Neodymium as an alternative contrast for uranium in electron microscopy

Uranyl acetate is the standard contrasting agent in electron microscopy (EM), but it is toxic and radioactive. We reasoned neodymium acetate might substitute uranyl acetate as a contrasting agent, and we find that neodymium acetate indeed can replace uranyl acetate in several routine applications. Since neodymium acetate is not toxic, not radioactive and easy to use, we foresee neodymium will replace uranyl in many EM sample preparation applications worldwide

Epithelial cell–cell junctions and plasma membrane domains

AbstractEpithelial cells form a barrier against the environment, but are also required for the regulated exchange of molecules between an organism and its surroundings. Epithelial cells are characterised by a remarkable polarization of their plasma membrane, evidenced by the appearance of structurally, compositionally, and functionally distinct surface domains. Here we consider the (in)dependence of epithelial cell polarisation and the function of smaller plasma membrane domains (e.g. adherens junctions, gap junctions, tight junctions, apical lipid rafts, caveolae, and clathrin-coated pits) in the development and maintenance of cell surface polarity. Recent evidence of cross-talk and/or overlap between the different cell–cell junction components and alternate functions of junction components, including gene expression regulation, are discussed in the context of cell surface polarity

Segmentation in large-scale cellular electron microscopy with deep learning: A literature survey

Electron microscopy (EM) enables high-resolution imaging of tissues and cells based on 2D and 3D imaging techniques. Due to the laborious and time-consuming nature of manual segmentation of large-scale EM datasets, automated segmentation approaches are crucial. This review focuses on the progress of deep learning-based segmentation techniques in large-scale cellular EM throughout the last six years, during which significant progress has been made in both semantic and instance segmentation. A detailed account is given for the key datasets that contributed to the proliferation of deep learning in 2D and 3D EM segmentation. The review covers supervised, unsupervised, and self-supervised learning methods and examines how these algorithms were adapted to the task of segmenting cellular and sub-cellular structures in EM images. The special challenges posed by such images, like heterogeneity and spatial complexity, and the network architectures that overcame some of them are described. Moreover, an overview of the evaluation measures used to benchmark EM datasets in various segmentation tasks is provided. Finally, an outlook of current trends and future prospects of EM segmentation is given, especially with large-scale models and unlabeled images to learn generic features across EM datasets

Nanobody-Based Probes for Subcellular Protein Identification and Visualization

Understanding how building blocks of life contribute to physiology is greatly aided by protein identification and cellular localization. The two main labeling approaches developed over the past decades are labeling with antibodies such as immunoglobulin G (IgGs) or use of genetically encoded tags such as fluorescent proteins. However, IgGs are large proteins (150 kDa), which limits penetration depth and uncertainty of target position caused by up to ∼25 nm distance of the label created by the chosen targeting approach. Additionally, IgGs cannot be easily recombinantly modulated and engineered as part of fusion proteins because they consist of multiple independent translated chains. In the last decade single domain antigen binding proteins are being explored in bioscience as a tool in revealing molecular identity and localization to overcome limitations by IgGs. These nanobodies have several potential benefits over routine applications. Because of their small size (15 kDa), nanobodies better penetrate during labeling procedures and improve resolution. Moreover, nanobodies cDNA can easily be fused with other cDNA. Multidomain proteins can thus be easily engineered consisting of domains for targeting (nanobodies) and visualization by fluorescence microscopy (fluorescent proteins) or electron microscopy (based on certain enzymes). Additional modules for e.g., purification are also easily added. These nanobody-based probes can be applied in cells for live-cell endogenous protein detection or may be purified prior to use on molecules, cells or tissues. Here, we present the current state of nanobody-based probes and their implementation in microscopy, including pitfalls and potential future opportunities

Tubulysin Synthesis Featuring Stereoselective Catalysis and Highly Convergent Multicomponent Assembly

A concise and modular total synthesis of the highly potent N14-desacetoxytubulysin H (1) has been accomplished in 18 steps in an overall yield of up to 30%. Our work highlights the complexity-augmenting and route-shortening power of diastereoselective multicomponent reaction (MCR) as well as the role of bulky ligands to perfectly control both the regioselective and diastereoselective synthesis of tubuphenylalanine in just two steps. The total synthesis not only provides an operationally simple and step economy but will also stimulate major advances in the development of new tubulysin analogues

Microscopic modulation and analysis of islets of Langerhans in living zebrafish larvae

Microscopic analysis of molecules and physiology in living cells and systems is a powerful tool in life sciences. While in vivo subcellular microscopic analysis of healthy and diseased human organs remains impossible, zebrafish larvae allow studying pathophysiology of many organs using in vivo microscopy. Here, we review the potential of the larval zebrafish pancreas in the context of islets of Langerhans and Type 1 diabetes. We highlight the match of zebrafish larvae with the expanding toolbox of fluorescent probes that monitor cell identity, fate and/or physiology in real time. Moreover, fast and efficient modulation and localization of fluorescence at a subcellular level, through fluorescence microscopy, including confocal and light sheet (single plane illumination) microscopes tailored to in vivo larval research, is addressed. These developments make the zebrafish larvae an extremely powerful research tool for translational research. We foresee that living larval zebrafish models will replace many cell line-based studies in understanding the contribution of molecules, organelles and cells to organ pathophysiology in whole organisms.ImPhys/Microscopy Instrumentation & Technique

Integrated Array Tomography for 3D Correlative Light and Electron Microscopy

Volume electron microscopy (EM) of biological systems has grown exponentially in recent years due to innovative large-scale imaging approaches. As a standalone imaging method, however, large-scale EM typically has two major limitations: slow rates of acquisition and the difficulty to provide targeted biological information. We developed a 3D image acquisition and reconstruction pipeline that overcomes both of these limitations by using a widefield fluorescence microscope integrated inside of a scanning electron microscope. The workflow consists of acquiring large field of view fluorescence microscopy (FM) images, which guide to regions of interest for successive EM (integrated correlative light and electron microscopy). High precision EM-FM overlay is achieved using cathodoluminescent markers. We conduct a proof-of-concept of our integrated workflow on immunolabelled serial sections of tissues. Acquisitions are limited to regions containing biological targets, expediting total acquisition times and reducing the burden of excess data by tens or hundreds of GBs.</p

Integrated Array Tomography for 3D Correlative Light and Electron Microscopy

Volume electron microscopy (EM) of biological systems has grown exponentially in recent years due to innovative large-scale imaging approaches. As a standalone imaging method, however, large-scale EM typically has two major limitations: slow rates of acquisition and the difficulty to provide targeted biological information. We developed a 3D image acquisition and reconstruction pipeline that overcomes both of these limitations by using a widefield fluorescence microscope integrated inside of a scanning electron microscope. The workflow consists of acquiring large field of view fluorescence microscopy (FM) images, which guide to regions of interest for successive EM (integrated correlative light and electron microscopy). High precision EM-FM overlay is achieved using cathodoluminescent markers. We conduct a proof-of-concept of our integrated workflow on immunolabelled serial sections of tissues. Acquisitions are limited to regions containing biological targets, expediting total acquisition times and reducing the burden of excess data by tens or hundreds of GBs

Endocytosis of Extracellular Vesicles and Release of Their Cargo from Endosomes

Extracellular vesicles (EVs), such as exosomes, can mediate long-distance communication between cells by delivering biomolecular cargo. It is speculated that EVs undergo back-fusion at multivesicular bodies (MVBs) in recipient cells to release their functional cargo. However, direct evidence is lacking. Tracing the cellular uptake of EVs with high resolution as well as acquiring direct evidence for the release of EV cargo is challenging mainly because of technical limitations. Here, we developed an analytical methodology, combining state-of-theart molecular tools and correlative light and electron microscopy, to identify the intracellular site for EV cargo release. GFP was loaded inside EVs through the expression of GFP-CD63, a fusion of GFP to the cytosolic tail of CD63, in EV producer cells. In addition, we genetically engineered a cell line which expresses anti-GFP fluobody that specifically recognizes the EV cargo (GFP). Incubation of anti-GFP fluobody- expressing cells with GFP-CD63 EVs resulted in the formation of fluobody punctae, designating cytosolic exposure of GFP. Endosomal damage was not observed in EV acceptor cells. Ultrastructural analysis of the underlying structures at GFP/ fluobody double-positive punctae demonstrated that EV cargo release occurs from endosomes/lysosomes. Finally, we show that neutralization of endosomal pH and cholesterol accumulation in endosomes leads to blockage of EV cargo exposure. In conclusion, we report that a fraction of internalized EVs fuse with the limiting membrane of endosomes/lysosomes in an acidification-dependent manner, which results in EV cargo exposure to the cell cytosol

EpCAM proteolysis:new fragments with distinct functions?

EpCAM [epithelial cell adhesion molecule; CD326 (cluster of differentiation 326)] is highly expressed on epithelium-derived tumours and can play a role in cell proliferation. Recently, RIP (regulated intramembrane proteolysis) has been implicated as the trigger for EpCAM-mediated proliferative signalling. However, RIP does not explain all EpCAM-derived protein fragments. To shed light on how proteolytic cleavage is involved in EpCAM signalling, we characterized the protein biochemically using antibodies binding to three different EpCAM domains. Using a newly generated anti-EpCAM antibody, we find that EpCAM can be cleaved at multiple positions within its ectodomain in addition to described peptides, revealing that EpCAM is processed via distinct proteolytic pathways. Here, we report on four new peptides, but also discuss the previously described cleavage products to provide a comprehensive picture of EpCAM cleavage at multiple positions. The complex regulation of EpCAM might not only result in the absence of full-length EpCAM, but the newly formed EpCAM-derived proteins may have their own signalling properties