Freeze Substitution and Low Temperature Embedding

The problems of conventional EM-preparation techniques based on chemical fixation may be overcome to a considerable extent by freeze substitution techniques. Although at present substitution cannot be performed at sufficiently low temperatures to prevent the recrystallization of vitrified aqueous specimens, thin sectioned biological samples show an improved information density. If freeze-substitution is combined with conventional embedding above 273 K (Epon/Araldite, Spurr\u27s resin) the substituting organic solvent must contain stabilizing agents such as osmiumtetroxide, glutaraldehyde or uranylions. In combination with low temperature embedding procedures (Lowicryl) completely unfixed samples are obtained, which are suitable for immunolabelling and electron spectroscopic experiments. Water in its different dynamic states is considered to be the most important factor in maintaining the structural and functional integrity. Thus, the main advantage of freeze substitution is a better control over the removal of the cellular water, necessary for subsequent plastic embedding

A Preparation Protocol for Postembedding Immunoelectron Microscopy of Dictyostelium discoideum Cells with Monoclonal Antibodies

On-section-labelling of Dictyostelium discoideum cells poses severe problems in retaining adequate morphology and antigenicity. Monoclonal antibodies are an essential tool in biochemistry and molecular biology because of their specificity and low. background staining. Unfortunately their advantage, the recognition of only one distinct epitope, is often a handicap for immunoelectron microscopy. Resin embedding and steric hindrance of the gold-tagged secondary antibodies further reduce the efficiency in detecting antigens making the localization of less abundant antigens difficult if not impossible. A successful preparation protocol should retain morphology and antigenicity, allow the antibodies easy access to the antigen and use a detection system which visualizes as many of the primary antibodies as possible. Fixation of Dictyostelium cells with buffered formaldehyde and picric acid, cryosectioning and the use of ultra-small gold conjugates enabled us to label most of the antigens under investigation

Towards Quantitative Simulations of High Power Proton Cyclotrons

PSI operates a cyclotron based high intensity proton accelerator routinely at an average beam power of 1.3MW. With this power the facility is at the worldwide forefront of high intensity proton accelerators. The beam current is practically limited by losses at extraction and the resulting activation of accelerator components. Further intensity upgrades and new projects aiming at an even higher average beam power, are only possible if the relative losses can be lowered in proportion, thus keeping absolute losses at a constant level. Maintaining beam losses at levels allowing hands-on maintenance is a primary challenge in any high power proton machine design and operation. In consequence, predicting beam halo at these levels is a great challenge and will be addressed in this paper. High power hadron driver have being used in many disciplines of science and, a growing interest in the cyclotron technology for high power hadron drivers are being observed very recently. This report will briefly introduce OPAL, a tool for precise beam dynamics simulations including 3D space charge. One of OPAL's flavors (OPAL-cycl) is dedicated to high power cyclotron modeling and is explained in greater detail. We then explain how to obtain initial conditions for our PSI Ring cyclotron which still delivers the world record in beam power of 1.3 MW continuous wave (cw). Several crucial steps are explained necessary to be able to predict tails at the level of 3\sigma ... 4\sigma in the PSI Ring cyclotron. We compare our results at the extraction with measurements, obtained with a 1.18 MW cw production beam. Based on measurement data, we develop a simple linear model to predict beam sizes of the extracted beam as a function of intensities and confirm the model with simulations.Comment: Corrections and new figur

Degradation of p0071 and p120-catenin during adherens junction disassembly by Leptospira interrogans

Leptospira interrogans disseminates hematogenously to reach the target organs by disrupting epithelial adherens junctions (AJs), thus causing leptospirosis, which is a globally neglected zoonotic disease. L. interrogans induces E-cadherin (E-cad) endocytosis and cytoskeletal rearrangement during AJ disassembly, but the detailed mechanism remains unknown. Elucidation of AJ disassembly mechanisms will guide new approaches to developing vaccines and diagnostic methods. In this study, we combine proteomic and imaging analysis with chemical inhibition studies to demonstrate that disrupting the AJs of renal proximal tubule epithelial cells involves the degradation of two armadillo repeat-containing proteins, p0071 and p120-catenin, that stabilize E-cad at the plasma membrane. Combining proteasomal and lysosomal inhibitors substantially prevented p120-catenin degradation, and monolayer integrity destruction without preventing p0071 proteolysis. In contrast, the pan-caspase inhibitor Z-VAD-FMK inhibited p0071 proteolysis and displacement of both armadillo repeat-containing proteins from the cell-cell junctions. Our results show that L. interrogans induces p120-catenin and p0071 degradation, which mutually regulates E-cad stability by co-opting multiple cellular degradation pathways. This strategy may allow L. interrogans to disassemble AJs and disseminate through the body efficiently

Named Entity Recognition for early-modern textual sources: a review of capabilities and challenges with strategies for the future

Purpose: By mapping-out the capabilities, challenges and limitations of named-entity recognition (NER), this article aims to synthesise the state of the art of NER in the context of the early modern research field and to inform discussions about the kind of resources, methods and directions that may be pursued to enrich the application of the technique going forward. // Design/methodology/approach: Through an extensive literature review, this article maps out the current capabilities, challenges and limitations of NER and establishes the state of the art of the technique in the context of the early modern, digitally augmented research field. It also presents a new case study of NER research undertaken by Enlightenment Architectures: Sir Hans Sloane's Catalogues of his Collections (2016–2021), a Leverhulme funded research project and collaboration between the British Museum and University College London, with contributing expertise from the British Library and the Natural History Museum. // Findings: Currently, it is not possible to benchmark the capabilities of NER as applied to documents of the early modern period. The authors also draw attention to the situated nature of authority files, and current conceptualisations of NER, leading them to the conclusion that more robust reporting and critical analysis of NER approaches and findings is required. // Research limitations/implications: This article examines NER as applied to early modern textual sources, which are mostly studied by Humanists. As addressed in this article, detailed reporting of NER processes and outcomes is not necessarily valued by the disciplines of the Humanities, with the result that it can be difficult to locate relevant data and metrics in project outputs. The authors have tried to mitigate this by contacting projects discussed in this paper directly, to further verify the details they report here. // Practical implications: The authors suggest that a forum is needed where tools are evaluated according to community standards. Within the wider NER community, the MUC and ConLL corpora are used for such experimental set-ups and are accompanied by a conference series, and may be seen as a useful model for this. The ultimate nature of such a forum must be discussed with the whole research community of the early modern domain. // Social implications: NER is an algorithmic intervention that transforms data according to certain rules-, patterns- or training data and ultimately affects how the authors interpret the results. The creation, use and promotion of algorithmic technologies like NER is not a neutral process, and neither is their output A more critical understanding of the role and impact of NER on early modern documents and research and focalization of some of the data- and human-centric aspects of NER routines that are currently overlooked are called for in this paper. // Originality/value: This article presents a state of the art snapshot of NER, its applications and potential, in the context of early modern research. It also seeks to inform discussions about the kinds of resources, methods and directions that may be pursued to enrich the application of NER going forward. It draws attention to the situated nature of authority files, and current conceptualisations of NER, and concludes that more robust reporting of NER approaches and findings are urgently required. The Appendix sets out a comprehensive summary of digital tools and resources surveyed in this article

Beam Dynamics in High Intensity Cyclotrons Including Neighboring Bunch Effects: Model, Implementation and Application

Space charge effects, being one of the most significant collective effects, play an important role in high intensity cyclotrons. However, for cyclotrons with small turn separation, other existing effects are of equal importance. Interactions of radially neighboring bunches are also present, but their combined effects has not yet been investigated in any great detail. In this paper, a new particle in cell based self-consistent numerical simulation model is presented for the first time. The model covers neighboring bunch effects and is implemented in the three-dimensional object-oriented parallel code OPAL-cycl, a flavor of the OPAL framework. We discuss this model together with its implementation and validation. Simulation results are presented from the PSI 590 MeV Ring Cyclotron in the context of the ongoing high intensity upgrade program, which aims to provide a beam power of 1.8 MW (CW) at the target destination

Subcellular investigation of photosynthesis-driven carbon assimilation in the symbiotic reef coral Pocillopora damicornis.

Reef-building corals form essential, mutualistic endosymbiotic associations with photosynthetic Symbiodinium dinoflagellates, providing their animal host partner with photosynthetically derived nutrients that allow the coral to thrive in oligotrophic waters. However, little is known about the dynamics of these nutritional interactions at the (sub)cellular level. Here, we visualize with submicrometer spatial resolution the carbon and nitrogen fluxes in the intact coral-dinoflagellate association from the reef coral Pocillopora damicornis by combining nanoscale secondary ion mass spectrometry (NanoSIMS) and transmission electron microscopy with pulse-chase isotopic labeling using [(13)C]bicarbonate and [(15)N]nitrate. This allows us to observe that (i) through light-driven photosynthesis, dinoflagellates rapidly assimilate inorganic bicarbonate and nitrate, temporarily storing carbon within lipid droplets and starch granules for remobilization in nighttime, along with carbon and nitrogen incorporation into other subcellular compartments for dinoflagellate growth and maintenance, (ii) carbon-containing photosynthates are translocated to all four coral tissue layers, where they accumulate after only 15 min in coral lipid droplets from the oral gastroderm and within 6 h in glycogen granules from the oral epiderm, and (iii) the translocation of nitrogen-containing photosynthates is delayed by 3 h. IMPORTANCE: Our results provide detailed in situ subcellular visualization of the fate of photosynthesis-derived carbon and nitrogen in the coral-dinoflagellate endosymbiosis. We directly demonstrate that lipid droplets and glycogen granules in the coral tissue are sinks for translocated carbon photosynthates by dinoflagellates and confirm their key role in the trophic interactions within the coral-dinoflagellate association

Direct Visualization and Silver Enhancement of Ultra-Small Antibody-Bound Gold Particles on Immunolabeled Ultrathin Resin Sections

Ultra-small gold colloids bound to immunolabeled ultrathin resin sections were visualized using transmission, scanning, and scanning transmission electron microscopy (TEM, SEM, STEM). The best marker contrast is obtained in a field emission STEM (200 kV) equipped with a high-angle annular dark-field (HAADF) detector. HAADF STEM renders possible the simultaneous visualization of ultra-small gold and ultrastructural details in unstained resin sections, and an overall presentation of a labeled E. coli cell. For routine work, an enhancement step is a prerequisite for easy detection of bound marker molecules. Five different silver enhancing solutions were tested for their suitability for ultra-small gold intensification. Enhancers lacking the protective colloid gum arabic exhibit lower quality with regard to efficiency and homogeneity of enhancement. This problem can be overcome by adding gum arabic. Silver enhancement generally results in heterogeneously sized particles. This is most probably due to the heterogeneous original gold colloid probe. In general, an estimation of enhancement efficiency is associated with difficulties depending on experimental conditions and the electron microscopic imaging modes used. Only a low number of the ultra-small gold particles seems to remain unenhanced or poorly enhanced when treated with high-quality enhancers. On-section labeling of ultrathin resin sections with silver-enhanced ultra-small gold markers also offers the possibility of high-resolution immunolabeling experiments at the light microscopic level

Cryo-FIB Machining: An Alternative to TEM Cryo-Sections Cut with Diamonds?

Extended abstract of a paper presented at Microscopy and Microanalysis 2011 in Nashville, Tennessee, USA, August 7-August 11, 201