Cryo-focused Ion Beam Milling Research Articles

Archaeal type six secretion system mediates contact-dependent antagonism.

Microbial communities are shaped by cell-cell interactions. Although archaea are often found in associations with other microorganisms, the mechanisms structuring these communities are poorly understood. Here, we report on the structure and function of haloarchaeal contractile injection systems (CISs). Using a combination of functional assays and time-lapse imaging, we show that Halogeometricum borinquense exhibits antagonism toward Haloferax volcanii by inducing cell lysis and inhibiting proliferation. This antagonism is contact-dependent and requires a functional CIS, which is encoded by a gene cluster that is associated with toxin-immunity pairs. Cryo-focused ion beam milling and imaging by cryo-electron tomography revealed that these CISs are bound to the cytoplasmic membrane, resembling the bacterial type six secretion systems (T6SSs). We show that related T6SS gene clusters are conserved and expressed in other haloarchaeal strains, which exhibit antagonistic behavior. Our data provide a mechanistic framework for understanding how archaea may shape microbial communities and affect the food webs they inhabit.

Visualizing the virus world inside the cell by cryo-electron tomography.

Structural studies on purified virus have revealed intricate architectures, but there is little structural information on how viruses interact with host cells in situ. Cryo-focused ion beam (FIB) milling and cryo-electron tomography (cryo-ET) have emerged as revolutionary tools in structural biology to visualize the dynamic conformational of viral particles and their interactions with host factors within infected cells. Here, we review the state-of-the-art cryo-ET technique for in situ viral structure studies and highlight exemplary studies that showcase the remarkable capabilities of cryo-ET in capturing the dynamic virus-host interaction, advancing our understanding of viral infection and pathogenesis.

Precision in situ cryogenic correlative light and electron microscopy of optogenetically positioned organelles.

Unambiguous targeting of cellular structures for in situ cryo-electron microscopy in the heterogeneous, dense and compacted environment of the cytoplasm remains challenging. Here, we have developed a cryogenic correlative light and electron microscopy (cryo-CLEM) workflow that utilizes thin cells grown on a mechanically defined substratum for rapid analysis of organelles and macromolecular complexes by cryo-electron tomography (cryo-ET). We coupled these advancements with optogenetics to redistribute perinuclear-localised organelles to the cell periphery, allowing visualisation of organelles that would otherwise be positioned in cellular regions too thick for cryo-ET. This reliable and robust workflow allows for fast in situ analyses without the requirement for cryo-focused ion beam milling. Using this protocol, cells can be frozen, imaged by cryo-fluorescence microscopy and be ready for batch cryo-ET within a day.

Extracellular filaments revealed by affinity capture cryo-electron tomography.

Cryogenic-electron tomography (cryo-ET) has provided an unprecedented glimpse into the nanoscale architecture of cells by combining cryogenic preservation of biological structures with electron tomography. Micropatterning of extracellular matrix proteins is increasingly used as a method to prepare adherent cell types for cryo-ET as it promotes optimal positioning of cells and subcellular regions of interest for vitrification, cryo-focused ion beam (cryo-FIB) milling, and data acquisition. Here we demonstrate a micropatterning workflow for capturing minimally adherent cell types, human T-cells and Jurkat cells, for cryo-FIB and cryo-ET. Our affinity capture system facilitated the nanoscale imaging of Jurkat cells, revealing extracellular filamentous structures. It improved workflow efficiency by consistently producing grids with a sufficient number of well positioned cells for an entire cryo-FIB session. Affinity capture can be extended to facilitate high resolution imaging of other adherent and non-adherent cell types with cryo-ET.

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’.

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

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. 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. 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.

P-095 Towards deciphering the molecular architecture of the human sperm connecting piece

Abstract Study question How can we make the human sperm connecting piece accessible for structural analysis by cryo-electron tomography (cryo-ET)? Summary answer Preparation of sperm by chemical thinning or production of lamellae via cryo-focused ion beam (cryo-FIB) milling allows direct imaging of the human sperm connecting piece. What is known already Previous studies from our group and others have demonstrated the versatility of cryo-ET to advance our understanding of sperm biology. Structures of macromolecular complexes have been resolved in vitro and in situ, enabling a functional assessment at molecular or even atomic level. Resolving structures at high resolution further allowed identification of proteins previously not known to have a role in mediating male fertility. Such insights are instrumental to further improve diagnosis and treatment of male infertility. Study design, size, duration In our research study we use sperm samples from patients undergoing IVF treatment or donor material, which we receive through an ongoing collaboration with the Division of Woman and Baby, University Medical Centre Utrecht (UMCU). These samples are processed in our own laboratory and subsequently imaged by cryo-ET at the Electron Microscopy Centre of Utrecht University. Participants/materials, setting, methods Due to its thickness, the connecting piece cannot be directly analyzed by cryo-ET. Thus, we designed two strategies to achieve thinning of this area and allow collection of tomograms for structural analysis. While the first approach involves incubation of sperm with a detergent, a reducing agent and heparin, the second approach utilizes cryo-FIB milling to produce thin lamellae of the connecting piece. Main results and the role of chance Both strategies developed to access the connecting piece with cryo-ET are effective and allowed us a glimpse of the molecular landscape of the link between sperm head and tail. Chemical thinning of the connecting piece enabled imaging of the proximal and distal centriole while still embedded in the segmented columns or outer dense fibers, respectively. Furthermore, electron density within the microtubules of both centrioles indicates that they are decorated by microtubule inner proteins. Data obtained from lamellae produced by cryo-FIB milling complements these findings, displaying regular densities along the microtubules extending from the proximal centriole. Analyzing tomograms collected from cryo-FIB lamellae we further found a regular protein density that appears to be connecting the nuclear envelope to the base plate. Moreover, cryo-ET of lamellae allowed a detailed view of nuclear pore complexes and the postacrosomal sheath in their native environment. Limitations, reasons for caution Altering the native state of the sperm cell by chemical preparation is a limitation of this approach. Cryo-FIB milling retains the native state of the cell but is limited in throughput. To improve our cryo-FIB milling approach, we will implement automated milling procedures. Wider implications of the findings Chemical preparation and cryo-FIB milling of sperm enable the in-depth analysis of the connecting piece by cryo-ET. These strategies can now be applied to provide structural details at the molecular level and thus advance our understanding of male infertility related to defects of the head-tail junction. Trial registration number not applicable

Visualizing the translation landscape in human cells at high resolution.

Obtaining comprehensive structural descriptions of macromolecules within their natural cellular context holds immense potential for understanding fundamental biology and improving health. Here, we present the landscape of protein synthesis inside human cells in unprecedented detail obtained using an approach which combines automated cryo-focused ion beam (FIB) milling and in situ single-particle cryo-electron microscopy (cryo-EM). With this in situ cryo-EM approach we resolved a 2.19 Å consensus structure of the human 80S ribosome and unveiled its 21 distinct functional states, nearly all higher than 3 Å resolution. In contrast to in vitro studies, we identified protein factors, including SERBP1, EDF1 and NAC/3, not enriched on purified ribosomes. Most strikingly, we observed that SERBP1 binds to the ribosome in almost all translating and non-translating states to bridge the 60S and 40S ribosomal subunits. These newly observed binding sites suggest that SERBP1 may serve an important regulatory role in translation. We also uncovered a detailed interface between adjacent translating ribosomes which can form the helical polysome structure. Finally, we resolved high-resolution structures from cells treated with homoharringtonine and cycloheximide, and identified numerous polyamines bound to the ribosome, including a spermidine that interacts with cycloheximide bound at the E site of the ribosome, underscoring the importance of high-resolution in situ studies in the complex native environment. Collectively, our work represents a significant advancement in detailed structural studies within cellular contexts.

Thinner is not always better: Optimizing cryo-lamellae for subtomogram averaging.

Cryo-electron tomography (cryo-ET) is a powerful method to elucidate subcellular architecture and to structurally analyze biomolecules in situ by subtomogram averaging, yet data quality critically depends on specimen thickness. Cells that are too thick for transmission imaging can be thinned into lamellae by cryo-focused ion beam (cryo-FIB) milling. Despite being a crucial parameter directly affecting attainable resolution, optimal lamella thickness has not been systematically investigated nor the extent of structural damage caused by gallium ions used for FIB milling. We thus systematically determined how resolution is affected by these parameters. We find that ion-induced damage does not affect regions more than 30 nanometers from either lamella surface and that up to ~180-nanometer lamella thickness does not negatively affect resolution. This shows that there is no need to generate very thin lamellae and lamella thickness can be chosen such that it captures cellular features of interest, thereby opening cryo-ET also for studies of large complexes.

Vimentin filaments integrate low-complexity domains in a complex helical structure

Intermediate filaments (IFs) are integral components of the cytoskeleton. They provide cells with tissue-specific mechanical properties and are involved in numerous cellular processes. Due to their intricate architecture, a 3D structure of IFs has remained elusive. Here we use cryo-focused ion-beam milling, cryo-electron microscopy and tomography to obtain a 3D structure of vimentin IFs (VIFs). VIFs assemble into a modular, intertwined and flexible helical structure of 40 α-helices in cross-section, organized into five protofibrils. Surprisingly, the intrinsically disordered head domains form a fiber in the lumen of VIFs, while the intrinsically disordered tails form lateral connections between the protofibrils. Our findings demonstrate how protein domains of low sequence complexity can complement well-folded protein domains to construct a biopolymer with striking mechanical strength and stretchability.

Lift-out cryo-FIBSEM and cryo-ET reveal the ultrastructural landscape of extracellular matrix.

The extracellular matrix (ECM) serves as a scaffold for cells and plays an essential role in regulating numerous cellular processes, including cell migration and proliferation. Due to limitations in specimen preparation for conventional room-temperature electron microscopy, we lack structural knowledge on how ECM components are secreted, remodeled, and interact with surrounding cells. We have developed a 3D-ECM platform compatible with sample thinning by cryo-focused ion beam milling, the lift-out extraction procedure, and cryo-electron tomography. Our workflow implements cell-derived matrices (CDMs) grown on EM grids, resulting in a versatile tool closely mimicking ECM environments. This allows us to visualize ECM for the first time in its hydrated, native context. Our data reveal an intricate network of extracellular fibers, their positioning relative to matrix-secreting cells, and previously unresolved structural entities. Our workflow and results add to the structural atlas of the ECM, providing novel insights into its secretion and assembly.

Serial Lift-Out: sampling the molecular anatomy of whole organisms

Cryo-focused ion beam milling of frozen-hydrated cells and subsequent cryo-electron tomography (cryo-ET) has enabled the structural elucidation of macromolecular complexes directly inside cells. Application of the technique to multicellular organisms and tissues, however, is still limited by sample preparation. While high-pressure freezing enables the vitrification of thicker samples, it prolongs subsequent preparation due to increased thinning times and the need for extraction procedures. Additionally, thinning removes large portions of the specimen, restricting the imageable volume to the thickness of the final lamella, typically <300 nm. Here we introduce Serial Lift-Out, an enhanced lift-out technique that increases throughput and obtainable contextual information by preparing multiple sections from single transfers. We apply Serial Lift-Out to Caenorhabditis elegans L1 larvae, yielding a cryo-ET dataset sampling the worm’s anterior–posterior axis, and resolve its ribosome structure to 7 Å and a subregion of the 11-protofilament microtubule to 13 Å, illustrating how Serial Lift-Out enables the study of multicellular molecular anatomy.

Molecular mechanism of glutaminase activation through filamentation and the role of filaments in mitophagy protection.

Glutaminase (GLS), which deaminates glutamine to form glutamate, is a mitochondrial tetrameric protein complex. Although inorganic phosphate (Pi) is known to promote GLS filamentation and activation, the molecular basis of this mechanism is unknown. Here we aimed to determine the molecular mechanism of Pi-induced mouse GLS filamentation and its impact on mitochondrial physiology. Single-particle cryogenic electron microscopy revealed an allosteric mechanism in which Pi binding at the tetramer interface and the activation loop is coupled to direct nucleophile activation at the active site. The active conformation is prone to enzyme filamentation. Notably, human GLS filaments form inside tubulated mitochondria following glutamine withdrawal, as shown by in situ cryo-electron tomography of cells thinned by cryo-focused ion beam milling. Mitochondria with GLS filaments exhibit increased protection from mitophagy. We reveal roles of filamentous GLS in mitochondrial morphology and recycling.

Preparing Arabidopsis thaliana root protoplasts for cryo electron tomography.

The use of protoplasts in plant biology has become a convenient tool for the application of transient gene expression. This model system has allowed the study of plant responses to biotic and abiotic stresses, protein location and trafficking, cell wall dynamics, and single-cell transcriptomics, among others. Although well-established protocols for isolating protoplasts from different plant tissues are available, they have never been used for studying plant cells using cryo electron microscopy (cryo-EM) and cryo electron tomography (cryo-ET). Here we describe a workflow to prepare root protoplasts from Arabidopsis thaliana plants for cryo-ET. The process includes protoplast isolation and vitrification on EM grids, and cryo-focused ion beam milling (cryo-FIB), with the aim of tilt series acquisition. The whole workflow, from growing the plants to the acquisition of the tilt series, may take a few months. Our protocol provides a novel application to use plant protoplasts as a tool for cryo-ET.

Locating cellular contents during cryoFIB milling using cellular secondary-electron imaging

Cryo-electron tomography (cryoET) is a powerful technology that allows in-situ observation of the molecular structure of tissues and cells. Cryo-focused ion beam (cryoFIB) milling plays an important role in the preparation of high-quality thin lamellar samples for cryoET studies, thus, promoting the rapid development of cryoET in recent years. However, locating the regions of interest in a large cell or tissue during cryoFIB milling remains a major challenge limiting cryoET applications on arbitrary biological samples. Here, we report an on-the-fly localization method based on cellular secondary electron imaging (CSEI), which is derived from a basic imaging function of the cryoFIB instruments and enables high-contrast imaging of the cellular contents of frozen-hydrated biological samples. Moreover, CSEI does not require fluorescent labels and additional devices. The present study discusses the imaging principles and settings for optimizing CSEI. Tests on several commercially available cryoFIB instruments demonstrated that CSEI was feasible on mainstream instruments to observe all types of cellular contents and reliable under different milling conditions. We established a simple milling-localization workflow and tested it using the basal body of Chlamydomonas reinhardtii.

HOPE-SIM, a cryo-structured illumination fluorescence microscopy system for accurately targeted cryo-electron tomography

Cryo-focused ion beam (cryo-FIB) milling technology has been developed for the fabrication of cryo-lamella of frozen native specimens for study by in situ cryo-electron tomography (cryo-ET). However, the precision of the target of interest is still one of the major bottlenecks limiting application. Here, we have developed a cryo-correlative light and electron microscopy (cryo-CLEM) system named HOPE-SIM by incorporating a 3D structured illumination fluorescence microscopy (SIM) system and an upgraded high-vacuum stage to achieve efficiently targeted cryo-FIB. With the 3D super resolution of cryo-SIM as well as our cryo-CLEM software, 3D-View, the correlation precision of targeting region of interest can reach to 110 nm enough for the subsequent cryo-lamella fabrication. We have successfully utilized the HOPE-SIM system to prepare cryo-lamellae targeting mitochondria, centrosomes of HeLa cells and herpesvirus assembly compartment of infected BHK-21 cells, which suggests the high potency of the HOPE-SIM system for future in situ cryo-ET workflows.

CryoFIB milling large tissue samples for cryo-electron tomography

Cryo-electron tomography (cryoET) is a powerful tool for exploring the molecular structure of large organisms. However, technical challenges still limit cryoET applications on large samples. In particular, localization and cutting out objects of interest from a large tissue sample are still difficult steps. In this study, we report a sample thinning strategy and workflow for tissue samples based on cryo-focused ion beam (cryoFIB) milling. This workflow provides a full solution for isolating objects of interest by starting from a millimeter-sized tissue sample and ending with hundred-nanometer-thin lamellae. The workflow involves sample fixation, pre-sectioning, a two-step milling strategy, and localization of the object of interest using cellular secondary electron imaging (CSEI). The milling strategy consists of two steps, a coarse milling step to improve the milling efficiency, followed by a fine milling step. The two-step milling creates a furrow–ridge structure with an additional conductive Pt layer to reduce the beam-induced charging issue. CSEI is highlighted in the workflow, which provides on-the-fly localization during cryoFIB milling. Tests of the complete workflow were conducted to demonstrate the high efficiency and high feasibility of the proposed method.

In situ cryo-electron tomography at the nucleus-cytoskeleton interface.

The linker of nucleoskeleton and cytoskeleton (LINC) is the primary node of force transduction at the nucleus - essential for regulating its morphology and position in mammalian cells. LINC spans both nucleus membranes; interacting with the nuclear lamina and chromatin within the nucleus, whilst also interacting with cytoskeletal filaments in the cytoplasm. Dysregulation of LINC and its interactions underlies numerous laminopathies with diverse manifestations, including deafness, muscular dystrophy and developmental disorders. Although a wealth of genetics and cell biology has described its large network of interactions, its architecture and mechanism of assembly is largely unknown. We present an approach to understand this interaction using cryo-focused ion beam milling (FIB-milling) and cryo-electron tomography (cryo-ET). By promoting actin polymerisation in the cytoplasm, we have determined the structure and architecture of actin in the vicinity of the nucleus. By visualizing LINC and actin in its native environment, our results shed new light on the role of actin-LINC in nucleus morphology and positioning.

Cryo-Electron Tomography: The Resolution Revolution and a Surge of In Situ Virological Discoveries.

The recent proliferation of cryo-electron tomography (cryo-ET) techniques has led to the cryo-ET resolution revolution. Meanwhile, significant efforts have been made to improve the identification of targets in the cellular context and the throughput of cryo-focused ion beam (FIB) milling. Together, these developments led to a surge of in situ discoveries on how enveloped viruses are assembled and how viruses interact with cells in infected hosts. In this article, we review the recent advances in cryo-ET, high-resolution insights into virus assembly, and the findings from inside infected eukaryotic and prokaryotic cells.

ELI trifocal microscope: a precise system to prepare target cryo-lamellae for in situ cryo-ET study

Cryo-electron tomography (cryo-ET) has become a powerful approach to study the high-resolution structure of cellular macromolecular machines in situ. However, the current correlative cryo-fluorescence and electron microscopy lacks sufficient accuracy and efficiency to precisely prepare cryo-lamellae of target locations for subsequent cryo-ET. Here we describe a precise cryogenic fabrication system, ELI-TriScope, which sets electron (E), light (L) and ion (I) beams at the same focal point to achieve accurate and efficient preparation of a target cryo-lamella. ELI-TriScope uses a commercial dual-beam scanning electron microscope modified to incorporate a cryo-holder-based transfer system and embed an optical imaging system just underneath the vitrified specimen. Cryo-focused ion beam milling can be accurately navigated by monitoring the real-time fluorescence signal of the target molecule. Using ELI-TriScope, we prepared a batch of cryo-lamellae of HeLa cells targeting the centrosome with a success rate of ~91% and discovered new in situ structural features of the human centrosome by cryo-ET.