structure of photosynthetic pigment-protein complexes and thylakoid membranes of various photosynthetic organisms was studied using electron microscopy.

The KdpFABC complex from E. coli functions as a high affinity K uptake system and belongs to the superfamily of P-type ATPases. So far, no information is available about the orientation of the subunits within the complex as well as its oligomeric state. By chemical crosslinking, gel filtration, electron transmission microscopy and single particle FRET analysis this study shows that the KdpFABC complex occurs as a homodimer with a dissociation constant between 30 to 50 nM. Furthermore, by means of single particle analysis of transmission electron micrographs, the solution structure of the complex at 1.9 nm resolution could be solved, thus providing the first structural analysis resolving all subunits of the holoenzyme. Based on crystal structures, it is generally assumed that P-type ATPases undergo large domain movements during catalysis. However, these conformational changes have never been shown directly. By use of single molecule FRET with alternating laser excitation, distance changes could be measured directly within KdpB during ATP hydrolysis. With this technique, distances and dwell times were determined for three conformational states in the working enzyme as well as in the orthovanadate- and the OCS-inhibited state.

The present work was carried out within the framework of the priority programme SPP 2045. Technical ultra–fine particle systems (< 10μm) from highly specific separation processes are to be investigated here with regard to multi–dimensional property distributions. Tomographic measurement methods allow a comprehensive 3D description of particle–discrete data sets of statistically relevant size. The focus of the work is on X–ray tomographic analysis by means of micro-computed tomography (micro–CT), which, if necessary, is extended to several size scales by including further measurement methods (nano–CT) and supplemented by suitable elemental analysis (FIB–SEM + EBSD, EDX). Two preparation methods (wax, epoxy resin) for different particle preparations are described methodically, which have already been published in a case study or are the subject of current studies in the outlook of the work. Finally, a networked multiple use of the generated data within an online particle database is shown and its application is explained using three concrete examples.:1 Outline 2 Description of Particle Properties 2.1 Integral or Class–Based Description 2.2 Particle–Discrete Description 2.2.1 2D Description 2.2.2 Full 3D Description 2.3 Multidimensional Characterization on Basis of Particle–Discrete 3D Data 2.3.1 Motivation 2.3.2 Kernel Density Approach 2.3.3 Copula Approach 3 X–ray Tomography 3.1 Historical Context 3.2 X–ray Physics 3.2.1 X–ray Generation 3.2.2 Polychromatic Spectrum 3.2.3 Interaction with Matter 3.3 Tomographic Imaging 3.3.1 Motivation 3.3.2 Basic Idea 3.3.3 X–ray Microscopy Measurement Setup andWorkflow 3.3.4 Tomographic Reconstruction via Filtered Back Projection 3.3.5 Region of Interest Tomography 3.4 Relevant Artefacts Related to Particle Measurement 3.4.1 Temperature Drift 3.4.2 Penumbral Blurring and Shadow 3.4.3 Cone Beam 3.4.4 Out–of–Field 3.4.5 Center Shift 3.4.6 Sample Drift 3.4.7 Beam Hardening 3.4.8 Rings 3.4.9 Noise 3.4.10 Partial Volume 3.4.11 Summary 4 Practical Implementation 4.1 Particle Sample Requirements 4.1.1 Geometry 4.1.2 Dispersity and Homogeneity 4.2 Statistics 4.2.1 Single Particle Properties 4.2.2 Properties of a Limited Number of Particles (10 to several 100) 4.2.3 Particle Populations with Distributed Properties 4.3 2D Validation 4.4 Measurement 4.4.1 X–ray Microscope 4.4.2 Source Filter 4.4.3 Detector Binning 4.4.4 Cone Beam Artefact Compensation 4.4.5 Center Shift Correction 4.4.6 Dynamic Ring Removal 5 Image Analysis 5.1 Image Quality 5.1.1 Grey Value Histogram 5.1.2 Resolution 5.1.3 Signal–to–Noise Ratio 5.1.4 Contrast and Dynamic Range 5.1.5 Sharpness 5.1.6 Summary 5.2 Basic Image Processing Strategies 5.2.1 Threshold–Based Segmentation 5.2.2 Machine Learning Assisted Segmentation 6 Correlative Tomography 6.1 Scouting Approach 6.2 Multiscale Approach 6.3 Multidisciplinary Approach 7 Data Management 7.1 Data Quality 7.2 Data Availability 7.2.1 Tomographic Datasets 7.2.2 Particle Database 8 Outlook on Further Research Activities 9 Publications 9.1 Copyright Declaration 9.2 Overview 9.3 List of Publications Paper A, Preparation techniques for micron–sized particulate samples in X–ray microtomography Paper B, Self–constructed automated syringe for preparation of micron–sized particulate samples in X–ray microtomography Paper C, Preparation strategy for statistically significant micrometer–sized particle systems suitable for correlative 3D imaging workflows on the example of X–ray microtomography Paper D, Multi–scale tomographic analysis for micron–sized particulate samples Paper E, PARROT: A pilot study on the open access provision of particle discrete tomographic datasets 10 Appendix 10.1 Application Example 1: Fracture Analysis 10.2 Application Example 2: 3D Contact Angle Measurement 10.3 Influence of the Source Filter 10.4 Influence of the X–rays on the Sample 10.5 Appropriate Filter Settings 10.6 Log File Parser / Die vorliegende Arbeit ist im Rahmen des Schwerpunktprogramms SPP 2045 entstanden. Technische Feinstpartikelsysteme (< 10μm) aus hochspezifischen Trennprozessen sollen hier hinsichtlich mehrdimensionaler Eigenschaftsverteilungen untersucht werden. Tomographische Messverfahren erlauben dabei eine vollständige 3D Beschreibung partikeldiskreter Datensätze statistisch relevanter Größe. Der Schwerpunkt der Arbeit liegt auf der röntgentomographischen Analyse mittels Mikro–Computertomographie (mikro–CT), die im Bedarfsfall unter Einbeziehung weiterer Messmethoden (nano–CT) auf mehrere Größenskalen erweitert und durch geeignete Elementanalytik (FIB–SEM + EBSD, EDX) ergänzt wird. Methodisch werden zwei Präparationsverfahren (Wachs, Epoxidharz) für unterschiedliche Partikelpräparate beschrieben, welche in einer Fallstudie bereits veröffentlicht bzw. im Ausblick der Arbeit Gegenstand aktueller Studien ist. Schließlich wird eine vernetzte Mehrfachnutzung der erzeugten Daten innerhalb einer online-Partikeldatenbank gezeigt und deren Anwendung an drei konkreten Beispielen erläutert.:1 Outline 2 Description of Particle Properties 2.1 Integral or Class–Based Description 2.2 Particle–Discrete Description 2.2.1 2D Description 2.2.2 Full 3D Description 2.3 Multidimensional Characterization on Basis of Particle–Discrete 3D Data 2.3.1 Motivation 2.3.2 Kernel Density Approach 2.3.3 Copula Approach 3 X–ray Tomography 3.1 Historical Context 3.2 X–ray Physics 3.2.1 X–ray Generation 3.2.2 Polychromatic Spectrum 3.2.3 Interaction with Matter 3.3 Tomographic Imaging 3.3.1 Motivation 3.3.2 Basic Idea 3.3.3 X–ray Microscopy Measurement Setup andWorkflow 3.3.4 Tomographic Reconstruction via Filtered Back Projection 3.3.5 Region of Interest Tomography 3.4 Relevant Artefacts Related to Particle Measurement 3.4.1 Temperature Drift 3.4.2 Penumbral Blurring and Shadow 3.4.3 Cone Beam 3.4.4 Out–of–Field 3.4.5 Center Shift 3.4.6 Sample Drift 3.4.7 Beam Hardening 3.4.8 Rings 3.4.9 Noise 3.4.10 Partial Volume 3.4.11 Summary 4 Practical Implementation 4.1 Particle Sample Requirements 4.1.1 Geometry 4.1.2 Dispersity and Homogeneity 4.2 Statistics 4.2.1 Single Particle Properties 4.2.2 Properties of a Limited Number of Particles (10 to several 100) 4.2.3 Particle Populations with Distributed Properties 4.3 2D Validation 4.4 Measurement 4.4.1 X–ray Microscope 4.4.2 Source Filter 4.4.3 Detector Binning 4.4.4 Cone Beam Artefact Compensation 4.4.5 Center Shift Correction 4.4.6 Dynamic Ring Removal 5 Image Analysis 5.1 Image Quality 5.1.1 Grey Value Histogram 5.1.2 Resolution 5.1.3 Signal–to–Noise Ratio 5.1.4 Contrast and Dynamic Range 5.1.5 Sharpness 5.1.6 Summary 5.2 Basic Image Processing Strategies 5.2.1 Threshold–Based Segmentation 5.2.2 Machine Learning Assisted Segmentation 6 Correlative Tomography 6.1 Scouting Approach 6.2 Multiscale Approach 6.3 Multidisciplinary Approach 7 Data Management 7.1 Data Quality 7.2 Data Availability 7.2.1 Tomographic Datasets 7.2.2 Particle Database 8 Outlook on Further Research Activities 9 Publications 9.1 Copyright Declaration 9.2 Overview 9.3 List of Publications Paper A, Preparation techniques for micron–sized particulate samples in X–ray microtomography Paper B, Self–constructed automated syringe for preparation of micron–sized particulate samples in X–ray microtomography Paper C, Preparation strategy for statistically significant micrometer–sized particle systems suitable for correlative 3D imaging workflows on the example of X–ray microtomography Paper D, Multi–scale tomographic analysis for micron–sized particulate samples Paper E, PARROT: A pilot study on the open access provision of particle discrete tomographic datasets 10 Appendix 10.1 Application Example 1: Fracture Analysis 10.2 Application Example 2: 3D Contact Angle Measurement 10.3 Influence of the Source Filter 10.4 Influence of the X–rays on the Sample 10.5 Appropriate Filter Settings 10.6 Log File Parser