Modular segmentation, spatial analysis and visualization of volume electron microscopy datasets.

Volume electron microscopy is the method of choice for the in situ interrogation of cellular ultrastructure at the nanometer scale, and with the increase in large raw image datasets generated, improving computational strategies for image segmentation and spatial analysis is necessary. Here we describe a practical and annotation-efficient pipeline for organelle-specific segmentation, spatial analysis and visualization of large volume electron microscopy datasets using freely available, user-friendly software tools that can be run on a single standard workstation. The procedures are aimed at researchers in the life sciences with modest computational expertise, who use volume electron microscopy and need to generate three-dimensional (3D) segmentation labels for different types of cell organelles while minimizing manual annotation efforts, to analyze the spatial interactions between organelle instances and to visualize the 3D segmentation results. We provide detailed guidelines for choosing well-suited segmentation tools for specific cell organelles, and to bridge compatibility issues between freely available open-source tools, we distribute the critical steps as easily installable Album solutions for deep learning segmentation, spatial analysis and 3D rendering. Our detailed description can serve as a reference for similar projects requiring particular strategies for single- or multiple-organelle analysis, which can be achieved with computational resources commonly available to single-user setups.

Host Subcellular Organelles: Targets of Viral Manipulation.

Viruses have evolved sophisticated mechanisms to manipulate host cell processes and utilize intracellular organelles to facilitate their replication. These complex interactions between viruses and cellular organelles allow them to hijack the cellular machinery and impair homeostasis. Moreover, viral infection alters the cell membrane's structure and composition and induces vesicle formation to facilitate intracellular trafficking of viral components. However, the research focus has predominantly been on the immune response elicited by viruses, often overlooking the significant alterations that viruses induce in cellular organelles. Gaining a deeper understanding of these virus-induced cellular changes is crucial for elucidating the full life cycle of viruses and developing potent antiviral therapies. Exploring virus-induced cellular changes could substantially improve our understanding of viral infection mechanisms.

Tools for assembling the cell: Towards the era of cell structural bioinformatics.

Cells consist of large components, such as organelles, that recursively factor into smaller systems, such as condensates and protein complexes, forming a dynamic multi-scale structure of the cell. Recent technological innovations have paved the way for systematic interrogation of subcellular structures, yielding unprecedented insights into their roles and interactions. In this workshop, we discuss progress, challenges, and collaboration to marshal various computational approaches toward assembling an integrated structural map of the human cell.

Genetically encoded multimeric tags for subcellular protein localization in cryo-EM.

Cryo-electron tomography (cryo-ET) allows for label-free high-resolution imaging of macromolecular assemblies in their native cellular context. However, the localization of macromolecules of interest in tomographic volumes can be challenging. Here we present a ligand-inducible labeling strategy for intracellular proteins based on fluorescent, 25-nm-sized, genetically encoded multimeric particles (GEMs). The particles exhibit recognizable structural signatures, enabling their automated detection in cryo-ET data by convolutional neural networks. The coupling of GEMs to green fluorescent protein-tagged macromolecules of interest is triggered by addition of a small-molecule ligand, allowing for time-controlled labeling to minimize disturbance to native protein function. We demonstrate the applicability of GEMs for subcellular-level localization of endogenous and overexpressed proteins across different organelles in human cells using cryo-correlative fluorescence and cryo-ET imaging. We describe means for quantifying labeling specificity and efficiency, and for systematic optimization for rare and abundant protein targets, with emphasis on assessing the potential effects of labeling on protein function.

DCX-EMAP is a core organizer for the ultrastructure of Drosophila mechanosensory organelles.

Mechanoreceptor cells develop specialized mechanosensory organelles (MOs), where force-sensitive channels and supporting structures are organized in an orderly manner to detect forces. It is intriguing how MOs are formed. Here, we address this issue by studying the MOs of fly ciliated mechanoreceptors. We show that the main structure of the MOs is a compound cytoskeleton formed of short microtubules and electron-dense materials (EDMs). In a knock-out mutant of DCX-EMAP, this cytoskeleton is nearly absent, suggesting that DCX-EMAP is required for the formation of the MOs and in turn fly mechanotransduction. Further analysis reveals that DCX-EMAP expresses in fly ciliated mechanoreceptors and localizes to the MOs. Moreover, it plays dual roles by promoting the assembly/stabilization of the microtubules and the accumulation of the EDMs in the MOs. Therefore, DCX-EMAP serves as a core ultrastructural organizer of the MOs, and this finding provides novel molecular insights as to how fly MOs are formed.

Ultrastructural Analysis and Quantification of Peroxisome-Organelle Contacts.

Transmission electron microscopy (TEM) has long been a vital technology to visualize the interaction of cellular compartments at the highest possible resolution. While this paved the way to describing organelles within the cellular context in detail, TEM has long been underused to generate quantitative data, analyzing those interactions as well as underlying mechanisms leading to their formation and modification. Here we describe a simple stereological method to unbiasedly assess the extent of organelle-organelle membrane contact sites, able to efficiently generate accurate and reproducible quantitative data from cultured mammalian cells prepared for TEM.

Morphology and ultrastructure of the Balbiani body in the oocytes of closely related bush cricket species. Shared features reveal important aspect of functioning.

Balbiani bodies (Bbs) are female germline-specific organelle assemblages usually composed of mitochondria, Golgi complexes, elements of endoplasmic reticulum and accumulations of fine granular material, termed the nuage. Here we present results of morphological and ultrastructural analysis of the Bb of four bush crickets nested in four subfamilies of the family Tettigonidae. This study has revealed that Bbs of closely related species (belonging to the defined evolutionary line) are morphologically rather different. In two species (Meconema meridionale and Pholidoptera griseoaptera) the Bb has the form of a hollow hemisphere that covers a part of the germinal vesicle surface. In contrast, the Bb of Conocephalus fuscus and Leptophyes albovittata is less distinct and surrounds the whole or the majority of the germinal vesicle surface. Aside from this difference, the Bbs of all four studied species are built of identical sets of organelles and, most importantly, share one significant feature: close association of mitochondria and nuage accumulations. We show additionally that mitochondria remaining in direct contact with the nuage are characterized by distinct morphologies e.g. elongated, dumbbell shaped or bifurcated. In the light of our results and literature survey, the ancestral function of the Bb is discussed.

Evaluation of the Role of Bacterial Amyloid on Nucleoid Structure Using Cryo-Soft X-Ray Tomography.

Bacterial chromosomal DNA is packed within a non-membranous structure, the nucleoid, thanks to nucleoid associated proteins (NAPs). The role of bacterial amyloid has recently emerged among these NAPs, particularly with the nucleoid-associated protein Hfq that plays a direct role in DNA compaction. In this chapter, we present a 3D imaging technique, cryo-soft X-ray tomography (cryo-SXT) to obtain a detailed 3D visualization of subcellular bacterial structures, especially the nucleoid. Cryo-SXT imaging of native unlabeled cells enables observation of the nucleoid in 3D with a high resolution, allowing to evidence in vivo the role of amyloids on DNA compaction. The precise experimental methods to obtain 3D tomograms will be presented.

In-resin CLEM of Epon-embedded cells using proximity labeling.

Biotin ligases have been developed as proximity biotinylation enzymes for analyses of the interactome. However, there has been no report on the application of proximity labeling for in-resin correlative light-electron microscopy of Epon-embedded cells. In this study, we established a proximity-labeled in-resin CLEM of Epon-embedded cells using miniTurbo, a biotin ligase. Biotinylation by miniTurbo was observed in cells within 10 min following the addition of biotin to the medium. Using fluorophore-conjugated streptavidin, intracellular biotinylated proteins were labeled after fixation of cells with a mixture of paraformaldehyde and glutaraldehyde. Fluorescence of these proteins was resistant to osmium tetroxide staining and was detected in 100-nm ultrathin sections of Epon-embedded cells. Ultrastructures of organelles were preserved well in the same sections. Fluorescence in sections was about 14-fold brighter than that in the sections of Epon-embedded cells expressing mCherry2 and was detectable for 14 days. When mitochondria-localized miniTurbo was expressed in the cells, mitochondria-like fluorescent signals were detected in the sections, and ultrastructures of mitochondria were observed as fluorescence-positive structures in the same sections by scanning electron microscopy. Proximity labeling using miniTurbo led to more stable and brighter fluorescent signals in the ultrathin sections of Epon-embedded cells, resulting in better performance of in-resin CLEM.

Perspective - ultrastructural analyses reflect the effects of sleep and sleep loss on neuronal cell biology.

Recent electron microscopic analyses of neurons in the Drosophila and rodent brain demonstrate that acute or chronic sleep loss can alter the structures of various organelles, including mitochondria, nucleus, and Golgi apparatus. Here, we discuss these findings in the context of biochemical findings from the sleep deprived brain, to clarify how these morphological changes may related to altered organelle function. We discuss how, taken together, the available data suggest that sleep loss (particularly chronic sleep loss) disrupts such fundamental cellular processes as transcription, translation, intracellular transport, and metabolism. A better understanding of these effects will have broad implications for understanding the biological importance of sleep, and the relationship of sleep loss to neuropathology.

Ultrastructure of selected life history stages of the parasitic dinoflagellate Euduboscquella cachoni.

Euduboscquella species differ from most other syndinean dinoflagellates by having mononucleate trophonts, but resemble species of Amoebophrya and Sphaeripara by episome-hyposome differentiation and cortical complexity. Cytology and development of Euduboscquella species are well characterized, but their ultrastructure remains essentially unexplored. Transmission electron microscopy of Euduboscquella cachoni trophonts, tomont, and sporocytes revealed previously unrecognized structures. Initially dense, fibrous chromosomes uncoiled during early infection, with condensed chromosomes absent over much of the growth cycle recondensing at trophont maturity. The hyposomal amphiesma was two appressed membranes, the episomal cortex was alveolate, and a supraepisomal cavity limited by membrane enclosed the episome. Pseudopod-like extensions of the hyposome during mid infection may facilitate osmotrophic nutrition. The pharyngeal lamina appears to lack ingestatory function; however, transcortical transport of particles occurred via the supraepisomal cavity and episomal micropores. Microtubules originating from the electron-opaque perinema bordering the episome, formed an episomal skeleton hypothesized to function with the pharyngeal lamina, perinema, and the paired membranes of the supraepisomal cavity to effect parasite egress and ingestion of host material. Trichocysts absent during early infection developed during late infection and reached maturity during sporogenesis, suggesting functional importance in spore survival or infection.

Electron microscopy for imaging organelles in plants and algae.

Recent developments in both instrumentation and image analysis algorithms have allowed three-dimensional electron microscopy (3D-EM) to increase automated image collections through large tissue volumes using serial block-face scanning EM (SEM) and to achieve near-atomic resolution of macromolecular complexes using cryo-electron tomography (cryo-ET) and sub-tomogram averaging. In this review, we discuss applications of cryo-ET to cell biology research on plant and algal systems and the special opportunities they offer for understanding the organization of eukaryotic organelles with unprecedently resolution. However, one of the most challenging aspects for cryo-ET is sample preparation, especially for multicellular organisms. We also discuss correlative light and electron microscopy (CLEM) approaches that have been developed for ET at both room and cryogenic temperatures.

High-speed fluorescence image-enabled cell sorting.

Fast and selective isolation of single cells with unique spatial and morphological traits remains a technical challenge. Here, we address this by establishing high-speed image-enabled cell sorting (ICS), which records multicolor fluorescence images and sorts cells based on measurements from image data at speeds up to 15,000 events per second. We show that ICS quantifies cell morphology and localization of labeled proteins and increases the resolution of cell cycle analyses by separating mitotic stages. We combine ICS with CRISPR-pooled screens to identify regulators of the nuclear factor κB (NF-κB) pathway, enabling the completion of genome-wide image-based screens in about 9 hours of run time. By assessing complex cellular phenotypes, ICS substantially expands the phenotypic space accessible to cell-sorting applications and pooled genetic screening.

Low-invasive 5D visualization of mitotic progression by two-photon excitation spinning-disk confocal microscopy.

Non-linear microscopy, such as multi-photon excitation microscopy, offers spatial localities of excitations, thereby achieving 3D cross-sectional imaging with low phototoxicity even in thick biological specimens. We had developed a multi-point scanning two-photon excitation microscopy system using a spinning-disk confocal scanning unit. However, its severe color cross-talk has precluded multi-color simultaneous imaging. Therefore, in this study, we introduced a mechanical switching system to select either of two NIR laser light pulses and an image-splitting detection system for 3- or 4-color imaging. As a proof of concept, we performed multi-color fluorescent imaging of actively dividing human HeLa cells and tobacco BY-2 cells. We found that the proposed microscopy system enabled time-lapse multi-color 3D imaging of cell divisions while avoiding photodamage. Moreover, the application of a linear unmixing method to the 5D dataset enabled the precise separation of individual intracellular components in multi-color images. We thus demonstrated the versatility of our new microscopy system in capturing the dynamic processes of cellular components that could have multitudes of application.

Symmetry breaking in the female germline cyst.

In mammals and flies, only one cell in a multicellular female germline cyst becomes an oocyte, but how symmetry is broken to select the oocyte is unknown. Here, we show that the microtubule (MT) minus end-stabilizing protein Patronin/CAMSAP marks the future Drosophila oocyte and is required for oocyte specification. The spectraplakin Shot recruits Patronin to the fusome, a branched structure extending into all cyst cells. Patronin stabilizes more MTs in the cell with the most fusome material. Our data suggest that this weak asymmetry is amplified by Dynein-dependent transport of Patronin-stabilized MTs. This forms a polarized MT network, along which Dynein transports oocyte determinants into the presumptive oocyte. Thus, Patronin amplifies a weak fusome anisotropy to break symmetry and select one cell to become the oocyte.

Whole-cell organelle segmentation in volume electron microscopy.

Cells contain hundreds of organelles and macromolecular assemblies. Obtaining a complete understanding of their intricate organization requires the nanometre-level, three-dimensional reconstruction of whole cells, which is only feasible with robust and scalable automatic methods. Here, to support the development of such methods, we annotated up to 35 different cellular organelle classes-ranging from endoplasmic reticulum to microtubules to ribosomes-in diverse sample volumes from multiple cell types imaged at a near-isotropic resolution of 4 nm per voxel with focused ion beam scanning electron microscopy (FIB-SEM)1. We trained deep learning architectures to segment these structures in 4 nm and 8 nm per voxel FIB-SEM volumes, validated their performance and showed that automatic reconstructions can be used to directly quantify previously inaccessible metrics including spatial interactions between cellular components. We also show that such reconstructions can be used to automatically register light and electron microscopy images for correlative studies. We have created an open data and open-source web repository, 'OpenOrganelle', to share the data, computer code and trained models, which will enable scientists everywhere to query and further improve automatic reconstruction of these datasets.

An open-access volume electron microscopy atlas of whole cells and tissues.

Understanding cellular architecture is essential for understanding biology. Electron microscopy (EM) uniquely visualizes cellular structures with nanometre resolution. However, traditional methods, such as thin-section EM or EM tomography, have limitations in that they visualize only a single slice or a relatively small volume of the cell, respectively. Focused ion beam-scanning electron microscopy (FIB-SEM) has demonstrated the ability to image small volumes of cellular samples with 4-nm isotropic voxels1. Owing to advances in the precision and stability of FIB milling, together with enhanced signal detection and faster SEM scanning, we have increased the volume that can be imaged with 4-nm voxels by two orders of magnitude. Here we present a volume EM atlas at such resolution comprising ten three-dimensional datasets for whole cells and tissues, including cancer cells, immune cells, mouse pancreatic islets and Drosophila neural tissues. These open access data (via OpenOrganelle2) represent the foundation of a field of high-resolution whole-cell volume EM and subsequent analyses, and we invite researchers to explore this atlas and pose questions.

Microstructure investigation of plant architecture with X-ray microscopy.

In recent years, the plant morphology has been well studied by multiple approaches at cellular and subcellular levels. Two-dimensional (2D) microscopy techniques offer imaging of plant structures on a wide range of magnifications for researchers. However, subcellular imaging is still challenging in plant tissues like roots and seeds. Here we use a three-dimensional (3D) imaging technology based on the X-ray microscope (XRM) and analyze several plant tissues from different plant species. The XRM provides new insights into plant structures using non-destructive imaging at high-resolution and high contrast. We also utilized a workflow aiming to acquire accurate and high-quality images in the context of the whole specimen. Multiple plant samples including rice, tobacco, Arabidopsis and maize were used to display the differences of phenotypes. Our work indicates that the XRM is a powerful tool to investigate plant microstructure in high-resolution scale. Our work also provides evidence that evaluate and quantify tissue specific differences for a range of plant species. We also characterize novel plant tissue phenotypes by the XRM, such as seeds in Arabidopsis, and utilize them for novel observation measurement. Our work represents an evaluated spatial and temporal resolution solution on seed observation and screening.

Molecular principles of recruitment and dynamics of guest proteins in liquid droplets.

Despite the continuous discovery of host and guest proteins in membraneless organelles, complex host-guest interactions hinder the understanding of the molecular grammar governing liquid-liquid phase separation. In this study, we characterized the localization and dynamic properties of guest proteins in liquid droplets using single-molecule fluorescence microscopy. Eighteen guest proteins of different sizes, structures, and oligomeric states were examined in host p53 liquid droplets. Recruitment did not significantly depend on the structural properties of the guest proteins, but was moderately correlated with their length, total charge, and number of R and Y residues. In contrast, the diffusion of disordered guest proteins was comparable to that of host p53, whereas that of folded proteins varied widely. Molecular dynamics simulations suggest that folded proteins diffuse within the voids of the liquid droplet while interacting weakly with neighboring host proteins, whereas disordered proteins adapt their structures to form tight interactions with the host proteins. Our study provides insights into the key molecular principles of the localization and dynamics of guest proteins in liquid droplets.

Ultrastructural Features of Membranous Replication Organelles Induced by Positive-Stranded RNA Viruses.

All intracellular pathogens critically depend on host cell organelles and metabolites for successful infection and replication. One hallmark of positive-strand RNA viruses is to induce alterations of the (endo)membrane system in order to shield their double-stranded RNA replication intermediates from detection by the host cell's surveillance systems. This spatial seclusion also allows for accruing host and viral factors and building blocks required for efficient replication of the genome and prevents access of antiviral effectors. Even though the principle is iterated by almost all positive-strand RNA viruses infecting plants and animals, the specific structure and the organellar source of membranes differs. Here, we discuss the characteristic ultrastructural features of the virus-induced membranous replication organelles in plant and animal cells and the scientific progress gained by advanced microscopy methods.