Montage electron cryo-tomography with square and rectangular beams.

Cryogenic single-particle photoluminescence (PL) spectroscopy has been used with great success to directly observe the heterogeneous photophysical states present in a population of luminescent particles. Cryogenic electron tomography provides complementary nanometer scale structural information to PL spectroscopy, but the two techniques have not been correlated due to technical challenges. Here, we present a method for correlating single-particle information from these two powerful microscopy modalities. We simultaneously observe PL brightness, emission spectrum, and in-plane excitation dipole orientation of CdSSe/ZnS quantum dots suspended in vitreous ice. Stable and fluctuating emitters were observed, as well as a surprising splitting of the PL spectrum into two bands with an average energy separation of 80 meV. In some cases, the onset of the splitting corresponded to changes in the in-plane excitation dipole orientation. These dynamics were assigned to structures of individual quantum dots and the excitation dipoles were visualized in the context of structural features.

This research presents a groundbreaking approach in virus-related research, addressing challenges in electron microscopy (EM). This imaging technique has been crucial in exploring virus structures; however, traditional methods involve complex sample preparations and the risk of contamination. Herein, we introduce an approach that overcomes these obstacles, enabling high-resolution virus imaging without toxic staining procedures. Focusing on Begomovirus particles, an economically significant plant virus genus, our images confirm their non-enveloped structure and their twin icosahedral symmetry. Our methods involve sample collection, purification, and crystallization, followed by transmission electron microscopy - selected area electron diffraction (TEM-SAED) analysis. Notably, this study achieves 2D and 3D virus imaging through standard TEM, providing a new avenue for virus structure analysis and advancing virus-related research. Remarkable high image quality stemmed from the crystallization process, offering exciting possibilities for improving virus research and diagnosis while eliminating staining limitations.

Knowledge of virus-host cell interactions is central to the formulation of antiviral therapies and vaccines. Because of their nanoscale size and dynamic nature, viruses are inherently difficult objects to investigate. Virus characterization, such as imaging viral structures, intracellular viral trafficking, and infection molecular mechanisms, has relied heavily on sophisticated imaging approaches. Classical light microscopy imaging, such as fluorescence and super-resolution microscopy, provides information on viral entry, replication, and protein localization within living cells. Electron microscopy (EM) techniques, such as Transmission Electron Microscopy (TEM), Scanning Electron Microscopy (SEM), and Cryo-Electron Microscopy (Cryo-EM), provide high-resolution structural information on the viruses and their replication compartments. Advances in correlative imaging techniques, which include light and electron microscopy, have improved our ability to study virus-induced cellular changes in three dimensions. But in comparison to the earlier developments, it remains challenging in virus imaging: a compromise between resolution and sample preparation, restrictions in labeling methods, the challenge in imaging rapid virus-host interactions, and biosafety limitations for highly pathogenic viruses. Solutions to these types of issues will be provided with the newer techniques such as AI-powered imaging analysis, nanotechnology-based imaging probes, and cryo-electron tomography. This review covers the present imaging methods in virology, their utilities and limitations, as well as future prospects, with an emphasis on microscopy to discern the interaction of viruses with cells electron microscopy.

Replica Microscopy and Scanning Electron Microscopy of Laser Impacts on the Skin

<p xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="first" dir="auto" id="d24807e72">Correlative cryo-FIB milling is a powerful sample preparation technique for <i>in situ</i> cryo electron tomography (cryo-ET). Using this technique, vitrified cells can be thinned down and cellular components can be directly visualized in their native context at near-atomic resolution. In recent years several solutions have been proposed to simplify the technique by integrating fluorescent microscopes (FLM) in the cryo-FIB [ <a class="xref-link" href="#r1">1</a>- <a class="xref-link" href="#r4">4</a>]. However, it remains challenging to precisely and accurately target the region of interest (ROI). Here we present a high-quality integrated fluorescent microscope, called METEOR, that allows precise and accurate targeting of the ROI. <p xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" dir="auto" id="d24807e83">To ensure accurate targeting the components of METEOR were carefully selected. We ensured it is compatible with objectives with high numerical apertures (NA), the highest being 0.9, providing a lateral resolution of at least 385 nm. The high-end camera brings background noise to an absolute minimum and provides an exceptional quantum efficiency allowing researchers to image challenging and dim samples. The system is equipped. Here we will present latest results and images of the METEOR system.

Processing has always been a key component in the development of new materials. Basic scientific understanding of the reactions and transformations that occur has obvious importance in guiding progress. Invaluable insight can be provided by observing the changes during processing, especially at high magnification by in situ microscopy. Now that this can be achieved at the atomic level by using high-resolution electron microscopy (HREM), atomic behavior can be seen directly. Accordingly, many deductions concerning reactions in materials at the atomic scale are possible.The purpose of this article is to illustrate the level reached by in situ HREM. The essential procedure is to form a high-resolution image of a standard transmission electron microscope (TEM) sample and then to alter the structure by some means in a controlled manner, such as by heating. Continual recording on videotape allows subsequent detailed analysis of the behavior, even on a frame-by-frame (1/30 second) basis. The most obvious advantage is to follow the atomic rearrangements directly in real time. However, in addition, by continuous recording no stages in a reaction are missed, which can often occur in a series of conventional ex situ annealed samples because of the limited number of samples that can realistically be examined by HREM. One can be sure that the same reaction, in the same area, is being studied. Furthermore, by changing the temperature systematically, extremely precise kinetic measurements can be made (e.g., for activation energies and kinetic laws) and the whole extent of a material transformation can be investigated in one sample, something that would take months of work if studied conventionally. The information provided by in situ HREM is often unique and so it can become an important technique for fundamental materials investigations.

Electron cryo-tomography (cryo-ET) is a powerful imaging tool that allows three-dimensional visualization of subcellular and molecular architecture without chemical fixation. Tomogram quality varies widely, particularly during large high-throughput data collections, and the most common strategy for initial quality assessment is empirical judgment by an expert. Tomograms may be collected for two distinct purposes: annotation of subcellular features and cellular morphology, typically performed at lower magnifications and higher defocus, and subtomogram averaging, at high magnifications, closer to focus. For the first purpose, contrast and the ability to distinguish cellular features of interest are key, whereas for subtomogram averaging, recoverable signal at high resolution is the key factor. We have developed "TomoScore" a deep-learning based tomogram screening tool targeting cellular annotation. This tool provides a single quantitative measure of the suitability of a tomogram for annotation of subcellular features, in terms of the scale of features that can be readily distinguished. We further explore the relationship between accumulated electron dose and resulting quality, suggesting an optimum dose range for cryo-ET data collection. Overall, our study streamlines data processing and reduces the need for human involvement during pre-selection for tomogram segmentation.

Cryo-Electron Tomography: Can it Reveal the Molecular Sociology of Cells in Atomic Detail?

Beyond resolution: Cryo-electron tomography of microbial nanomachines in native host context.

Standard and high resolution transmission electron microscopy (TEM) and advanced post processing of the TEM images have been applied for quantitative characterization of the hardening particle structure of an Al-Mg-Si alloy optimized for formation of the metastable phase β’. The relation between the structural characterization and the mechanical properties has been developed. A first attempt is presented on visualization and quantification of the coherency strain field of β’, based on a combination of Vienna Ab-initio Simulation Package (VASP) calculations and continuum mechanical modelling.

In modern structural biology, there are three major methods for structural biologists to obtain structural information of macromolecules: cryo-electron microscopy (cryo-EM), nuclear magnetic resonance (NMR), and X-ray crystallography. Cryo-EM, in comparison with the other two methods, allows structural biologists to obtain the structures of various macromolecules in a more native and less perturbed system. Over the past decade, cryo-EM has enabled scientists to determine the structures of protein complexes at atomic resolution and made a profound impact in molecular bioscience and pharmaceutical sectors. Along with cryo-EM, another emerging technique called cryo-electron tomography (cryo-ET) has gained increasing importance in structural biology. It has the potential to visualize macromolecular complexes and assemblies in their native environments at high resolution, but there are still some challenges for small, sparse subjects and in approaching atomic resolution in situ. This chapter summarizes the major steps involved in structure determination using cryo-EM and cryo-ET and highlights the major challenges for in situ cryo-ET. We also present a few examples of near-atomic resolution structure determination of macromolecular assemblies both in purified systems in vitro and in native contexts in situ. Future perspectives are discussed as well.

Enveloped viruses utilize membrane glycoproteins on their surface to mediate entry into host cells. Three-dimensional structural analysis of these glycoprotein 'spikes' is often technically challenging but important for understanding viral pathogenesis and in drug design. Here, a protocol is presented for viral spike structure determination through computational averaging of electron cryo-tomography data. Electron cryo-tomography is a technique in electron microscopy used to derive three-dimensional tomographic volume reconstructions, or tomograms, of pleomorphic biological specimens such as membrane viruses in a near-native, frozen-hydrated state. These tomograms reveal structures of interest in three dimensions, albeit at low resolution. Computational averaging of sub-volumes, or sub-tomograms, is necessary to obtain higher resolution detail of repeating structural motifs, such as viral glycoprotein spikes. A detailed computational approach for aligning and averaging sub-tomograms using the Jsubtomo software package is outlined. This approach enables visualization of the structure of viral glycoprotein spikes to a resolution in the range of 20-40 Å and study of the study of higher order spike-to-spike interactions on the virion membrane. Typical results are presented for Bunyamwera virus, an enveloped virus from the family Bunyaviridae. This family is a structurally diverse group of pathogens posing a threat to human and animal health.

Transmission electron microscopy can be used for the characterisation of a wide range of thin specimens, but soft matter and aqueous samples such as gels, nanoparticle dispersions, and emulsions will dry out and collapse under the microscope vacuum, therefore losing information on their native state and ultimately limiting the understanding of the sample. This study examines commonly used techniques in transmission electron microscopy when applied to the characterisation of cryogenically frozen Pickering emulsion samples. Oil-in-water Pickering emulsions stabilised by 3 to 5nm platinum nanoparticles were cryogenically frozen by plunge-freezing into liquid ethane to retain the native structure of the system without inducing crystallisation of the droplet oil cores. A comparison between the droplet morphology following different sample preparation methods has confirmed the effectiveness of using plunge-freezing to prepare these samples. Scanning transmission electron microscopy imaging showed that dry droplets collapse under the microscope vacuum, changing their shape and size (average apparent diameter: ∼342nm) compared to frozen samples (average diameter: ∼183nm). Cryogenic electron tomography was used to collect additional information of the 3D shape and size of the emulsion droplets, and the position of the stabilising nanoparticles relative to the droplet surface. Cryogenic energy dispersive X-ray and electron energy loss spectroscopy were used to successfully obtain elemental data and generate elemental maps to identify the stabilising nanoparticles and the oil phase. Elemental maps generated from spectral data were used in conjunction with electron tomography to obtain 3D information of the oil phase in the emulsion droplets. Beam-induced damage to the ice was the largest limiting factor to the sample characterisation, limiting the effective imaging resolution and signal-to-noise ratio, though careful consideration of the imaging parameters used allowed for the characterisation of the samples presented in this study. Ultimately this study shows that cryo-methods are effective for the representative characterisation of Pickering emulsions.

Size distributions of cellulose nanocrystals (CNCs), extracted from softwood pulp via strong sulfuric acid hydrolysis, exhibit large variability when analyzed from transmission electron microscopy (TEM) images. In this article, the causes of this variability are studied and discussed. In order to obtain results comparable with those reported, a reference material of CNCs (CNCD-1) was used to evaluate size distribution. CNC TEM specimens were prepared as-stained and dried with a rapid-flushing staining method or hydrated and embedded in vitreous ice with the plunge-freezing method. Several sets of bright-field TEM (BF-TEM), annular dark-field scanning TEM (ADF-STEM) and cryogenic-TEM (cryo-TEM) images were acquired for size distribution analysis to study the contributing factors. The rapid-flushing staining method was found to be the most effective for contrast enhancement of CNCs, not only revealing the helical structure of single CNCs but also resolving the laterally jointed CNCs. During TEM specimen preparation, CNCs were fractionated on TEM grids driven by the coffee-ring effect, as observed from contrast variation of CNCs with a stain-depth gradient. From the edge to the center of the TEM grids, the width of CNCs increases, while the aspect ratio (length to width) decreases. This fractionated dispersion of CNCs suggests that images taken near the center of a droplet would give a larger mean width. In addition to particle fractionation driven by the coffee-ring effect, the arrangement and orientation of CNC particles on the substrate significantly affect the size measurement when CNC aggregation cannot be resolved in images. The coexistence of asymmetric cross-section CNC particles introduces a large variation in size measurement, as TEM images of CNCs are mixed projections of the width and height of particles. As a demonstration of how this contributes to inflated size measurement, twisted CNC particles, rectangular cross-section particles and end-to-end jointed CNCs were revealed in reconstructed three-dimensional (3D) micrographs by electron tomography (ET).

Cryo-electron tomography (CET) of fully hydrated, vitrified biological specimens has emerged as a vital tool for biological research. For cellular studies, the conventional imaging modality of transmission electron microscopy places stringent constraints on sample thickness because of its dependence on phase coherence for contrast generation. Here we demonstrate the feasibility of using scanning transmission electron microscopy for cryo-tomography of unstained vitrified specimens (CSTET). We compare CSTET and CET for the imaging of whole bacteria and human tissue culture cells, finding favorable contrast and detail in the CSTET reconstructions. Particularly at high sample tilts, the CSTET signals contain more informative data than energy-filtered CET phase contrast images, resulting in improved depth resolution. Careful control over dose delivery permits relatively high cumulative exposures before the onset of observable beam damage. The increase in acceptable specimen thickness broadens the applicability of electron cryo-tomography.