Conventional Electron Microscopy Research Articles

Heterovalent semiconductor structures and devices grown by molecular beam epitaxy

Heterovalent structures consisting of group II-VI/group III-V compound semiconductors offer attractive properties, such as a very broad range of bandgaps, large conduction band offsets, high electron and hole mobilities, and quantum-material properties such as electric-field-induced topological insulator states. These properties and characteristics are highly desirable for many electronic and optoelectronic devices as well as potential condensed-matter quantum-physics applications. Here, we provide an overview of our recent studies of the MBE growth and characterization of zincblende II-VI/III-V heterostructures as well as several novel device applications based on different sets of these materials. By combining materials with small lattice mismatch, such as ZnTe/GaSb (Δa/a ∼ 0.13%), CdTe/InSb (Δa/a ∼ 0.05%), and ZnSe/GaAs (Δa/a ∼ 0.26%), epitaxial films of excellent crystallinity were grown once the growth conditions had been optimized. Cross-sectional observations using conventional and atomic-resolution electron microscopy revealed coherent interfaces and close to defect-free heterostructures. Measurements across CdTe/InSb interfaces indicated a limited amount (∼1.5 nm) of chemical intermixing. Results for ZnTe/GaSb distributed Bragg reflectors, CdTe/MgxCd1−xTe double heterostructures, and CdTe/InSb two-color photodetectors are briefly presented, and the growth of a rock salt/zincblende PbTe/CdTe/InSb heterostructure is also described.

A Method for Spatial Quantification of Water in Microporous Layers of Polymer Electrolyte Fuel Cells by X-ray Tomographic Microscopy.

A microporous layer (MPL) is typically added to the gas diffusion layer of polymer electrolyte fuel cells (PEFCs) to promote cell performance and water management. The transport mechanism of the water through the MPL is, however, not well understood due to its small pores (20-500 nm). Here, we demonstrate that polychromatic X-ray tomographic microscopy (XTM) can be used to determine the porosity and the spatial distribution of water in nanoporous materials and can quantitatively map the liquid water saturation of MPLs. The presented technique requires no a priori knowledge of the composition of the MPL and has a field of view on the millimeter scale, which is orders of magnitude larger than conventional electron microscopy techniques for nanoscale materials. The available field of view is compatible with existing operando cells for X-ray tomography, paving the way for the analysis of water transport in MPLs during operation.

Direct imaging and electronic structure modulation of moir\xe9 superlattices at the 2D/3D interface

The atomic structure at the interface between two-dimensional (2D) and three-dimensional (3D) materials influences properties such as contact resistance, photo-response, and high-frequency electrical performance. Moiré engineering is yet to be utilized for tailoring this 2D/3D interface, despite its success in enabling correlated physics at 2D/2D interfaces. Using epitaxially aligned MoS2/Au{111} as a model system, we demonstrate the use of advanced scanning transmission electron microscopy (STEM) combined with a geometric convolution technique in imaging the crystallographic 32 Å moiré pattern at the 2D/3D interface. This moiré period is often hidden in conventional electron microscopy, where the Au structure is seen in projection. We show, via ab initio electronic structure calculations, that charge density is modulated according to the moiré period, illustrating the potential for (opto-)electronic moiré engineering at the 2D/3D interface. Our work presents a general pathway to directly image periodic modulation at interfaces using this combination of emerging microscopy techniques.

Morphometry of SARS-CoV and SARS-CoV-2 particles in ultrathin plastic sections of infected Vero cell cultures

SARS-CoV-2 is the causative of the COVID-19 disease, which has spread pandemically around the globe within a few months. It is therefore necessary to collect fundamental information about the disease, its epidemiology and treatment, as well as about the virus itself. While the virus has been identified rapidly, detailed ultrastructural analysis of virus cell biology and architecture is still in its infancy. We therefore studied the virus morphology and morphometry of SARS-CoV-2 in comparison to SARS-CoV as it appears in Vero cell cultures by using conventional thin section electron microscopy and electron tomography. Both virus isolates, SARS-CoV Frankfurt 1 and SARS-CoV-2 Italy-INMI1, were virtually identical at the ultrastructural level and revealed a very similar particle size distribution with a median of about 100 nm without spikes. Maximal spike length of both viruses was 23 nm. The number of spikes per virus particle was about 30% higher in the SARS-CoV than in the SARS-CoV-2 isolate. This result complements a previous qualitative finding, which was related to a lower productivity of SARS-CoV-2 in cell culture in comparison to SARS-CoV.

Visualization of the structure of native human pulmonary mucus

Human respiratory mucus lining the airway epithelium forms a challenging barrier to inhalation therapeutics. Therefore, structural elucidation of hydrated mucus is essential for an efficient drug delivery development. The structure of mucus has been primarily investigated by conventional electron microscopy techniques, which operate under vacuum conditions and require sample preparation steps that might alter the structure of mucus. In this study we investigated the impact of dehydration on mucus and analyzed the structure of mucus in its hydrated state. Cryo-scanning electron microscopy (Cryo-SEM) analysis of mucus showed, that during the process of sublimation, non-porous structure of mucus is transformed into a porous network. Similarly, images acquired by environmental scanning electron microscopy (ESEM), revealed a non-porous structure of hydrated mucus, while further observation at decreasing pressure demonstrated the strong influence of dehydration on mucus structure. We could successfully visualize the structural organization of the major gel forming mucin MUC5B in its hydrated state by employing stimulated emission depletion (STED) microscopy, which allowed resolving the nano-scale patterns of mucin macromolecules within the essentially pore-free mucus structure. The general structural organization of mucus components was addressed by confocal laser scanning microscopy (CLSM), which revealed the heterogeneous and composite structure of mucus. These results provide a novel view on the native structure of mucus and will affect drug delivery development.

Nuclear Import of Adeno-Associated Viruses Imaged by High-Speed Single-Molecule Microscopy.

Understanding the detailed nuclear import kinetics of adeno-associated virus (AAV) through the nuclear pore complex (NPC) is essential for the application of AAV capsids as a nuclear delivery instrument as well as a target for drug development. However, a comprehensive understanding of AAV transport through the sub-micrometer NPCs in live cells calls for new techniques that can conquer the limitations of conventional fluorescence microscopy and electron microscopy. With recent technical advances in single-molecule fluorescence microscopy, we are now able to image the entire nuclear import process of AAV particles and also quantify the transport dynamics of viral particles through the NPCs in live human cells. In this review, we initially evaluate the necessity of single-molecule live-cell microscopy in the study of nuclear import for AAV particles. Then, we detail the application of high-speed single-point edge-excitation sub-diffraction (SPEED) microscopy in tracking the entire process of nuclear import for AAV particles. Finally, we summarize the major findings for AAV nuclear import by using SPEED microscopy.

Cross-Sectional Chemical Nanoimaging of Composite Polymer Nanoparticles by Infrared Nanospectroscopy

Nanoscale Fourier transform infrared spectroscopy (nano-FTIR) is an emerging technique that allows for label-free chemical nanoimaging. Here, we introduce its application to studying multicomponent nanostructured polymer particles synthesized by emulsion polymerization and demonstrate its excellent sensitivity to local chemical composition via hyperspectral infrared nanoimaging and subsequent chemometric analysis. To that end, we developed a method for preparing thin particle slices without any embedding material, thus avoiding chemical contamination. In particular, we studied particles composed of acrylic and fluoroacrylate monomers. Nano-FTIR reveals an unexpected composition for each of the phases that forms the complex core–shell–shell morphology, which provides unprecedented chemical insights beyond conventional electron microscopy analysis of the same particles. Label-free chemical nanoimaging and infrared nanochemometrics of individual phases inside multicomponent polymer particles will benefit the understanding of particle formation and the development of novel synthesis recipes and strategies.

A Workflow for High-pressure Freezing and Freeze Substitution of the Caenorhabditis elegans Embryo for Ultrastructural Analysis by Conventional and Volume Electron Microscopy.

The free-living nematode Caenorhabditis elegans is a popular model system for studying developmental biology. Here we describe a detailed protocol to high-pressure freeze the C. elegans embryo (either ex vivo after dissection, or within the intact worm) followed by quick freeze substitution. Processed samples are suitable for ultrastructural analysis by conventional electron microscopy (EM) or newer volume EM (vEM) approaches such as Focused Ion Beam Scanning Electron Microscopy (FIB-SEM). The ultrastructure of cellular features such as the nuclear envelope, chromosomes, endoplasmic reticulum and mitochondria are well preserved after these experimental procedures and yield accurate 3D models for visualization and analysis ( Chang et al., 2020 ). This protocol was used in the 3D reconstruction of membranes and chromosomes after pronuclear meeting in the C. elegans zygote ( Rahman et al., 2020 ).

High-throughput imaging of fresh-frozen plant reproductive samples in a variable pressure SEM

Conventional light and electron microscopy are the most widely used techniques for examining plant reproductive tissues; however, they are time-consuming or expensive. The anther is the male part of the plant reproductive system. Structural changes drive development, and any structural defect may lead to an increase in fertility or cause sterility; thus, quick detection of structural changes is crucial in reproductive biology. We optimized an existing low-temperature SEM alternative to examine the internal structure of hydrated, fresh-frozen anthers. In contrast with the original technique, our method does not require precooling adhesion (ethanol to fix the specimen), and the cryo-sectioning can be conducted at atmospheric pressure. In addition to enabling the differentiation between aerial and liquid-filled intercellular spaces, this method is expected to facilitate the detection of quick (during a day) developmental changes in plant reproductive tissues, which is a current challenge using conventional approaches.•This method allows the high-throughput imaging of fresh-frozen plant reproductive samples collected every 10 min, which is important for developmental studies.•The cryo-images of samples with thickness ranging from 0.2 to 3 mm can be well-preserved at 800X magnification.•This method does not require chemical processing, critical point drying, customized cryo-accessories, controlled temperature cold stages, or metal coating. This simplified method does not require highly skilled personnel, and it is suitable in most microscopy laboratories.

Microgel Dynamics Characterization Using SEM

A methodology for imaging the dynamics of individual microgel particles using high resolution scanning electron microscopy (SEM) is presented. To enable this, the microgels are dispersed in an ionic liquid, which due to its low vapor pressure allows them to remain in suspension even under the high vacuum conditions present in a typical electron gun. Thus, compared with conventional electron microscopy studies of microgels, no sample drying or freezing is necessary which preserves their morphology while also allowing to probe microgel dynamics in solution. The results based on the individually tracked particles are compared with results from dynamic light scattering (DLS) which measures the mean size and diffusion properties of large collectives of particles. For the size measurements, the SEM and DLS data are in general agreement. For the particle dynamics, monitoring individual microgel motion reveals complex dynamics in which, aside from the expected thermal motion, one observes effects such as clustering, rotation and drift. This is in contrast with a control sample of hard sphere-like silica particles where the motion is primarily diffusional in good agreement with DLS studies.

Getting Started with In Situ Cryo-Electron Tomography.

Cryo-electron tomography (cryo-ET) is an extremely powerful tool which is used to image cellular features in their close-to-native environment at a resolution where both protein structure and membrane morphology can be revealed. Compared to conventional electron microscopy methods for biology, cryo-ET does not include the use of potentially artifact generating agents for sample fixation or visualization. Despite its obvious advantages, cryo-ET has not been widely adopted by cell biologists. This might originate from the overwhelming and constantly growing number of complex ways to record and process data as well as the numerous methods available for sample preparation. In this chapter, we will take one step back and guide the reader through the essential steps of sample preparation using mammalian cells, as well as the basic steps involved in data recording and processing. The described protocol will allow the reader to obtain data that can be used for morphological analysis and precise measurements of biological structures in their cellular environment. Furthermore, this data can be used for more elaborate structural analysis by applying further image processing steps like subtomogram averaging, which is required to determine the structure of proteins.

In situ Electron Microscopy of Complex Biological and Nanoscale Systems: Challenges and Opportunities

In situ imaging for direct visualization is important for physical and biological sciences. Research endeavors into elucidating dynamic biological and nanoscale phenomena frequently necessitate in situ and time-resolved imaging. In situ liquid cell electron microscopy (LC-EM) can overcome certain limitations of conventional electron microscopies and offer great promise. This review aims to examine the status-quo and practical challenges of in situ LC-EM and its applications, and to offer insights into a novel correlative technique termed microfluidic liquid cell electron microscopy. We conclude by suggesting a few research ideas adopting microfluidic LC-EM for in situ imaging of biological and nanoscale systems.

Polarization‐resolved SHG imaging as a fast screening method for collagen alterations during aging: Comparison with light and electron microscopy

Our previous study on rat skin showed that cumulative oxidative pressure induces profound structural and ultrastructural alterations in both rat skin epidermis and dermis during aging. Here, we aimed to investigate the biophotonic properties of collagen as a main dermal component in the function of chronological aging. We used second harmonic generation (SHG) and two-photon excited fluorescence (TPEF) on 5 μm thick skin paraffin sections from 15-day-, 1-month- and 21-month-old rats, respectively, to analyze collagen alterations, in comparison to conventional light and electron microscopy methods. Obtained results show that polarization-resolved SHG (PSHG) images can detect collagen fiber alterations in line with chronological aging and that this method is consistent with light and electron microscopy. Moreover, the β coefficient calculated from PSHG images points out that delicate alterations lead to a more ordered structure of collagen molecules due to oxidative damage. The results of this study also open the possibility of successfully applying this fast and label-free method to previously fixed samples.

Determination of mitophagy by electron microscope.

Mitophagy is an autophagic mechanism for targeting damaged or unnecessary mitochondria and responsible for mitochondria quality control. Emerging evidence revealed that mitophagy is associated with many physiological processes and cellular activities. Therefore, the determination of mitophagy may provide insights into human physiological and pathological processes. Electron microscopy, one of the best approaches, can directly provide the ultrastructure evidence for mitophagy. Here, we detail an experiment protocol for electron microscopy preparation, so as to detect mitophagy in biological samples. Compare with other biochemical techmology, conventional electron microscopy are still essential for strengthening or replacing biochemical methods, and a better understanding of this method could be important to investigate mitophagy.

3D-Reconstructed Retinal Pigment Epithelial Cells Provide Insights into the Anatomy of the Outer Retina.

The retinal pigment epithelium (RPE) is located between the neuroretina and the choroid, and plays a critical role in vision. RPE cells internalise outer segments (OS) from overlying photoreceptors in the daily photoreceptor renewal. Changes to RPE structure are linked with age and retinopathy, which has been described in the past by conventional 2D electron microscopy. We used serial block face scanning electron microscopy (SBF-SEM) to reconstruct RPE cells from the central mouse retina. Three-dimensional-reconstructed OS revealed the RPE to support large numbers of photoreceptors (90–216 per RPE cell). Larger bi-nucleate RPE maintained more photoreceptors, although their cytoplasmic volume was comparable to smaller mono-nucleate RPE supporting fewer photoreceptors. Scrutiny of RPE microvilli and interdigitating OS revealed the angle and surface area of contact between RPE and photoreceptors. Bi-nucleate RPE contained more mitochondria compared to mono-nucleate RPE. Furthermore, bi-nucleate cells contained larger sub-RPE spaces, supporting a likely association with disease. Use of perfusion-fixed tissues ensured the highest possible standard of preservation, providing novel insights into the 3D RPE architecture and changes linked with retinopathy. This study serves as a benchmark for comparing retinal tissues from donor eyes with age-related macular degeneration (AMD) and other retinopathies.

Satellite Glial Cells of the Dorsal Root Ganglion: A New "Guest/Physiopathological Target" in ALS.

Introduction: Amyotrophic lateral sclerosis (ALS) might not only be circumscribed to the motor system but also involves other neuronal systems including sensory abnormalities. In line with this notion, we aimed to assess the pathophysiology of sensory disturbances in the SOD1G93A mouse model of ALS, focusing on the satellite glial cells (SGCs) at the dorsal root ganglion (DRG) as a new potential target of the disease.Material and Methods: The presence of sensory disturbances was evaluated using von Frey, hot plate, and hot water tail immersion tests at 75 days old, which represented the motor-pre-symptomatic stage. Cell biology analysis was performed at 75 and 95 days old and included conventional histology, immunofluorescence, and electron microscopy of sensory neuron-SGC unit dissociates as a well as western blotting from DRG lysates.Results: At 75 days old, von Frey and hot plate tests demonstrated clear thermoalgesic disturbances in ALS transgenic mice. Histological studies of the SN-SGC units revealed abnormal SOD1 accumulation, which was associated with nitro-oxidative stress and biogenesis of lipid droplets in SGCs. Interestingly, these alterations led to a progressive lysosomal storage disorder and occasionally vacuolar degeneration in SGCs.Conclusions: SGCs emerge as a primary pathophysiological target in the SOD1 transgenic murine model of ALS, clearly reinforcing the pathogenic role of glial cells in motor neuron disease. Presymptomatic alterations of SGCs, might not only be responsible of sensory disturbances in ALS, but due to spinal cord sensory-motor circuits could also contribute to anterior horn motor disturbances.

Cryo-fluorescence microscopy of high-pressure frozen C. elegans enables correlative FIB-SEM imaging of targeted embryonic stages in the intact worm.

Rapidly changing features in an intact biological sample are challenging to efficiently trap and image by conventional electron microscopy (EM). For example, the model organism C. elegans is widely used to study embryonic development and differentiation, yet the fast kinetics of cell division makes the targeting of specific developmental stages for ultrastructural study difficult. We set out to image the condensed metaphase chromosomes of an early embryo in the intact worm in 3-D. To achieve this, one must capture this transient structure, then locate and subsequently image the corresponding volume by EM in the appropriate context of the organism, all while minimizing a variety of artifacts. In this methodological advance, we report on the high-pressure freezing of spatially constrained whole C. elegans hermaphrodites in a combination of cryoprotectants to identify embryonic cells in metaphase by in situ cryo-fluorescence microscopy. The screened worms were then freeze substituted, resin embedded and further prepared such that the targeted cells were successfully located and imaged by focused ion beam scanning electron microscopy (FIB-SEM). We reconstructed the targeted metaphase structure and also correlated an intriguing punctate fluorescence signal to a H2B-enriched putative polar body autophagosome in an adjacent cell undergoing telophase. By enabling cryo-fluorescence microscopy of thick samples, our workflow can thus be used to trap and image transient structures in C. elegans or similar organisms in a near-native state, and then reconstruct their corresponding cellular architectures at high resolution and in 3-D by correlative volume EM.

Gephyrin-mediated formation of inhibitory postsynaptic density sheet via phase separation.

Inhibitory synapses are also known as symmetric synapses due to their lack of prominent postsynaptic densities (PSDs) under a conventional electron microscope (EM). Recent cryo-EM tomography studies indicated that inhibitory synapses also contain PSDs, albeit with a rather thin sheet-like structure. It is not known how such inhibitory PSD (iPSD) sheet might form. Here, we demonstrate that the key inhibitory synapse scaffold protein gephyrin, when in complex with either glycine or GABAA receptors, spontaneously forms highly condensed molecular assemblies via phase separation both in solution and on supported membrane bilayers. Multivalent and specific interactions between the dimeric E-domain of gephyrin and the glycine/GABAA receptor multimer are essential for the iPSD condensate formation. Gephyrin alone does not form condensates. The linker between the G- and E-domains of gephyrin inhibits the iPSD condensate formation via autoinhibition. Phosphorylation of specific residues in the linker or binding of target proteins such as dynein light chain to the linker domain regulates gephyrin-mediated glycine/GABAA receptor clustering. Thus, analogous to excitatory PSDs, iPSDs are also formed by phase separation-mediated condensation of scaffold protein/neurotransmitter receptor complexes.

Direct comparison of line edge roughness measurements by SEM and a metrological tilting-atomic force microscopy for reference metrology

Background: Conventional scanning electron microscopy (SEM) that is used for 2D top-view metrology, a classical line edge roughness (LER) measurement technique, is incapable of measuring 3D structures of a nanoscale line pattern. For LER measurements, SEM measurement generates a single line-edge profile for the 3D sidewall roughness, although the line-edge profile differs at each height in the 3D sidewall. Aim: To develop an evaluation method of SEM-based LER measurement techniques and to verify how the 3D sidewall shape is reflected in the SEM’s 2D result. Approach: Direct comparison by measuring an identical location of a line pattern by SEM and an atomic force microscopy (AFM) with the tip-tilting technique that is capable of measuring the 3D sidewall. The line pattern has vertical stripes on the sidewall due to its fabrication process. Measured line edge profiles were analyzed using power spectral density, height-height correlation function, and autocorrelation function. Results: Line edge profiles measured by SEM and AFM were well matched except for noise level. Frequency and scaling analyses showed that SEM profile contained high noise and had lost a property of self-affine fractals in contrast to AFM. Conclusions: In the case of the line pattern with vertical stripes on the sidewall, SEM profile is generally consistent with 3D sidewall shape. The AFM-based LER measurement technique is useful as LER reference metrology to evaluate other LER measurement techniques.

Cryo-electron tomography of cardiac myofibrils reveals a 3D lattice spring within the Z-discs

The Z-disc forms a boundary between sarcomeres, which constitute structural and functional units of striated muscle tissue. Actin filaments from adjacent sarcomeres are cross-bridged by α-actinin in the Z-disc, allowing transmission of tension across the myofibril. Despite decades of studies, the 3D structure of Z-disc has remained elusive due to the limited resolution of conventional electron microscopy. Here, we observed porcine cardiac myofibrils using cryo-electron tomography and reconstructed the 3D structures of the actin-actinin cross-bridging complexes within the Z-discs in relaxed and activated states. We found that the α-actinin dimers showed contraction-dependent swinging and sliding motions in response to a global twist in the F-actin lattice. Our observation suggests that the actin-actinin complex constitutes a molecular lattice spring, which maintains the integrity of the Z-disc during the muscle contraction cycle.