CryoVesNet: A dedicated framework for synaptic vesicle segmentation in cryo-electron tomograms.

Cryo-electron tomography (cryo-ET) has the potential to reveal cell structure down to atomic resolution. Nevertheless, cellular cryo-ET data is highly complex, requiring image segmentation for visualization and quantification of subcellular structures. Due to noise and anisotropic resolution in cryo-ET data, automatic segmentation based on classical computer vision approaches usually does not perform satisfactorily. Communication between neurons relies on neurotransmitter-filled synaptic vesicle (SV) exocytosis. Cryo-ET study of the spatial organization of SVs and their interconnections allows a better understanding of the mechanisms of exocytosis regulation. Accurate SV segmentation is a prerequisite to obtaining a faithful connectivity representation. Hundreds of SVs are present in a synapse, and their manual segmentation is a bottleneck. We addressed this by designing a workflow consisting of a convolutional network followed by post-processing steps. Alongside, we provide an interactive tool for accurately segmenting spherical vesicles. Our pipeline can in principle segment spherical vesicles in any cell type as well as extracellular and in vitro spherical vesicles.

Non-averaged single-molecule tertiary structures reveal RNA self-folding through individual-particle cryo-electron tomography.

Large-scale and continuous conformational changes in the RNA self-folding process present significant challenges for structural studies, often requiring trade-offs between resolution and observational scope. Here, we utilize individual-particle cryo-electron tomography (IPET) to examine the post-transcriptional self-folding process of designed RNA origami 6-helix bundle with a clasp helix (6HBC). By avoiding selection, classification, averaging, or chemical fixation and optimizing cryo-ET data acquisition parameters, we reconstruct 120 three-dimensional (3D) density maps from 120 individual particles at an electron dose of no more than 168 e-Å-2, achieving averaged resolutions ranging from 23 to 35 Å, as estimated by Fourier shell correlation (FSC) at 0.5. Each map allows us to identify distinct RNA helices and determine a unique tertiary structure. Statistical analysis of these 120 structures confirms two reported conformations and reveals a range of kinetically trapped, intermediate, and highly compacted states, demonstrating a maturation folding landscape likely driven by helix-helix compaction interactions.

Serialized on-grid lift-in sectioning for tomography (SOLIST) enables a biopsy at the nanoscale.

Cryo-focused ion beam milling has substantially advanced our understanding of molecular processes by opening windows into cells. However, applying this technique to complex samples, such as tissues, has presented considerable technical challenges. Here we introduce an innovative adaptation of the cryo-lift-out technique, serialized on-grid lift-in sectioning for tomography (SOLIST), addressing these limitations. SOLIST enhances throughput, minimizes ice contamination and improves sample stability for cryo-electron tomography. It thereby facilitates the high-resolution imaging of a wide range of specimens. We illustrate these advantages on reconstituted liquid-liquid phase-separated droplets, brain organoids and native tissues from the mouse brain, liver and heart. With SOLIST, cellular processes can now be investigated at molecular resolution directly in native tissue. Furthermore, our method has a throughput high enough to render cryo-lift-out a competitive tool for structural biology. This opens new avenues for unprecedented insights into cellular function and structure in health and disease, a 'biopsy at the nanoscale'.

Small LEA proteins mitigate air-water interface damage to fragile cryo-EM samples during plunge freezing.

Air-water interface (AWI) interactions during cryo-electron microscopy (cryo-EM) sample preparation cause significant sample loss, hindering structural biology research. Organisms like nematodes and tardigrades produce Late Embryogenesis Abundant (LEA) proteins to withstand desiccation stress. Here we show that these LEA proteins, when used as additives during plunge freezing, effectively mitigate AWI damage to fragile multi-subunit molecular samples. The resulting high-resolution cryo-EM maps are comparable to or better than those obtained using existing AWI damage mitigation methods. Cryogenic electron tomography reveals that particles are localized at specific interfaces, suggesting LEA proteins form a barrier at the AWI. This interaction may explain the observed sample-dependent preferred orientation of particles. LEA proteins offer a simple, cost-effective, and adaptable approach for cryo-EM structural biologists to overcome AWI-related sample damage, potentially revitalizing challenging projects and advancing the field of structural biology.

In Situ Synthesis and Visualization of Membrane SNAP25 Nano-Organization.

Cryo-electron tomography (cryo-ET) can provide insights into the structure and states of natural membrane environments to explore the role of SNARE proteins at membrane fusion and understand the relationship between their subcellular localization/formation and action mechanism. Nevertheless, the identification of individual molecules in crowded and low signal-to-noise ratio membrane environments remains a significant challenge. In this study, cryo-ET is employed to image near-physiological state 293T cell membranes, specifically utilizing in situ synthesized gold nanoparticles (AuNPs) bound with cysteine-rich protein tags to single-molecularly labeled synaptosomal-associated protein 25 (SNAP25) on the membrane surface. The high-resolution images reveal that SNAP25 is predominantly located in regions of high molecular density within the cell membrane and aggregates into smaller clusters, which may increase the fusion efficiency. Remarkably, a zigzag arrangement of SNAP25 is observed on the cell membrane. These findings provide valuable insights into the functional mechanisms of SNARE proteins.

Zooming in and out: Exploring RNA Viral Infections with Multiscale Microscopic Methods.

RNA viruses, being submicroscopic organisms, have intriguing biological makeups and substantially impact human health. Microscopic methods have been utilized for studying RNA viruses at a variety of scales. In order of observation scale from large to small, fluorescence microscopy, cryo-soft X-ray tomography (cryo-SXT), serial cryo-focused ion beam/scanning electron microscopy (cryo-FIB/SEM) volume imaging, cryo-electron tomography (cryo-ET), and cryo-electron microscopy (cryo-EM) single-particle analysis (SPA) have been employed, enabling researchers to explore the intricate world of RNA viruses, their ultrastructure, dynamics, and interactions with host cells. These methods evolve to be combined to achieve a wide resolution range from atomic to sub-nano resolutions, making correlative microscopy an emerging trend. The developments in microscopic methods provide multi-fold and spatial information, advancing our understanding of viral infections and providing critical tools for developing novel antiviral strategies and rapid responses to emerging viral threats.

MiLoPYP: self-supervised molecular pattern mining and particle localization in situ.

Cryo-electron tomography allows the routine visualization of cellular landscapes in three dimensions at nanometer-range resolutions. When combined with single-particle tomography, it is possible to obtain near-atomic resolution structures of frequently occurring macromolecules within their native environment. Two outstanding challenges associated with cryo-electron tomography/single-particle tomography are the automatic identification and localization of proteins, tasks that are hindered by the molecular crowding inside cells, imaging distortions characteristic of cryo-electron tomography tomograms and the sheer size of tomographic datasets. Current methods suffer from low accuracy, demand extensive and time-consuming manual labeling or are limited to the detection of specific types of proteins. Here, we present MiLoPYP, a two-step dataset-specific contrastive learning-based framework that enables fast molecular pattern mining followed by accurate protein localization. MiLoPYP's ability to effectively detect and localize a wide range of targets including globular and tubular complexes as well as large membrane proteins, will contribute to streamline and broaden the applicability of high-resolution workflows for in situ structure determination.

Protocol for subtomogram averaging of helical filaments in cryo-electron tomography.

Helical filaments are essential macromolecular elements in cellular organization and dynamics. Recent advances in cryo-electron tomography allow faithful imaging of isolated or in-cell filaments. Here, we present a protocol to generate density maps at sub-nanometer resolution of helical filaments by subtomogram averaging, exemplified with isolated mumps virus nucleocapsids and their in-cell form as an extension of the protocol. We detail procedures from pre-processing of tilt-series movie frames to refinement of reconstructed averages for streamlined data processing of helical filaments. For complete details on the use and execution of this protocol, please refer to Zhang et al.1.

Smart parallel automated cryo-electron tomography.

In situ cryo-electron tomography enables investigation of macromolecules in their native cellular environment. Samples have become more readily available owing to recent software and hardware advancements. Data collection, however, still requires an experienced operator and appreciable microscope time to carefully select targets for high-throughput tilt series acquisition. Here, we developed smart parallel automated cryo-electron tomography (SPACEtomo), a workflow using machine learning approaches to fully automate the entire cryo-electron tomography process, including lamella detection, biological feature segmentation, target selection and parallel tilt series acquisition, all without the need for human intervention. This degree of automation will be essential for obtaining statistically relevant datasets and high-resolution structures of macromolecules in their native context.

Observation of Vacuoles in Arabidopsis Developing Pollen Grains with Whole-Cell Electron Tomography.

Arabidopsis thaliana developing pollen grains serve as an excellent system for studying vacuole dynamics. Here, we present a methodological approach that utilizes the serial tomography package in Etomo software from IMOD to generate whole-cell tomograms on A. thaliana developing pollens for visualizing vacuoles on the whole-cell scale. In order to understand the vacuole dynamics along with the pollen maturation, we also introduce a sampling method aimed at harvesting the pollen grains at various stages, marked by the vegetative nucleus or generative cell. The cryo-fixation/freeze-substitution technique can then be applied to preserve the fine structures of the pollen grains and facilitate detailed ultrastructure examination. Through this method, large-volume whole-cell electron tomograms regarding vacuolar morphologies and ultrastructural changes during pollen development and maturation have been obtained. Overall, the method presented here provides valuable insights into the dynamic nature of vacuoles in Arabidopsis developing pollen.

Whole-Cell Electron Tomography Analysis of Vacuole Biogenesis.

Vacuoles in plant cells are the most prominent organelles that harbor distinctive features, including lytic function, storage of proteins and sugars, balance of cell volume, and defense responses. Despite their dominant size and functional versatility, the nature and biogenesis of vacuoles in plants per se remain elusive and several models have been proposed. Recently, we used the whole-cell 3D electron tomography (ET) technique to study vacuole formation and distribution at nanometer resolution and demonstrated that small vacuoles are derived from multivesicular body maturation and fusion. Good sample preparation is a critical step to get high-quality electron tomography images. In this chapter, we provide detailed sample preparation methods for high-resolution ET in Arabidopsis thaliana root cells, including high-pressure freezing, subsequent freeze-substitution fixation, embedding, and serial sectioning.

High-Pressure Freezing and Low-Temperature Processing of Seeds for Electron Microscopy and Electron Tomography.

High-pressure freezing/freeze substitution has been used to preserve biological samples for ultrastructure study instead of chemical fixation. For most plant samples, the water content is too high and cannot be properly preserved during cryofixation. Additionally, the cell wall is a barrier that prevents the substitution of water with the resin. In this chapter, we will discuss modified high-pressure freezing and subsequent processing protocols based on our routinely used methodology for examining Arabidopsis seeds in transmission electron microscopy and electron tomography.

Combining array tomography with electron tomography provides insights into leakiness of the blood-brain barrier in mouse cortex.

Like other volume electron microscopy approaches, automated tape-collecting ultramicrotomy (ATUM) enables imaging of serial sections deposited on thick plastic tapes by scanning electron microscopy (SEM). ATUM is unique in enabling hierarchical imaging and thus efficient screening for target structures, as needed for correlative light and electron microscopy. However, SEM of sections on tape can only access the section surface, thereby limiting the axial resolution to the typical size of cellular vesicles with an order of magnitude lower than the acquired xy resolution. In contrast, serial-section electron tomography (ET), a transmission electron microscopy-based approach, yields isotropic voxels at full EM resolution, but requires deposition of sections on electron-stable thin and fragile films, thus making screening of large section libraries difficult and prone to section loss. To combine the strength of both approaches, we developed 'ATUM-Tomo, a hybrid method, where sections are first reversibly attached to plastic tape via a dissolvable coating, and after screening detached and transferred to the ET-compatible thin films. As a proof-of-principle, we applied correlative ATUM-Tomo to study ultrastructural features of blood-brain barrier (BBB) leakiness around microthrombi in a mouse model of traumatic brain injury. Microthrombi and associated sites of BBB leakiness were identified by confocal imaging of injected fluorescent and electron-dense nanoparticles, then relocalized by ATUM-SEM, and finally interrogated by correlative ATUM-Tomo. Overall, our new ATUM-Tomo approach will substantially advance ultrastructural analysis of biological phenomena that require cell- and tissue-level contextualization of the finest subcellular textures.

Cell-to-cell interactions revealed by cryo-tomography of a DPANN co-culture system.

DPANN is a widespread and diverse group of archaea characterized by their small size, reduced genome, limited metabolic pathways, and symbiotic existence. Known DPANN species are predominantly obligate ectosymbionts that depend on their host for proliferation. The structural and molecular details of host recognition, host-DPANN intercellular communication, and host adaptation in response to DPANN attachment remain unknown. Here, we use electron cryotomography (cryo-ET) to show that the Microcaldus variisymbioticus ARM-1 may interact with its host, Metallosphaera javensis AS-7 through intercellular proteinaceous nanotubes. Combining cryo-ET and sub-tomogram averaging, we show the in situ architectures of host and DPANN S-layers and the structures of the nanotubes in their primed and extended states. In addition, comparative proteomics and genomic analyses identified host proteomic changes in response to DPANN attachment. These results provide insights into the structural basis of host-DPANN communication and deepen our understanding of the host ectosymbiotic relationships.

Quantitative Analysis of Rhodobacter sphaeroides Storage Organelles via Cryo-Electron Tomography and Light Microscopy.

Bacterial cytoplasmic organelles are diverse and serve many varied purposes. Here, we employed Rhodobacter sphaeroides to investigate the accumulation of carbon and inorganic phosphate in the storage organelles, polyhydroxybutyrate (PHB) and polyphosphate (PP), respectively. Using cryo-electron tomography (cryo-ET), these organelles were observed to increase in size and abundance when growth was arrested by chloramphenicol treatment. The accumulation of PHB and PP was quantified from three-dimensional (3D) segmentations in cryo-tomograms and the analysis of these 3D models. The quantification of PHB using both segmentation analysis and liquid chromatography and mass spectrometry (LCMS) each demonstrated an over 10- to 20-fold accumulation of PHB. The cytoplasmic location of PHB in cells was assessed with fluorescence light microscopy using a PhaP-mNeonGreen fusion-protein construct. The subcellular location and enumeration of these organelles were correlated by comparing the cryo-ET and fluorescence microscopy data. A potential link between PHB and PP localization and possible explanations for co-localization are discussed. Finally, the study of PHB and PP granules, and their accumulation, is discussed in the context of advancing fundamental knowledge about bacterial stress response, the study of renewable sources of bioplastics, and highly energetic compounds.

Recent technical advances in cellular cryo-electron tomography.

Understanding the in situ structure, organization, and interactions of macromolecules is essential for elucidating their functions and mechanisms of action. Cellular cryo-electron tomography (cryo-ET) is a cutting-edge technique that reveals in situ molecular-resolution architectures of macromolecules in their lifelike states. It also provides insights into the three-dimensional distribution of macromolecules and their spatial relationships with various subcellular structures. Thus, cellular cryo-ET bridges the gap between structural biology and cell biology. With rapid advancements, this technique achieved substantial improvements in throughput, automation, and resolution. This review presents the fundamental principles and methodologies of cellular cryo-ET, highlighting recent developments in sample preparation, data collection, and image processing. We also discuss emerging trends and potential future directions. As cellular cryo-ET continues to develop, it is set to play an increasingly vital role in structural cell biology.

Automated fiducial-based alignment of cryo-electron tomography tilt series in Dynamo.

With the advent of modern technologies for cryo-electron tomography (cryo-ET), high-quality tilt series are more rapidly acquired than processed and analyzed. Thus, a robust and fast-automated alignment for batch processing in cryo-ET is needed. While different software packages have made available several approaches for automated marker-based alignment of tilt series, manual user intervention remains necessary for many datasets, thus preventing high-throughput tomography. We have developed a MATLAB-based framework integrated into the Dynamo software package for automatic detection of fiducial markers that generates a robust alignment model with minimal input parameters. This approach allows high-throughput, unsupervised volume reconstruction. This new module extends Dynamo with a large repertory of tools for tomographic alignment and reconstruction, as well as specific visualization browsers to rapidly assess the biological relevance of the dataset. Our approach has been successfully tested on a broad range of datasets that include diverse biological samples and cryo-ET modalities.

The GoldX Fiducial Eraser.

Gold nanoparticles with sizes in the range of 5-15 nm are a standard method of providing fiducial markers to assist with alignment during reconstruction in cryogenic electron tomography. However, due to their high electron density and resulting contrast when compared to standard cellular or biological samples, they introduce artifacts such as streaking in the reconstructed tomograms. Here, we demonstrate a tool that automatically detects these nanoparticles and suppresses them by replacing them with a local background as a post-processing step, providing a cleaner tomogram without removing any sample relevant information or introducing new artifacts or edge effects from uniform density replacements.

Cryo-electron tomography elucidates annular intraluminal configurations in Caenorhabditis elegans microtubules.

BACKGROUND INFORMATION: Microtubules serve as integral components in cellular operations such as cell division, intracellular trafficking, and cellular architecture. Composed of tubulin protein subunits, these hollow tubular structures have been increasingly elucidated through advanced cryo-electron microscopy (Cryo-EM), which has unveiled the presence of microtubule inner proteins (MIPs) within the microtubular lumen. RESULTS: In the present investigation, we employ a synergistic approach incorporating high-pressure freezing, cryo-focused ion beam milling, and Cryo-electron tomography (Cryo-ET) to interrogate the in situ architecture of microtubules in Caenorhabditis elegans larvae. Our Cryo-ET assessments across neuronal cilia and diverse tissue types consistently demonstrate the formation of annular configurations within the microtubular lumen. CONCLUSIONS: In concert with recently characterized MIPs, our in situ observations within a living organism corroborate the hypothesis that intricate luminal assemblages exist within microtubule scaffolds. These findings necessitate further exploration into the molecular constituents and functional ramifications of these internal microtubular configurations in both cellular physiology and pathophysiology.