Abstract Upon infection, arteriviruses, coronaviruses, and other nidoviruses transform endoplasmic reticulum membranes into viral replication organelles. These include large numbers of double-membrane vesicles (DMVs) whose interior is considered the primary site of viral RNA synthesis. Early studies characterized nidovirus DMVs as sealed compartments, leaving it unclear how newly synthesized viral RNA could be exported to the cytosol. The discovery of DMV-spanning pore complexes in coronavirus-infected cells provided a plausible solution for this topological challenge. However, their structural organization, functional features, and evolutionary conservation across the nidovirus order, have remained unclear. Here, we investigated the macromolecular architecture of DMVs induced by two prototypic arteriviruses using cellular cryo-electron tomography. Despite the substantial evolutionary distance separating arteriviruses and coronaviruses, we observed DMV-spanning pore complexes with striking structural similarities to those previously described in coronaviruses. These pores appear to facilitate both export and encapsidation of viral RNA. In the absence of viral RNA synthesis, ectopic expression of the arterivirus transmembrane nonstructural proteins nsp2 and nsp3 sufficed to induce the formation of pore-containing DMVs. Together, our findings reveal the conservation of key structural features of DMV pores across two distantly related nidovirus families and support a central role for these pores in nidovirus replication.

In various medical and biological modalities, in particular, electron microscopy (EM), visualization of large fields of view requires acquisition of multiple overlapping frames with their subsequent reconstruction into a single panoramic image. Such reconstruction process is hampered by several factors, including different intensity scaling and imperfect localization of the acquired frames, intensity inhomogeneity within each frame, and large content variability between different frames. This poses a significant challenge not only for visualization, but also for further quantification of such panoramic images. In this work, we present a simple yet efficient data-driven algorithm that improves reconstruction of the large panoramic views using aminimal set of assumptions. More precisely, our approach fully relies on the information from the overlap regions of the neighbouring frames. Such formulation results in a linear system of equations that can be solved numerically, when supported by proper constraints. We validated our approach on a large set of highly-diverse in-house EMpanoramic views and demonstrated improved performance with respect to traditional metrics as well as network training capacity.

Pluripotent stem cell-derived kidney organoids hold great promise as a potential auxiliary transplant tissue for individuals with end-stage renal disease and as a platform for studying kidney diseases and drug discovery. To establish accurate models, it is crucial to thoroughly characterize the morphological features and maturation stages of the cellular components within these organoids. Nephrons, the functional units of the kidney, possess distinct morphological structures that directly correlate with their specific functions. High spatial resolution imaging emerges as a powerful technique for capturing ultrastructural details that may go unnoticed with other methods such as immunofluorescent imaging and scRNA sequencing. In our study, we have applied software capable of seamlessly stitching virtual slides generated from electron microscopy, resulting in high-definition overviews of tissue slides. With this technology, we can comprehensively characterize the development and maturation of kidney organoids when transplanted under the renal capsule of mice. These organoids exhibit advanced ultrastructural developments upon transplantation, including the formation of the filtration barrier in the renal corpuscle, the presence of microvilli in the proximal tubule, and various types of cell sub-segmentation in the connecting tubule similarly to those seen in the adult kidney. Such ultrastructural characterization provides invaluable insights into the structural development and functional morphology of nephron segments within kidney organoids and how to advance them by interventions such as a transplantation. Research Highlights High-resolution imaging is crucial to determine morphological maturation of hiPSC-derived kidney organoids. Upon transplantation, refined ultrastructural development of nephron segments was observed, such as the development of the glomerular filtration barrier.

CRB1 gene mutations can cause early- or late-onset retinitis pigmentosa, Leber congenital amaurosis, or maculopathy. Recapitulating human CRB1 phenotypes in animal models has proven challenging, necessitating the development of alternatives. We generated human induced pluripotent stem cell (iPSC)-derived retinal organoids of patients with retinitis pigmentosa caused by biallelic CRB1 mutations and evaluated them against autologous gene-corrected hiPSCs and hiPSCs from healthy individuals. Patient organoids show decreased levels of CRB1 and NOTCH1 expression at the retinal outer limiting membrane. Proximity ligation assays show that human CRB1 and NOTCH1 can interact via their extracellular domains. CRB1 patient organoids feature increased levels of WDFY1+ vesicles, fewer RAB11A+ recycling endosomes, decreased VPS35 retromer complex components, and more degradative endolysosomal compartments relative to isogenic control organoids. Taken together, our data demonstrate that patient-derived retinal organoids enable modeling of retinal degeneration and highlight the importance of CRB1 in early endosome maturation receptor recycling in the retina.

Cryogenic electron tomography (cryo-ET) combined with subtomogram averaging, allows in situ visualization and structure determination of macromolecular complexes at subnanometre resolution. Cryogenic focused ion beam (cryo-FIB) micromachining is used to prepare a thin lamella-shaped sample out of a frozen-hydrated cell for cryo-ET imaging, but standard cryo-FIB fabrication is blind to the precise location of the structure or proteins of interest. Fluorescence-guided focused ion beam (FIB) milling at target locations requires multiple sample transfers prone to contamination, and relocation and registration accuracy is often insufficient for 3D targeting. Here, we present in situ fluorescence microscopy-guided FIB fabrication of a frozen-hydrated lamella to address this problem: we built a coincident three-beam cryogenic correlative microscope by retrofitting a compact cryogenic microcooler, custom positioning stage, and an inverted widefield fluorescence microscope (FM) on an existing FIB scanning electron microscope. We show FM controlled targeting at every milling step in the lamella fabrication process, validated with transmission electron microscope tomogram reconstructions of the target regions. The ability to check the lamella during and after the milling process results in a higher success rate in the fabrication process and will increase the throughput of fabrication for lamellae suitable for high-resolution imaging.

AreTomo, an abbreviation for Alignment and Reconstruction for Electron Tomography, is a GPU accelerated software package that fully automates motion-corrected marker-free tomographic alignment and reconstruction in a single package. By correcting in-plane rotation, translation, and importantly, the local motion resulting from beam-induced motion from tilt to tilt, AreTomo can produce tomograms with sufficient accuracy to be directly used for subtomogram averaging. Another major application is the on-the-fly reconstruction of tomograms in parallel with tilt series collection to provide users with real-time feedback of sample quality allowing users to make any necessary adjustments of collection parameters. Here, the multiple alignment algorithms implemented in AreTomo are described and the local motions measured on a typical tilt series are analyzed. The residual local motion after correction for global motion was found in the range of ±80 Å, indicating that the accurate correction of local motion is critical for high-resolution cryo-electron tomography (cryoET).

Journal Article A Cryogenic Fluorescence Microscope Retrofittable in Coincidence with a FIB/SEM Get access Daan B Boltje, Daan B Boltje Department of Imaging Physics, Delft University of Technology, Lorentzweg 1, 2628 CJ Delft, The NetherlandsDelmic B.V., Kanaalweg 4, 2326 EB Delft, The Netherlands Corresponding author: boltje@delmic.com Search for other works by this author on: Oxford Academic Google Scholar Jacob P Hoogenboom, Jacob P Hoogenboom Department of Imaging Physics, Delft University of Technology, Lorentzweg 1, 2628 CJ Delft, The Netherlands Search for other works by this author on: Oxford Academic Google Scholar Arjen J Jakobi, Arjen J Jakobi Kavli Institute of Nanoscience, Delft University of Technology, Van der Maasweg 9, 2629 HZ Delft, The Netherlands Search for other works by this author on: Oxford Academic Google Scholar Grant J Jensen, Grant J Jensen California Institute of Technology, Pasadena, CA 91125, United States Search for other works by this author on: Oxford Academic Google Scholar Caspar TH Jonker, Caspar TH Jonker Delmic B.V., Kanaalweg 4, 2326 EB Delft, The Netherlands Search for other works by this author on: Oxford Academic Google Scholar Abraham J Koster, Abraham J Koster Department of Cell and Chemical Biology, Leiden University Medical Center, Einthovenweg 20, 2333 ZC Leiden, The Netherlands Search for other works by this author on: Oxford Academic Google Scholar Mart GF Last, Mart GF Last Delmic B.V., Kanaalweg 4, 2326 EB Delft, The Netherlands Search for other works by this author on: Oxford Academic Google Scholar Jürgen M Plitzko, Jürgen M Plitzko Department of Molecular Structural Biology, Max Planck Institute of Biochemistry, 82152 Planegg-Martinsried, Germany Search for other works by this author on: Oxford Academic Google Scholar Stefan Raunser, Stefan Raunser Department of Structural Biochemistry, Max Planck Institute of Molecular Physiology, Otto-Hahn-Str. 11, 44227 Dortmund, Germany Search for other works by this author on: Oxford Academic Google Scholar Sebastian Tacke, Sebastian Tacke Department of Structural Biochemistry, Max Planck Institute of Molecular Physiology, Otto-Hahn-Str. 11, 44227 Dortmund, Germany Search for other works by this author on: Oxford Academic Google Scholar ... Show more Roger Wepf, Roger Wepf Centre for Microscopy and Microanalysis, The University of Queensland, St. Lucia Queensland 4072, Brisbane, Australia Search for other works by this author on: Oxford Academic Google Scholar Sander den Hoedt Sander den Hoedt Delmic B.V., Kanaalweg 4, 2326 EB Delft, The Netherlands Search for other works by this author on: Oxford Academic Google Scholar Microscopy and Microanalysis, Volume 28, Issue S1, 1 August 2022, Pages 1276–1278, https://doi.org/10.1017/S1431927622005256 Published: 01 August 2022

Image processing in cryogenic electron tomography (cryoET) is currently at a similar state as Single Particle Analysis (SPA) in cryogenic electron microscopy (cryoEM) was a few years ago. Its data processing workflows are far from being well defined and the user experience is still not smooth. Moreover, file formats of different software packages and their associated metadata are not standardized, mainly since different packages are developed by different groups, focusing on different steps of the data processing pipeline. The Scipion framework, originally developed for SPA (de la Rosa-Trevín et al., 2016), has a generic python workflow engine that gives it the versatility to be extended to other fields, as demonstrated for model building (Martínez et al., 2020). In this article, we provide an extension of Scipion based on a set of tomography plugins (referred to as ScipionTomo hereafter), with a similar purpose: to allow users to be focused on the data processing and analysis instead of having to deal with multiple software installation issues and the inconvenience of switching from one to another, converting metadata files, managing possible incompatibilities, scripting (writing a simple program in a language that the computer must convert to machine language each time the program is run), etcetera. Additionally, having all the software available in an integrated platform allows comparing the results of different algorithms trying to solve the same problem. In this way, the commonalities and differences between estimated parameters shed light on which results can be more trusted than others. ScipionTomo is developed by a collaborative multidisciplinary team composed of Scipion team engineers, structural biologists, and in some cases, the developers whose software packages have been integrated. It is open to anyone in the field willing to contribute to this project. The result is a framework extension that combines the acquired knowledge of Scipion developers in close collaboration with third-party developers, and the on-demand design of functionalities requested by beta testers applying this solution to actual biological problems.

The speed and efficiency of data collection and image processing in cryo-electron microscopy have increased over the last decade. However, cryo specimen preparation techniques have lagged and faster, more reproducible specimen preparation devices are needed. Here, we present a vitrification device with highly automated sample handling, requiring only limited user interaction. Moreover, the device allows inspection of thin films using light microscopy, since the excess liquid is removed through suction by tubes, not blotting paper. In combination with dew-point control, this enables thin film preparation in a controlled and reproducible manner. The advantage is that the quality of the prepared cryo specimen is characterized before electron microscopy data acquisition. The practicality and performance of the device are illustrated with experimental results obtained by vitrification of protein suspensions, lipid vesicles, bacterial and human cells, followed by imaged using single particle analysis, cryo-electron tomography, and cryo correlated light and electron microscopy.

Correlative light and electron microscopy (CLEM) entails a group of multimodal imaging techniques that are combined to pinpoint to the location of fluorescently labeled molecules in the context of their ultrastructural cellular environment. Here we describe a detailed workflow for STORM-CLEM, in which STochastic Optical Reconstruction Microscopy (STORM), an optical super-resolution technique, is correlated with transmission electron microscopy (TEM). This protocol has the advantage that both imaging modalities have resolution at the nanoscale, bringing higher synergies on the information obtained. The sample is prepared according to the Tokuyasu method followed by click-chemistry labeling and STORM imaging. Then, after heavy metal staining, electron microscopy imaging is performed followed by correlation of the two images. The case study presented here is on intracellular pathogens, but the protocol is versatile and could potentially be applied to many types of samples.

Tuberculosis (TB) is the most prevalent bacterial infectious disease in the world, caused by the pathogen Mycobacterium tuberculosis (Mtb). In this study, we have used Mycobacterium marinum (Mm) infection in zebrafish larvae as an animal model for this disease to study the role of the myeloid differentiation factor 88 (Myd88), the key adapter protein of Toll-like receptors. Previously, Myd88 has been shown to enhance innate immune responses against bacterial infections, and in the present study, we have investigated the effect of Myd88 deficiency on the granuloma morphology and the intracellular distribution of bacteria during Mm infection. Our results show that granulomas formed in the tail fin from myd88 mutant larvae have a more compact structure and contain a reduced number of leukocytes compared to the granulomas observed in wild-type larvae. These morphological differences were associated with an increased bacterial burden in the myd88 mutant. Electron microscopy analysis showed that the majority of Mm in the myd88 mutant are located extracellularly, whereas in the wild type, most bacteria were intracellular. In the myd88 mutant, intracellular bacteria were mainly present in compartments that were not electron-dense, suggesting that these compartments had not undergone fusion with a lysosome. In contrast, approximately half of the intracellular bacteria in wild-type larvae were found in electron-dense compartments. These observations in a zebrafish model for tuberculosis suggest a role for Myd88-dependent signalling in two important phenomena that limit mycobacterial growth in the infected tissue. It reduces the number of leukocytes at the site of infection and the acidification of bacteria-containing compartments inside these cells.

Intensity of acquired electron microscopy data is subjected to large variability due to the interplay of many different factors, such as microscope and camera settings used for data acquisition, sample thickness, specimen staining protocol and more. In this work, we developed an efficient method for performing intensity inhomogeneity correction on a single set of combined transmission electron microscopy (TEM) images and demonstrated its positive impact on training a neural network on these data. In addition, we investigated what impact different intensity standardization methods have on the training performance, both for data originating from a single source as well as from several different sources. As a concrete example, we considered the problem of segmenting mitochondria from EM data and demonstrated that we were able to obtain promising results when training our network on a large array of highly-variable in-house TEM data.

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Booklet Date: Friday November 19, 2021 09.15 am – 5 pm Location: Lecture hall S003 UniS, University of Berne Schanzeneckstrasse 1 3012 Bern Registration: https://www.mic.unibe.ch/events/mic_symposium_2021 Organized by the Microscopy Imaging Center (MIC) of the University of Bern The 2021 MIC Symposium will be dedicated to three-dimensional electron microscopy (EM). In recent years, a broad range of EM applications in life sciences has seen major advances. New technologies and hardware solutions are driving tremendous developments, notably with respect to the elucidation of three-dimensional structures of macromolecular assemblies. Protein structure determination by single-particle cryoEM is revolutionising structural biology. Cryo-electron tomography is offering unprecedented views into the macromolecular landscape of cells. Large volume reconstructions by scanning EM visualise tissue organisation in great detail. This year’s MIC Symposium program will offer cuttingedge scientific talks in all these areas to provide a glimpse on the current and future opportunities that EM offers to life sciences. Application is approved for accreditation of the Symposium 2021 for 0.5 day credit for continued education in animal experimentation in the Canton of Bern, Switzerland. MIC thankfully acknowledges financial support of the MIC Symposium 2021 by: The PhD Program Cutting Edge Microscopy and our sponsors from industry Bern, November 19, 2021, MIC Symposium 2021 Abstract Booklet Seite 4/10 Viral replication organelles revisited by cellular cryo-tomography

An exclusive feature of dendritic cells (DCs) is their capacity to present exogenous antigens by MHC class I molecules, called cross-presentation. Here, we show that protein antigen can be conserved in mature murine DCs for several days in a lysosome-like storage compartment, distinct from MHC class II and early endosomal compartments, as an internal source for the supply of MHC class I ligands. Using two different uptake routes via Fcγ receptors and C-type lectin receptors, we could show that antigens were routed towards the same endolysosomal compartments after 48 h. The antigen-containing compartments lacked co-expression of molecules involved in MHC class I processing and presentation including TAP and proteasome subunits as shown by single-cell imaging flow cytometry. Moreover, we observed the absence of cathepsin S but selective co-localization of active cathepsin X with protein antigen in the storage compartments. This indicates cathepsin S-independent antigen degradation and a novel but yet undefined role for cathepsin X in antigen processing and cross-presentation by DCs. In summary, our data suggest that these antigen-containing compartments in DCs can conserve protein antigens from different uptake routes and contribute to long-lasting antigen cross-presentation.

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We have recently identified a novel plasticity protein, doublecortin-like (DCL), that is specifically expressed in the shell of the mouse suprachiasmatic nucleus (SCN). DCL is implicated in neuroplastic events, such as neurogenesis, that require structural rearrangements of the microtubule cytoskeleton, enabling dynamic movements of cell bodies and dendrites. We have inspected DCL expression in the SCN by confocal microscopy and found that DCL is expressed in GABA transporter-3 (GAT3)-positive astrocytes that envelope arginine vasopressin (AVP)-expressing cells. To investigate the role of these DCL-positive astrocytes in circadian rhythmicity, we have used transgenic mice expressing doxycycline-induced short-hairpin (sh) RNA's targeting DCL mRNA (DCL knockdown mice). Compared with littermate wild type (WT) controls, DCL-knockdown mice exhibit significant shorter circadian rest-activity periods in constant darkness and adjusted significantly faster to a jet-lag protocol. As DCL-positive astrocytes are closely associated with AVP-positive cells, we analyzed AVP expression in DCL-knockdown mice and in their WT littermates by 3D reconstructions and transmission electron microscopy (TEM). We found significantly higher numbers of AVP-positive cells with increased volume and more intensity in DCL-knockdown mice. We found alterations in the numbers of dense core vesicle-containing neurons at ZT8 and ZT20 suggesting that the peak and trough of neuropeptide biosynthesis is dampened in DCL-knockdown mice compared to WT littermates. Together, our data suggest an important role for the astrocytic plasticity in the regulation of circadian rhythms and point to the existence of a specific DCL+ astrocyte-AVP+ neuronal network located in the dorsal SCN implicated in AVP biosynthesis.

Mutations in the Crumbs homologue 1 (CRB1) gene cause inherited retinal dystrophies, such as early-onset retinitis pigmentosa and Leber congenital amaurosis. A Brown Norway rat strain was reported with a spontaneous insertion-deletion (indel) mutation in exon 6 of Crb1. It has been reported that these Crb1 mutant rats show vascular abnormalities associated with retinal telangiectasia and possess an early-onset retinal degenerative phenotype with outer limiting membrane breaks and focal loss of retinal lamination at 2 months of age. Here, we further characterized the morphological phenotype of new-born and adult Crb1 mutant rats in comparison with age-matched Brown Norway rats without a mutation in Crb1. A significantly decreased retinal function and visual acuity was observed in Crb1 mutant rats at 1 and 3 months of age, respectively. Moreover, in control rats, the subcellular localization of canonical CRB1 was observed at the subapical region in Müller glial cells while CRB2 was observed at the subapical region in both photoreceptors and Müller glial cells by immuno-electron microscopy. CRB1 localization was lost in the Crb1 mutant rats, whereas CRB2 was still observed. In addition, we determined the tropism of subretinal or intravitreally administered AAV5-, AAV9- or AAV6-variant ShH10Y445F vectors in new-born control and Crb1 mutant rat retinas. We showed that subretinal injection of AAV5 and AAV9 at postnatal days 5 (P5) or 8 (P8) predominantly infected the retinal pigment epithelium (RPE) and photoreceptor cells; while intravitreal injection of ShH10Y445F at P5 or P8 resulted in efficient infection of mainly Müller glial cells. Using knowledge of the subcellular localization of CRB1 and the ability of ShH10Y445F to infect Müller glial cells, canonical hCRB1 and hCRB2 AAV-mediated gene therapy were explored in new-born Crb1 mutant rats. Enhanced retinal function after gene therapy delivery in the Crb1 rat was not observed. No timely rescue of the retinal phenotype was observed using retinal function and visual acuity, suggesting the need for earlier onset of expression of recombinant hCRB proteins in Müller glial cells to rescue the severe retinal phenotype in Crb1 mutant rats.

Cathepsin S is a lysosomal cysteine protease highly expressed in immune cells such as dendritic cells, B cells and macrophages. Its functions include extracellular matrix breakdown and cleavage of cell adhesion molecules to facilitate immune cell motility, as well as cleavage of the invariant chain during maturation of major histocompatibility complex II. The identification of these diverse specific functions has brought the challenge of delineating cathepsin S activity with great spatial precision, relative to related enzymes and substrates. Here, the development of a potent and highly selective two-step activity-based probe for cathepsin S and the application in multicolor bio-orthogonal correlative light-electron microscopy is presented. LHVS, which has been reported as a selective inhibitor of cathepsin S with nanomolar potency, formed the basis for our probe design. However, in competitive activity-based protein profiling experiments LHVS showed significant cross-reactivity toward Cat L. Introduction of an azide group in the P2 position expanded the selectivity window for cathepsin S, but rendered the probe undetectable, as demonstrated in bio-orthogonal competitive activity-based protein profiling. Incorporation of an additional azide handle for click chemistry on the solvent-exposed P1 position allowed for selective labeling of cathepsin S. This highlights the influence of click handle positioning on probe efficacy. This probe was utilized in multicolor bio-orthogonal confocal and correlative light-electron microscopy to investigate the localization of cathepsin S activity at an ultrastructural level in bone marrow-derived dendritic cells. The tools developed in this study will aid the characterization of the variety of functions of cathepsin S throughout biology.