454 research outputs found
Update of the risk assessment of hexabromocyclododecanes (HBCDDs) in food
No full textPanel members: Margherita Bignami, Laurent Bodin, James Kevin Chipman, Jesus del Mazo, BettinaGrasl-Kraupp, Christer Hogstrand, Laurentius (Ron) Hoogenboom, Jean-Charles Leblanc, Carlo StefanoNebbia, Elsa Nielsen, Evangelia Ntzani, Annette Petersen, Salomon Sand, Dieter Schrenk, TanjaSchwerdtle, Christiane Vleminckx and Heather Wallace Requestor: European Commission Question number: EFSA‐Q‐2018‐00433 Acknowledgements: The Panel wishes to thank the hearing experts: Cathy Fernandes and Henri Schroeder, and EFSA staff members: Kelly Niermans and Federico Cruciani, for the support provided to this scientific output. The Panel wishes to acknowledge all European competent institutions and Member State bodies that provided consumption and occurrence data for this scientific output. Data: All annexes on occurrence and exposure data, as well as the protocol used to produce this Scientific opinion, cross‐referenced in the text, are available on the EFSA Knowledge Junction at: https://doi.org/10.5281/zenodo.4475651Peer reviewe
Need for Speed: Imaging Biological Ultrastructure with the 64-beams FAST-EM
No full textGreen Open Access added to TU Delft Institutional Repository 'You share, we take care!' - Taverne project https://www.openaccess.nl/en/you-share-we-take-care Otherwise as indicated in the copyright section: the publisher is the copyright holder of this work and the author uses the Dutch legislation to make this work public.ImPhys/Hoogenboom grou
Electron Beam-Induced Fluorescence Localization : Implementation and Feasibility in Integrated Light-Electron Microscopy
No full textCorrelative light-electron microscopy (CLEM) combines the molecular specificity of fluorescence microscopy (FM) with the ultrastructural resolution of electron microscopy (EM) to provide functional information in the context of structural detail. However, the correlation between the two modalities is hindered by a 100-fold resolution gap. Superresolution fluorescence microscopy (SR-FM) has enabled more accurate correlation in CLEM, aided by advancements in sample preparation, bimodal registration, and optimized workflows. As the resolution of FM approaches that of EM, SR-FM becomes increasingly challenging due to the need for accurate registration and preservation of fluorophore properties during EM sample preparation. Integrated microscopes can remove the need for external alignment markers and achieve high registration accuracies, eliminating sample deformations and facilitating SR-CLEM. Yet, this necessitates samples that are simultaneously amenable to both FM and EM. While advancements are being made to engineer fixation-resistant fluorescent proteins and develop preparation protocols for preserving in-resin fluorescence, it is worth exploring the possibilities of the unified platform that integrated light-electron microscopy offers, especially for SR-FM. In caseswhere traditional SR-FM cannot be used due to vacuum or other limitations, the combination of both light and electron microscopy can provide valuable multi-modal information. It also enables central control of the experimental system, thereby offering new ways to manipulate, process, and interpret fluorescence data. This thesis aims to investigate and utilize luorescent response to electron irradiation using integrated light-electronmicroscopy....ImPhys/Hoogenboom grou
On Introducing Imperfection in the Non-Linear Analysis of Buckling of Thin Shell Structures
No full textThis master thesis details the investigation of the effect of geometrical imperfection on thin shell structures using general FEM software packages. The author proposes a finite element based method for the analysis and design of thin shell structures, and describes the implementation of such a procedure on four different FEM packages. The procedure involves the assessment of imperfection sensitivity of a design, and imposing geometrical imperfection in the shape of the first buckling mode prior to a geometrically non-linear analysis. Starting with thin metallic cylinders, by incorporating imperfection to the surface, the author shows that ANSYS is capable of reproducing resulting similar to Koiter's asymptotic theory and to experimental data. The author further demonstrates the robustness and easy-to-use nature of the imperfection procedure by implementing it on four simple yet realistic structures with both symmetric and asymmetric load cases. For extremely imperfection sensitive structures such as an axially loaded cylinder. The author then introduces four different types of imperfection that may be imposed in place of the first buckling mode, and gauges the effectiveness of each with a modified knock-down factor. By varying the vertical curvature, it is discovered that an axisymmetric imperfection shape governs the ultimate buckling capacity of any near-cylindrical shells. An theory is being developed that explains the effect of the axisymmetric shape. Change in Gaussian curvature is calculated as the end of every load step in the FEM analysis. By plotting the change in Gaussian curvature, the onset of buckling can be readily defined. Finally, physical non-linearities are introduced to the FEM models to gauge the effect of yielding and cracking on a steel cylinder, and a reinforced concrete (RC) cooling tower respectively. It is found that metal buckles within the elastic range, and yielding eliminates post-buckling capacity without altering the ultimate capacity. With RC cracking, it is discovered that instability occurs soon after cracks develop at the buckles of the imperfection shape, therefore reducing the capacity by as much as 8 times from that of the elastic model.Structural MechanicsStructural EngineeringCivil Engineering and Geoscience
Depth-dependent scaling of axial distances in light microscopy
No full textIn volume fluorescence microscopy, refractive index matching is essential to minimize aberrations. There are, however, common imaging scenarios where a refractive index mismatch (RIM) between immersion and a sample medium cannot be avoided. This RIM leads to an axial deformation in the acquired image data. Over the years, different axial scaling factors have been proposed to correct for this deformation. While some reports have suggested a depth-dependent axial deformation, so far none of the scaling theories has accounted for a depth-dependent, non-linear scaling. Here, we derive an analytical theory based on determining the leading constructive interference band in the objective lens pupil under RIM. We then use this to calculate a depth-dependent re-scaling factor as a function of the numerical aperture (NA), the refractive indices n1 and n2, and the wavelength λ. We compare our theoretical results with wave-optics calculations and experimental results obtained using a measurement scheme for different values of NA and RIM. As a benchmark, we recorded multiple datasets in different RIM conditions, and corrected these using our depth-dependent axial scaling theory. Finally, we present an online web applet that visualizes the depth-dependent axial re-scaling for specific optical setups. In addition, we provide software that will help microscopists to correctly re-scale the axial dimension in their imaging data when working under RIM.ImPhys/Hoogenboom groupImPhys/Geertsema grou
Development, validation and routine application of the in vitro REA and DR-CALUX reporter gene bioassays for the screening of estrogenic compounds and dioxins in food and feed
No full textA dedicated cell-line was developed by the Department of Toxicology of Wageningen University in a joined project with the University of California in Davis and the RIKILT-WUR - Institute of Food Safety in Wageningen. This DR-CALUX ® bioassay was tested, optimised and validated for its use to determine low elevated levels of dioxins in bovine milk around the existing limits. It was shown that this mammalian cell based test is very sensitive for 2,3,7,8-substituted dioxins and related PCBs, thereby reflecting the relative potencies (TEF) of these compounds as set by the World Health Organisation (WHO). These toxic equivalency factors (WHO-TEFs) express the toxicity of a compound in comparison to the most toxic compoundcongener 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, TEF=1). The response obtained with a mixture of dioxins was additive, in accordance with the TEQ-principle. Milk fat was isolated by centrifugation followed by clean-up of the fat with n-pentane, removal of the fat on a 33% H2SO4silica column, and determination of Ah receptor agonist activity with the DR-CALUX ® bioassay. To investigate the performance of this 33% H2SO4silica method, milk fat was cleaned with activated carbon and spiked with a mixture of 17 different 2,3,7,8-substituted PCDD and PCDF congeners at 1, 3, 6, 9, 12 and 15 pg WHO-TEQ per g fat, as confirmed by GC/MS. In this concentration range, the method showed a recovery of TEQs around 67% (58-87%). The reproducibility showed a coefficient of variation (CV) varying between 4% and 54%, with the exception of the sample spiked at 1 pg WHO-TEQ/g (CV 97%). The repeatability determined with the sample spiked at 6 pg WHO-TEQ per g showed a CV of 10% These results clearly demonstrate that the reproducibility of the silica-CALUX procedure with samples containing more than 1 pg WHO-TEQ per g fat is relatively good, in particular regarding the fact that no internal standards could be used in the assay for correction of data for varying recoveries. The fact that the CV was much higher for the sample spiked at the lowest level, confirmed the calculated limit of quantification of 1 pg WHO-TEQ per g fat. The current tolerance limit for bovine milk in the EU is 3 pg WHO-TEQ per g fat, with an action limit of 2 pg WHO-TEQ per g fat. Therefore, the DR-CALUX ® bioassay can be a useful pre-screening tool for selecting milk samples that may contain dioxin levels exceeding this tolerance limit. This was supported by the results obtained with 22 field samples, since all five samples exceeding the 2 pg WHO-TEQ per g fat concentration gave a higher response in the DR-CALUX ® bioassay.The DR-CALUX ® bioassay in combination with the 33% H2SO4clean-up procedure results in a specific test for the determination of dioxins and dioxin-like PCBs, allowing the screening of relatively large sets of samples for the presence of unacceptable high levels of these compounds. This results in a reduction of costs involved in the analysis of food for the presence of these compounds, enabling more intense monitoring programs.Following the successful optimisation and validation of the test for milk fat, the bioassay was first used at RIKILT in the food and feed area during the 1998 Brazilian citrus pulp incident. The test procedure was subsequently optimised and validated for animal feed. During the German bakery waste incident in 2003, animal feed was contaminated with dioxins due to the use of waste wood for drying of the material. Besides Germany, the material was also shipped to the Netherlands. Levels up to 12 ng WHO-TEQ/kg were detected, being about 15 times over the current limit of 0.75 ng WHO-TEQ/kg. A combined strategy of screening with the DRCALUX ® -bioassay and the HRGC/HRMS confirmatory method was used in the Netherlands to rapidly control the incident. Pigs were contaminated by the incident but only to a very limited extent. Despite the rather low limits for pig meat (1 pg WHO-TEQ per g fat), the DR-CALUX ® bioassay, in combination with an extra acid pre-treatment of the fat samples, showed excellent performance, confirming once again the value of this bioassay. Shown during the recent incidents with kaolinic clay (2004) and the contaminated HCl used for gelatine production (2006), the assay is still the best availablescreening test for dioxins and dioxin-like PCBs.The second aim of the research in this thesis was to develop, validate and apply a new recombinant yeast screen to detect chemicals with an estrogenic mode of action in animal feed, urine and illegal preparations. A recombinant yeast cell that stably expresses the human estrogen receptor α (hERα) and yeast enhanced green fluorescent protein (yEGFP) as a reporter protein in response to estrogens was developed at the RIKILT. The EC50 revealed by the RIKILT yeast Estrogen bioAssay (REA) was 0.5 nM for 17b-estradiol and was comparable with reported EC50 values for yeast estrogen bioassays that contain β-galactosidase as a reporter. However, the yEGFP assay can be performed completely in 96 well plates within 4 hours and does not need require cell wall disruption, nor does it need the addition of a substrate. This makes the test sensitive, rapid and convenient with high reproducibility and small variation. The robustness and ease of the yeast cells in combination with the qualities ofyEGFP,ensure that the assay will be suited to be used as a high through put system.The properties of the RIKILT yeast Estrogen bioAssay expressing thehERα(REA) were further studied by testing a series of estrogenic compounds. In addition, a similar assay was developed based on the stable expression of human estrogen receptor β (hERβ). When exposed to 17b-estradiol, the maximum transcriptional activity of the hERb cytosensor was only about 40% of the activity observed with hERa, but the concentration where half-maximal activation is reached (EC50), was about 5 times lower. The relative estrogenic potencies (REP), defined as the ratio between the EC50 of 17b-estradiol and the EC50 of the compound, of the synthetic hormones dienestrol, hexestrol and especially mestranol were higher with ERa than with ERβ, while DES was slightly more potent with ERb. The gestagens progesterone and medroxyprogesterone-acetate showed no response, whereas the androgen testosterone showed a very weak response and only at high concentrations. The isoflavones genistein, genistin, daidzein and daidzin, the coumestran coumestrol and the flavonoid naringenin were relatively more potent with ERb than with ERa. Coumestrol and genistein were by far the most potent of these compounds with ERb. However, 8-prenylnaringenin, a phytoestrogen present in hops, was relatively more potent with ERa than with ERb and was actually the most potent phytoestrogen with ERa. The data demonstrate that the REA shows clear dose-response curves when exposed to estrogenic compounds. Since good dose-response curves can be obtained after only 4 h of exposure, the often questioned permeability of the yeast cell wall does not seem to be an obstacle in our yeast estrogen bioassays.The RIKILT yeast Estrogen bioAssay stably expressing human estrogen receptor α (REA) was validated as a qualitative screening method for the determination of estrogenic activity in calf urine and animal feed. These validations were performed according to EC Decision 2002/657, which prescribes the determination of the detection capability (CCb), the specificity and the stability. To determine these performance characteristics, twenty blank urine samples of 19 week old calves were collected and spiked with 17b-estradiol (E2b) at 1 ng/ml-1, diethylstilbestrol (DES) at 1 ng/ml-1, 17a-ethynylestradiol (EE2) at 1 ngml-1a-zearalanol at 50 ngml-1 or mestranol at 10 ng/ml-1. Following enzymatic deconjugation and solid phase extraction, 100 ml equivalents of these blank and spiked urine samples were screened for estrogenic activity in a 96 well plate using the REA. All of these blank and low estrogen spiked feed samples fulfilled the CCα and CCβ criterions, meaning that all 20 blank urine samples gave a signal below the determined decision limit CCα and were thus classified as compliant and at least 19 out of the 20 spiked samples gave a signal above this CCα (β=5%) and were thus classified as suspect. The specificity of the method was determined with blank urine samples spiked with a high dose of testosterone or progesterone (1000 ng/ml-1). No response to these substances was detected in the REA. There was also no interference of a high dose of testosterone or progesterone on the response of a low dose of the estrogens. Stability of urine samples was checked with spiked urine samples that were kept frozen for up to 90 days, showing that urine samples could be stored at -20°C for up to 60 days without changing the screening result of the assay. The assay was validated for animal feed in a simalar way, using twenty blank animal feed samples, including milk replacers and wet and dry feed samples.As all the performance characteristics met the criteria that were put forward in EC Decision 2002/657 for validation of a qualitative screening method, the described clean-up/yeast estrogen bioassay procedures were proven to be valid for the determination of estrogenic activity in calf urine and animal feed. The clean-up procedures for urine and feed samples are relatively simple and the yeast estrogen bioassay, using yEGFP as a reporter protein, is sensitive, rapid, convenient and reproducible. Due to the good sensitivity of the bioassay, only 2 ml of urine or 1 gram of feed were enough to be processed. Combined this resulted in a low cost bioassay that is suited to be used as a high through-put system for the screening of estrogenic activity in calf urine and animal feed. Like the DR CALUX ® assay, Tthe method acquired an ISO 17025 accreditation status in the Netherlands for both of these matrices. The examples of the MPA-incident with wet pig feed and the fishfeed,described in Chapter 7 of the thesis, demonstrate the applicability of the bioassay method as an early warning system for pharmaceutical waste and hormone use respectively. This is the first successful example of a developed, validated and applied bioassay for the screening of hormonal substances in feed. At present this method has been in routine use at RIKILT for more than two years.Overall the work presented in this thesis shows that bioassays are valuable tools for rapid and high throughput screening of samples for both known and unknown compounds. As such they may contribute to an earlier detection of new emerging risks and prevent the use of illegal growth-promoting agents with thus far unknown identity
Malachite green in food
No full textThe Panel wishes to thank the members of the Standing Working Group on non-allowed pharmacologically active substances in food and feed and their reference points for action (2015–2018): Metka Filipič, Peter Fürst, Laurentius (Ron) Hoogenboom, Anne-Katrine Lundebye, Carlo Stefano Nebbia, Michael O'Keeffe and Rolaf Van Leeuwen for the preparatory work on this scientific output, the hearing expert: Eva Persson, and EFSA staff members: Katleen Baert and Sofia Ioannidou for the support provided to this scientific opinion. The CONTAM Panel acknowledges all European competent institutions and other stakeholders that provided occurrence data on malachite green and leucomalachite green in food, and supported the data collection for the Comprehensive European Food Consumption Database.Peer reviewe
Cathodoluminescence Microscopy of Nanostructures on Transparent Substrates
No full textCathodoluminescence (CL), the excitation of light by an electron beam, has gained attention as an analysis tool for investigating the optical response of a structure, at a resolution that approaches that in electron microscopy, in the nanometer range. However, the application possibilities are limited because the use of transparent substrates, one of the most common sample substrates for optical characterizations in multiple research fields, is normally avoided in CL microscopy, since these materials generate a strong signal that contributes as a background to the measurement. The main goal in this thesis is to achieve cathodoluminescence detection of nanostructures on glass-based substrates. For that purpose, a CL system with enhanced collection efficiency and confocal detection of the signal was developed, built and tested. The design is based on an integrated Scanning Electron and Optical Microscope, a setup that offers simultaneous correlated acquisition of the electron and light signals. Besides cathodoluminescence, other interesting applications derive from the combination of these techniques, but they are out of the scope of this thesis. Chapter 1 intends to give general introduction to cathodoluminescence as a microscopy analysis tool. First we discuss its generation principle: considering the excitation and emission mechanisms, electron-hole recombination, transition radiation and surface plasmon polariton radiative outcoupling are identified as the main CL sources in the structures investigated in this thesis. In bulk samples, the emission is not restricted to the nanometer size spot where the incoming electron beam is focused, but it extends to a region that spreads below it, where electrons scatter and interact with the host material. Cathodoluminescence is potentially generated throughout this volume, the size of which increases dramatically with the electron beam energy. Therefore it should be considered as an extended excitation, although its size can be modulated by a spatially confined generation yield. Most of the CL setups are incorporated in the vacuum chamber of an electron microscope, where the light collector is a parabolic mirror placed on top of the sample. The advantages, challenges and improvement examples of these standard setups are discussed. An overview of applications in cell and molecular biology, geosciences and nanophotonics emphasizes the increasing interest on applying the technique at the nanometer regime. The chapter ends by summarizing the main challenges that cathodoluminescence microscopy encounters for successful imaging of nanostructures on glass, which define the design criteria for our setup. The system details are presented in chapter 2: a brief description of the integrated electron light microscope functionalities and the implementation of the confocal detection path are presented. Explanation of the available acquisition modes, alignment procedures and typical imaging examples serve to establish an operation routine. The effect of the pinhole can be observed by comparing unfiltered and confocal CL images on the same region of a sample. Additionally, the filtering is evaluated without using the electron beam: a laser excitation path included in the setup allows acquiring confocal fluorescence images of a sample with luminescent beads on a glass substrate, for different sizes of the pinhole diameter. Besides efficient CL detection, potential applications of the setup could include: (i) emission localization for excitations with long propagation length, (ii) simultaneous light and electron excitation, (iii) monitoring the effect of electron excitation with subsequent light microscopy, and (iv) the incorporation of light or electron pulses for time-resolved characterization. The use of low energies for the electron excitation probe is proposed in chapter 3 as a strategy to reduce the background CL contribution. This is further investigated with Monte Carlo simulations that show the dependence of the electron interaction volume on the electron beam acceleration voltage. We observe however, that to detect nanostructures with a weak cathodoluminescence signal it is necessary to increase the electron current, which in the low acceleration voltage regime may compromise the spatial resolution. With the low energy approach, individual 30nm phosphor particles are resolved and the high order resonant modes of a gold nanowire on an indium tin oxide (ITO) covered glass microscope slide are detected. For high electron energies, the substrate cathodoluminescence is too strong and overwhelms the signal. Chapter 4 demonstrates confocal filtering as an effective tool for background rejection at high acceleration voltages. The filtering achieved for a given pinhole size is estimated with simulations of the electron interaction volume and measurements of the axial intensity distribution of a phosphor nanoparticle, which acts as a point source. As an illustrative example, a series of CL confocal sections of a gold nanowire on a transparent substrate shows a contrast inversion at the plane where the nanowire is in focus. Here, the highest CL intensity is detected at the position of the structure. The need of a high resolution electron probe is evidenced by acquiring the CL spectral distribution of a gold triangle nano plate, which shows a strong sensitivity to the excitation probe position. Both of the strategies presented in this thesis, the use of low energy excitation and confocal filtering are applicable not only for transparent substrates but for any highly cathodoluminescent material. Chapter 5 explores the use of quantum dots as cathodoluminescent biological markers. In cellular biology, investigation of cellular interactions requires imaging the specific functional proteins on top of the organelles ultrastructure. Therefore, direct correlation between electron and light optical information is a key element for understanding cell function at a molecular level. Among other potential cathodoluminescent markers, quantum dots have the additional advantage that they are already routinely incorporated as bio-labels in fluorescence and consequently, many different bio functionalization possibilities are currently available. Here, we report on the cathodoluminescence detection of bio-functionalized quantum-dots embedded in cells. A high similarity between the fluorescence and cathodoluminescence signals is observed, but the cathodoluminescence signal originates from a smaller sample volume defined by the electron penetration depth. We observe a bleaching of the quantum dots emission under high electron irradiation dose, which so far prevents high magnification imaging. However, recording the fluorescence emission after incremental low dose electron irradiation reveals a complicated dependence of the emission intensity on electron dose, featuring even a regime wherein intensity slightly increases. The origin of this behavior is discussed as a charging mechanism, building on existing models that are also used to explain photo blinking, -bleaching and -brightening of fluorescence from quantum dots. The results presented support the use of cathodoluminescence as a high resolution imaging technique for optical characterization of biological systems. Finally, the main findings on the cathodoluminescence emitted from ITO-covered glass slides, the substrate through this work, are summarized in chapter 6. A dynamic behavior of the intensity and spectral distribution of the emission is observed. Cathodoluminescence measurements at different electron doses reveal a faster cathodoluminescence bleaching with increasing dose, but also the appearance and growth of a new intensity peak at a different position in the spectra. Secondary electron images of the irradiated areas suggest that deposition may be involved in this process. Additionally, experiments with different thicknesses for the ITO conductive layer point to glass as the main responsible for the background emission in our measurements. The results reinforce the importance of sample pre-exposure and confocal filtering for CL characterization at high electron energies.Imaging PhysicsApplied Science
High Speed Electron Microscopy: Engineering of a commercial multi-beam scanning electron microscope with transmission imaging
No full textIn this thesis, the design and engineering considerations for a multi-beam scanning electron microscope (MBSEM) are discussed. This microscope can benefit biological research in various ways. It can give new insights into the inner workings of a multitude of biological systems that were hard to get using previously existing instrumentation. For instance, a higher throughput gives the option to do statistical analysis of multiple samples instead of drawing conclusions from only one. The goal of this thesis was to get from a proof of principle to a final system that can actually be used to do the research. It is divided into 5 chapters showing a step-by-step process of getting to the final system as it is now on the market. Chapter 1 is an introduction to the subject showing the current state of the art with respect to high throughput imaging. Chapter 2 Describes a novel imaging method in scanning electron microscopes. This chapter does not focus on the multi-beam application but shows it in the context of the often-used backscatter imaging. In this method, we place the tissue section directly on top of a thin scintillator screen (thinner than 200 μm) which is coated with a conductive layer. The light signal generated by the electrons transmitted through the sample is collected by a high NA objective lens and the light is imaged onto a photon detector outside of the vacuumchamber. A noise model is created to calculate the signal-to-noise ratio and the contrast-tonoise ratio of this imaging method. It shows that the best images are generated around a landing energy of about 5keV. There are some dependencies on sample thickness, staining level, and light collection efficiency which are also explored. This method does lower the resolution in the image to some extent (by a factor of 2 at low energies and thick sections), which is shown at the end of the chapter. Chapter 3 Goes into the considerations that have to be taken into account when dealing with the imaging method from chapter 2. This chapter is applicable to a single beam SEM as much as anMBSEM. A list of possible light detectors is given from which silicon photomultipliers are selected as the best candidate for the MBSEM. Combined with the light detector, multiple options for a scintillator were discussed, from which YAG:CE is selected. Organic scintillators are discarded due to their bleaching behavior due to electron beam irradiation. The surface of the scintillator and the coating layer are shown to have a large impact on image quality. For this reason, the scintillators are ion-beam polished and coated with a Boron layer. Unexpected behavior in the form of scintillator saturation is observed which is then described by a model and connected to the noise model fromchapter 2. Chapter 4 Gives an analysis of all the hardware requirements for a MBSEM. First a measurement of the crosstalk as a function of landing energy and pitch. It is found that a crosstalk of at least 10 % is to be expected in the system. Next, an overview is given for all the parameters that are related to the stage and the light optics. These are then related to the final throughput of the system. Two imaging strategies are described, in one the beam scans in one direction and the stage in the other. In the other strategy, the beams scan like in a regular SEM and are subsequently descanned in the light-optical system. It is found that with a step and scan approach in combination with planned beamshifts, the maximum throughput that can be achieved is around 420 mpix/s. Chapter 5 Shows results from the final prototype system. Alignments are of great importance in any SEMbut even more so in theMBSEM. Therefore a large part of this chapter is dedicated to describing this alignment. This starts with the electron optical alignment of the source and the beam through the column. The grid of beams has to be optimized to show as little as possible distortions to improve system throughput. The scan and descan have to be aligned to the grid axes and the amplitude has to be precisely correct. The beams have to be perfectly aligned to the detector array. On the processing side, a description of how can be compensated for varying dark and gain levels in the detector array. In the end, a final image is shown, consisting of 400 megapixels. Chapter 6 Describes the valorization of the project and all the challenges and choices that were involved.ImPhys/Hoogenboom grou
- …
