Practical Guide for Implementing Cryogenic Electron Microscopy Structure Determination in Dermatology Research.

Cryo-electron microscopy (cryo-EM) and cryo-electron tomography (cryo-ET) have transformed structural biology by enabling the visualization of biological macromolecules at near-atomic resolution. Since its establishment in 2002, the Electron Microscopy Data Bank (EMDB) has played a pivotal role in this “Resolution Revolution,” serving as a central repository for cryo-EM data. Over the past decade, major advances in detector technology, image processing, and analytical methodologies have led to a surge in cryo-EM structure depositions. This study provides a comprehensive analysis of temporal trends in the EMDB, highlighting the growing number of entries, improvements in resolution, and the evolution of detector usage. It emphasizes the rise of single-particle analysis (SPA) as the dominant cryo-EM technique and examines how high-resolution detectors have enhanced data quality. Additionally, it explores resolution standards across leading scientific journals, underscoring the role of EMDB not only as a data archive but also as a catalyst for innovation in structural biology.

Advances in cryogenic electron microscopy (cryoEM) single particle analysis have revolutionized structural biology by facilitating the in vitro determination of atomic- and near-atomic-resolution structures for fully hydrated macromolecular complexes exhibiting compositional and conformational heterogeneity across a wide range of sizes. Cryogenic electron tomography (cryoET) and subtomogram averaging are rapidly progressing toward delivering similar insights for macromolecular complexes in situ, without requiring tags or harsh biochemical purification. Furthermore, cryoET enables the visualization of cellular and tissue phenotypes directly at molecular, nanometric resolution without chemical fixation or staining artifacts. This forward-looking review covers recent developments in cryoEM/ET and related technologies such as cryogenic focused ion beam milling scanning electron microscopy and correlative light microscopy, increasingly enhanced and supported by artificial intelligence algorithms. Their potential application to emerging concepts is discussed, primarily the prospect of complementing medical histopathology analysis. Machine learning solutions are poised to address current challenges posed by "big data" in cryoET of tissues, cells, and macromolecules, offering the promise of enabling novel, quantitative insights into disease processes, which may translate into the clinic and lead to improved diagnostics and targeted therapeutics.

Cryogenic electron microscopy (cryo-EM) is a powerful tool for imaging liquid and semiliquid systems. While cryogenic transmission electron microscopy (cryo-TEM) is a standard technique in many fields, cryogenic scanning electron microscopy (cryo-SEM) is still not that widely used and is far less developed. The vast majority of systems under investigation by cryo-EM involve either water or organic components. In this paper, we introduce the use of novel cryo-TEM and cryo-SEM specimen preparation and imaging methodologies, suitable for highly acidic and very reactive systems. Both preserve the native nanostructure in the system, while not harming the expensive equipment or the user. We present examples of direct imaging of single-walled, multiwalled carbon nanotubes and graphene, dissolved in chlorosulfonic acid and oleum. Moreover, we demonstrate the ability of these new cryo-TEM and cryo-SEM methodologies to follow phase transitions in carbon nanotube (CNT)/superacid systems, starting from dilute solutions up to the concentrated nematic liquid-crystalline CNT phases, used as the 'dope' for all-carbon-fibre spinning. Originally developed for direct imaging of CNTs and graphene dissolution and self-assembly in superacids, these methodologies can be implemented for a variety of highly acidic systems, paving a way for a new field of nonaqueous cryogenic electron microscopy.

Cryogenic electron microscopy (cryo-EM) has been an important technique for determining the structures of macromolecules at near-atomic resolution in their native states. In recent years, the structures of some respiratory syncytial virus (RSV) protein complexes, such as the RSV polymerase (L:P complex) and nucleoprotein-RNA complex, have been determined by cryo-EM, providing insights into the mechanisms of viral RNA synthesis and facilitating antiviral development. Here, we describe the step-by-step protocols for the negative-stain and cryo-EM analysis of the RSV protein complexes.

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.

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.

Physical and chemical processes at solid-liquid interfaces play a crucial role in many natural and technological phenomena, including catalysis, solar energy and fuel generation, and electrochemical energy storage. Nanoscale characterization of such interfaces has recently been achieved using cryogenic electron microscopy, thereby providing a new path to advancing our fundamental understanding of interface processes. This contribution provides a practical guide to mapping the structure and chemistry of solid-liquid interfaces in materials and devices using an integrated cryogenic electron microscopy approach. In this approach, we pair cryogenic sample preparation which allows stabilization of solid-liquid interfaces with cryogenic focused ion beam (cryo-FIB) milling to create cross-sections through these complex buried structures. Cryogenic scanning electron microscopy (cryo-SEM) techniques performed in a dual-beam FIB/SEM enable direct imaging as well as chemical mapping at the nanoscale. We discuss practical challenges, strategies to overcome them, as well as protocols for obtaining optimal results. While we focus in our discussion on interfaces in energy storage devices, the methods outlined are broadly applicable to a range of fields where solid-liquid interface play a key role.

While lithium ion batteries with electrodes based on intercalation compounds have dominated the portable energy storage market for decades, the energy density of these materials is fundamentally limited. Today, rapidly growing demand for this type of energy storage is driving research into materials that utilize alternative reaction mechanisms to enable higher energy densities. Transition metal compounds are one such class of materials, with storage enabled by “conversion” reactions, where the material is converted to new compound upon lithiation. MoS2 is one example of this type of material that has generated a large amount of interest recently due to its high theoretical lithium storage capacity compared to graphite. Here, cryogenic scanning transmission electron microscopy techniques are used to reveal the atomic‐scale processes that occur during reaction of a model monolayer MoS2 system by enabling the unaltered atomic structure to be determined at various levels of lithiation. It is revealed that monolayer MoS2 can undergo a conversion reaction even with no substrate, and that the resulting particles are smaller than those that form in bulk MoS2, likely due to the more limited 2D diffusion. Additionally, while bilayer MoS2 undergoes intercalation with a corresponding phase transition before conversion, monolayer MoS2 does not.

Understanding of the construction and function of the HIV capsid has advanced considerably in the last decade. This is due in large part to the development of more sophisticated structural techniques, particularly cryo-electron microscopy (cryoEM) and cryo-electron tomography (cryoET). The capsid is known to be a pleomorphic fullerene cone comprised of capsid protein monomers arranged into 200–250 hexamers and 12 pentamers. The latter of these induce high curvature necessary to close the cone at both ends. CryoEM/cryoET, NMR, and X-ray crystallography have collectively described these interactions to atomic or near-atomic resolutions. Further, these techniques have helped to clarify the role the HIV capsid plays in several parts of the viral life cycle, from reverse transcription to nuclear entry and integration into the host chromosome. This includes visualizing the capsid bound to host factors. Multiple proteins have been shown to interact with the capsid. Cyclophilin A, nucleoporins, and CPSF6 promote viral infectivity, while MxB and Trim5α diminish the viral infectivity. Finally, structural insights into the intra- and intermolecular interactions that govern capsid function have enabled development of small molecules, peptides, and truncated proteins to disrupt or stabilize the capsid to inhibit HIV replication. The most promising of these, GS6207, is now in clinical trial.

Structural features of the salivary gland hypertrophy virus of the tsetse fly revealed by cryo-electron microscopy and tomography

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.

As the resolution revolution in CryoEM expands to encompass all manner of macromolecular complexes, an important new frontier is the implementation of cryogenic time resolved EM (cryoTREM). Biological macromolecular complexes are dynamic systems that undergo conformational changes on timescales from microseconds to minutes. Understanding the dynamic nature of biological changes is critical to understanding function. To realize the full potential of CryoEM, time resolved methods will be integral in coupling static structures to dynamic functions. Here, we present an LED-based photo-flash system as a core part of the sample preparation phase in CryoTREM. The plug-and-play system has a wide range of operational parameters, is low cost and ensures uniform irradiation and minimal heating of the sample prior to plunge freezing. The complete design including electronics and optics, manufacturing, control strategies and operating procedures are discussed for the Thermo Scientific™ Vitrobot and Leica™ EM GP2 plunge freezers. Possible adverse heating effects on the biological sample are also addressed through theoretical as well as experimental studies.

Cryogenic electron microscopy is one of the fastest and most robust methods for capturing high-resolution images of proteins, but stringent sample preparation, imaging conditions, and in situ radiation damage inflicted during data acquisition directly affect the resolution and ability to capture dynamic details, thereby limiting its broader utilization and adoption for protein studies. We addressed these drawbacks by introducing synthesized giant carbon nano-test tubes (GCNTTs) as radiation-insulating materials that lessen the irradiation impact on the protein during data acquisition, physical molecular concentrators that localize the proteins within a nanoscale field of view, and vessels that create a microenvironment for solution-phase imaging. High-resolution electron microscopy images of single and aggregated hemoglobin molecules within GCNTTs in both solid and solution states were acquired. Subsequent scanning transmission electron microscopy, small-angle neutron scattering, and fluorescence studies demonstrated that the GCNTT vessel protected the hemoglobin molecules from electron irradiation-, light-, or heat-induced denaturation. To demonstrate the robustness of GCNTT as an imaging platform that could potentially augment the study of proteins, we demonstrated the robustness of the GCNTT technique to image an alternative protein, d-fructose dehydrogenase, after cyclic voltammetry experiments to review encapsulation and binding insights. Given the simplicity of the material synthesis, sample preparation, and imaging technique, GCNTT is a promising imaging companion for high-resolution, single, and dynamic protein studies under electron microscopy.

Cryo-electron tomography: The power of seeing the whole picture

Structural insight into transmissive mutant huntingtin species by correlative light and electron microscopy and cryo-electron tomography