Electron Tomography Research Articles

Three-Dimensional Reconstruction of Interface Roughness and Alloy Disorder in Ge/GeSi Asymmetric Coupled Quantum Wells Using Electron Tomography.

Interfaces play an essential role in the performance of ever-shrinking semiconductor devices, making comprehensive determination of their three-dimensional (3D) structural properties increasingly important. This becomes even more relevant in compositional interfaces, as is the case for Ge/GeSi heterostructures, where chemical intermixing is pronounced in addition to their morphology. We use the electron tomography method to reconstruct buried interfaces and layers of asymmetric coupled Ge/Ge0.8Si0.2 multiquantum wells, which are considered a potential building block in THz quantum cascade lasers. The three-dimensional reconstruction is based on a series of high-angle annular dark-field scanning transmission electron microscopy images. It allows chemically sensitive investigation of a relatively large interfacial area of about (80 × 80) nm2 with subnanometer resolution as well as the analysis of several interfaces within the multiquantum well stack. Representing the interfaces as iso-concentration surfaces in the tomogram and converting them to topographic height maps allows the determination of their morphological roughness as well as layer thicknesses, reflecting low variations in either case. Simulation of the reconstructed tomogram intensities using a sigmoidal function provides in-plane-resolved maps of the chemical interface widths showing a relatively large spatial variation. The more detailed analysis of the intermixed region using thin slices from the reconstruction and additional iso-concentration surfaces provides an accurate picture of the chemical disorder of the alloy at the interface. Our comprehensive three-dimensional image of Ge/Ge0.8Si0.2 interfaces reveals that in the case of morphologically very smooth interfaces─depending on the scale considered─the interface alloy disorder itself determines the overall characteristics, a result that is fundamental for highly miscible material systems.

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.

Inferring biophysical properties of membranes during endocytosis using machine learning.

Endocytosis is a fundamental cellular process in eukaryotic cells that facilitates the transport of molecules into the cell. With the help of fluorescence microscopy and electron tomography, researchers have accumulated extensive geometric data of membrane shapes during endocytosis. These data contain rich information about the mechanical properties of membranes, which are hard to access via experiments due to the small dimensions of the endocytic patch. In this study, we propose an approach that combines machine learning with the Helfrich theory of membranes to infer the mechanical properties of membranes during endocytosis from a dataset of membrane shapes extracted from electron tomography. Our results demonstrate that machine learning can output solutions that both match the experimental profile and satisfy the membrane shape equations derived from Helfrich theory. The learning results show that during the early stage of endocytosis, the inferred membrane tension is negative, indicating the presence of strong compressive forces at the boundary of the endocytic invagination. Our method presents a generic framework for extracting membrane information from super-resolution imaging.

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.

Controlling nanoparticle placement in Au/TiO2 inverse opal photocatalysts.

Gold nanoparticle-loaded titania (Au/TiO2) inverse opals are highly ordered three-dimensional photonic structures with enhanced photocatalytic properties. However, fine control over the placement of the Au nanoparticles in the inverse opal structures remains challenging with traditional preparative methods. Here, we present a multi-component co-assembly strategy to prepare high-quality Au/TiO2 inverse opal films in which Au nanoparticles are either located on, or inside the TiO2 matrix, as verified using electron tomography. We report that Au nanoparticles embedded in the TiO2 support exhibit enhanced thermal and mechanical stability compared to non-embedded nanoparticles that are more prone to both leaching and sintering.

Three-dimensional quantitative analysis of porosity evolution and inheritance from biomass to biochar through pyrolysis

Electron tomography is employed to conduct a comprehensive porosity analysis of the biomass tobacco and its derived carbon materials.

Correlative cryo-microscopy pipelines for in situ cellular studies.

Correlative cryo-microscopy pipelines combining light and electron microscopy and tomography in cryogenic conditions (cryoCLEM) on the same sample are powerful methods for investigating the structure of specific cellular targets identified by a fluorescent tag within their unperturbed cellular environment. CryoCLEM approaches circumvent one of the inherent limitations of cryo EM, and specifically cryo electron tomography (cryoET), of identifying the imaged structures in the crowded 3D environment of cells. Whereas several cryoCLEM approaches are based on thinning the sample by cryo FIB milling, here we present detailed protocols of two alternative cryoCLEM approaches for in situ studies of adherent cells at the single-cell level without the need for such cryo-thinning. The first approach is a complete cryogenic pipeline in which both fluorescence and electronic imaging are performed on frozen-hydrated samples, the second is a hybrid cryoCLEM approach in which fluorescence imaging is performed at room temperature, followed by rapid freezing and subsequent cryoEM imaging. We provide a detailed description of the two methods we have employed for imaging fluorescently labeled cellular structures with thickness below 350-500nm, such as cell protrusions and organelles located in the peripheral areas of the cells.

Internal structure of Mycoplasma mobile gliding machinery analyzed by negative staining electron tomography.

Mycoplasma mobile is a parasitic bacterium that forms gliding machinery on the cell pole and glides on a solid surface in the direction of the cell pole. The gliding machinery consists of both internal and surface structures. The internal structure is divided into a bell at the front and chain structure extending from the bell. In this study, the internal structures prepared under several conditions were analyzed using negative-staining electron microscopy and electron tomography. The chains were constructed by linked motors containing two complexes similar to ATP synthase. A cylindrical spacer with a maximum diameter of 6 nm and a height of 13 nm, and anonymous linkers with a diameter of 0.9-8.3 nm and length of 14.7±6.9 nm were found between motors. The bell is bowl-shaped and features a honeycomb surface with a periodicity of 8.4 nm. The chains of the motor are connected to the rim of the bell through a wedge-shaped structure. These structures may play roles in the assembly and cooperation of gliding machinery units.

Study of super-efficient defective MoVSbO catalysts used for ethane oxidative dehydrogenation by HAADF-STEM and of their thermal evolution by environmental electron microscopy and tomography

Scanning transmission electron microscopy and in situ electron tomography have been used to understand the role of structure, morphology and porosity of a remarkably active M1 phase based MoVSbO oxide catalyst for ethane oxidative dehydrogenation.

TomoNet: A streamlined cryogenic electron tomography software pipeline with automatic particle picking on flexible lattices.

Cryogenic electron tomography (cryoET) is capable of determining in situ biological structures of molecular complexes at near-atomic resolution by averaging half a million subtomograms. While abundant complexes/particles are often clustered in arrays, precisely locating and seamlessly averaging such particles across many tomograms present major challenges. Here, we developed TomoNet, a software package with a modern graphical user interface to carry out the entire pipeline of cryoET and subtomogram averaging to achieve high resolution. TomoNet features built-in automatic particle picking and three-dimensional (3D) classification functions and integrates commonly used packages to streamline high-resolution subtomogram averaging for structures in 1D, 2D, or 3D arrays. Automatic particle picking is accomplished in two complementary ways: one based on template matching and the other using deep learning. TomoNet's hierarchical file organization and visual display facilitate efficient data management as required for large cryoET datasets. Applications of TomoNet to three types of datasets demonstrate its capability of efficient and accurate particle picking on flexible and imperfect lattices to obtain high-resolution 3D biological structures: virus-like particles, bacterial surface layers within cellular lamellae, and membranes decorated with nuclear egress protein complexes. These results demonstrate TomoNet's potential for broad applications to various cryoET projects targeting high-resolution in situ structures.

Improving electron tomography of mesoporous silica structures by Ga intrusion

Improving electron tomography of mesoporous silica structures by Ga intrusion

Unraveling nanosprings: morphology control and mechanical characterization.

Nanosprings demonstrate promising mechanical characteristics, positioning them as pivotal components in a diverse array of potential nanoengineering applications. To unlock the full potential of these nanosprings, ongoing research is concentrated on emulating springs at the nanoscale in terms of both morphology and function. This review underscores recent advancements in the field and provides a comprehensive overview of the diverse methods employed for nanospring preparation. Understanding the general mechanism behind nanospring formation lays the groundwork for the informed design of nanosprings. The synthesis section delineates four prominent methods employed for nanospring fabrication: vapor phase synthesis, templating methods, post-treatment techniques, and molecular engineering. Each method is critically analyzed, highlighting its strengths, limitations, and potential for scalability. Mechanical properties of nanosprings are explored in depth, discussing their response to external stimuli and their potential applications in various fields such as sensing, energy storage, and biomedical engineering. The interplay between nanospring morphology and mechanical behavior is elucidated, providing insights into the design principles for tailored functionality. Additionally, we anticipate that the evolution of state-of-the-art characterization tools, such as in situ transmission electron microscopy, 3D electron tomography, and machine learning, will significantly contribute to both the study of nanospring mechanisms and their applications.

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.

Resolving the In Situ Three-Dimensional Structure of Fly Mechanosensory Organelles Using Serial Section Electron Tomography.

Mechanosensory organelles (MOs) are specialized subcellular entities where force-sensitive channels and supporting structures (e.g., microtubule cytoskeleton) are organized in an orderly manner. The delicate structure of MOs needs to be resolved to understand the mechanisms by which they detect forces and how they are formed. Here, we describe a protocol that allows obtaining detailed information about the nanoscopic ultrastructure of fly MOs by using serial section electron tomography (SS-ET). To preserve fine structural details, the tissues are cryo-immobilized using a high-pressure freezer followed by freeze-substitution at low temperature and embedding in resin at room temperature. Then, sample sections are prepared and used to acquire the dual-axis tilt series images, which are further processed for tomographic reconstruction. Finally, tomograms of consecutive sections are combined into a single larger volume using microtubules as fiducial markers. Using this protocol, we managed to reconstruct the sensory organelles, which provide novel molecular insights as to how fly mechanosensory organelles work and are formed. Based on our experience, we think that, with minimal modifications, this protocol can be adapted to a wide range of applications using different cell and tissue samples. Key features • Resolving the high-resolution 3D ultrastructure of subcellular organelles using serial section electron tomography (SS-ET). • Compared with single-axis tilt series, dual-axis tilt series provides a much wider coverage of Fourier space, improving resolution and features in the reconstructed tomograms. • The use of high-pressure freezing and freeze-substitution maximally preserves the fine structural details.

M-SIS Software: Automatic tilted series acquisition for environmental (gas, liquid and temperature) multi-scale electron tomography

M-SIS Software: Automatic tilted series acquisition for environmental (gas, liquid and temperature) multi-scale electron tomography

Deep image prior for limited-angle electron tomography

Deep image prior for limited-angle electron tomography

Real-time undersampling optimization during electron tomography of beam-sensitive samples using golden ratio scanning and RECAST3D

Real-time undersampling optimization during electron tomography of beam-sensitive samples using golden ratio scanning and RECAST3D

Single-crystal and pentatwinned nanorods reverse the handedness of chiral plasmonic nanocrystals: An electron tomography study

Single-crystal and pentatwinned nanorods reverse the handedness of chiral plasmonic nanocrystals: An electron tomography study

Analytical electron tomography, fine structure EELS and nanoscale X-ray CT of nanoporous Copper materials

Analytical electron tomography, fine structure EELS and nanoscale X-ray CT of nanoporous Copper materials

Multi-scale electron tomography in liquid state and its application to the study of beam-sensitive nanomaterials

Multi-scale electron tomography in liquid state and its application to the study of beam-sensitive nanomaterials