Complementary imaging of nanoclusters interacting with mitochondria via stimulated emission depletion and scanning transmission electron microscopy.

The emerging stress caused by nanomaterials in the environment is of great concern because they can have toxic effects on organisms. However, thorough study of the interactions between cells and diverse nanoparticles (NPs) using a unified approach is challenging. Here, we present a novel approach combining stimulated emission depletion (STED) microscopy and scanning transmission electron microscopy (STEM) for quantitative assessment, real-time tracking, and in situ imaging of the intracellular behavior of gold-silver nanoclusters (AuAgNCs), based on their fluorescence and electron properties. The results revealed an aggregated state of AuAgNCs within the mitochondria and an increase in sulfur content in AuAgNCs, presumably owing to their reaction with thiol-containing molecules inside the mitochondria. Moreover, AuAgNCs (100 µg/mL) induced a 75% decline in mitochondrial membrane potential and a 12-fold increase of mitochondrial reactive oxygen species in comparison to control. This mitochondrial damage may be triggered by the reaction of AuAgNCs with thiol, which provides direct imaging evidence for uncovering the action mechanism of AuAgNCs on the mitochondria. The proposed dual-imaging strategy using STED and STEM is a potential tool to offer valuable insights into cytotoxicity between subcellular structures and diverse NPs, and can serve as a key strategy for nanomaterial biosafety assessment.

Statistical 3D morphology characterization of vaterite microspheres produced by engineered Escherichia coli.

Hollow vaterite microspheres are important materials for biomedical applications such as drug delivery and regenerative medicine owing to their biocompatibility, high specific surface area, and ability to encapsulate a large number of bioactive molecules and compounds. We demonstrated that hollow vaterite microspheres are produced by an Escherichia coli strain engineered with a urease gene cluster from the ureolytic bacteria Sporosarcina pasteurii in the presence of bovine serum albumin. We characterized the 3D nanoscale morphology of five biogenic hollow vaterite microspheres using 3D high-angle annular dark field scanning transmission electron microscopy (HAADF-STEM) tomography. Using automated high-throughput HAADF-STEM imaging across several sample tilt orientations, we show that the microspheres evolved from a smaller more ellipsoidal shape to a larger more spherical shape while the internal hollow core increased in size and remained relatively spherical, indicating that the microspheres produced by this engineered strain likely do not contain the bacteria. The statistical 3D morphology information demonstrates the potential for using biogenic calcium carbonate mineralization to produce hollow vaterite microspheres with controlled morphologies. STATEMENT OF SIGNIFICANCE: The nanoscale 3D structures of biomaterials determine their physical, chemical, and biological properties, however significant efforts are required to obtain a statistical understanding of the internal 3D morphology of materials without damaging the structures. In this study, we developed a non-destructive, automated technique that allows us to understand the nanoscale 3D morphology of many unique hollow vaterite microspheres beyond the spectroscopy methods that lack local information and microscopy methods that cannot interrogate the full 3D structure. The method allowed us to quantitatively correlate the external diameters and aspect ratios of vaterite microspheres with their hollow internal structures at the nanoscale. This work demonstrates the opportunity to use automated transmission electron microscopy to characterize nanoscale 3D morphologies of many biomaterials and validate the chemical and biological functionality of these materials.

Visualizing the intracellular aggregation behavior of gold nanoclusters via structured illumination microscopy and scanning transmission electron microscopy.

Given the growing concerns about nanotoxicity, numerous studies have focused on providing mechanistic insights into nanotoxicity by imaging the intracellular fate of nanoparticles. A suitable imaging strategy is necessary to uncover the intracellular behavior of nanoparticles. Although each conventional technique has its own limitations, scanning transmission electron microscopy (STEM) and three-dimensional structured illumination microscopy (3D-SIM) combine the advantages of chemical element mapping, ultrastructural analysis, and cell dynamic tracking. Gold nanoclusters (AuNCs), synthesized using 6-aza-2 thiothymine (ATT) and L-arginine (Arg) as reducing and protecting ligands, referred to as Arg@ATT-AuNCs, have been widely used in biological sensing and imaging, medicine, and catalyst yield. Based on their intrinsic fluorescence and high electron density, Arg@ATT-AuNCs were selected as a model. STEM imaging showed that both the single-particle and aggregated states of Arg@ATT-AuNCs were compartmentally distributed within a single cell. Real-time 3D-SIM imaging showed that the fluorescent Arg@ATT-AuNCs gradually aggregated after being located in the lysosomes of living cells, causing lysosomal damage. The aggregate formation of Arg@ATT-AuNCs was triggered by the low-pH medium, particularly in the lysosomal acidic environment. The proposed dual imaging strategy was verified using other types of AuNCs, which is valuable for studying nano-cell interactions and any associated cytotoxicity, and has the potential to be a useful approach for exploring the interaction of cells with various nanoparticles.

An introduction to scanning transmission electron microscopy for the study of protozoans.

Since its inception in the 1930s, transmission electron microscopy (TEM) has been a powerful method to explore the cellular structure of parasites. TEM usually requires samples of <100 nm thick and with protozoans being larger than 1 µm, their study requires resin embedding and ultrathin sectioning. During the past decade, several new methods have been developed to improve, facilitate, and speed up the structural characterisation of biological samples, offering new imaging modalities for the study of protozoans. In particular, scanning transmission electron microscopy (STEM) can be used to observe sample sections as thick as 1 µm thus becoming an alternative to conventional TEM. STEM can also be performed under cryogenic conditions in combination with cryo-electron tomography providing access to the study of thicker samples in their native hydrated states in 3D. This method, called cryo-scanning transmission electron tomography (cryo-STET), was first developed in 2014. This review presents the basic concepts and benefits of STEM methods and provides examples to illustrate the potential for new insights into the structure and ultrastructure of protozoans.

Distribution Analysis of Iron and Copper by STEM-EDX Spectroscopy of Hemosiderin Particles in the Liver of Rats Overloaded With Iron.

BACKGROUND/AIM: Our recent studies have indicated that trace copper co-existed with iron in hemosiderin particles of human genetic iron overload. To understand this phenomenon, we analyzed hemosiderin particles in iron-overloaded rat liver by using scanning transmission electron microscopy - energy-dispersive X-ray (STEM-EDX) spectroscopy. MATERIALS AND METHODS: Samples for STEM-EDX spectroscopy were prepared from the liver of rats administered an intraperitoneal injection of dextran iron. RESULTS: The micro-domain analysis with STEM-EDX spectroscopy showed that dense bodies contained high levels of iron and trace copper. Quantitative analysis of copper levels in the liver specimen using atomic spectrophotometry showed that copper concentration in the liver was not increased by iron overload. These findings suggest that the overload of iron induced distribution of trace copper to hemosiderin particles without changing cellular copper levels. CONCLUSION: Co-existence of copper with iron was observed in hemosiderin particles of the liver of an experimental model of iron overload, suggesting that iron overload induced distribution of trace copper into hemosiderin particles.

Dynamical scattering in ice-embedded proteins in conventional and scanning transmission electron microscopy.

Structure determination of biological macromolecules using cryogenic electron microscopy is based on applying the phase object (PO) assumption and the weak phase object (WPO) approximation to reconstruct the 3D potential density of the molecule. To enhance the understanding of image formation of protein complexes embedded in glass-like ice in a transmission electron microscope, this study addresses multiple scattering in tobacco mosaic virus (TMV) specimens. This includes the propagation inside the molecule while also accounting for the effect of structural noise. The atoms in biological macromolecules are light but are distributed over several nanometres. Commonly, PO and WPO approximations are used in most simulations and reconstruction models. Therefore, dynamical multislice simulations of TMV specimens embedded in glass-like ice were performed based on fully atomistic molecular-dynamics simulations. In the first part, the impact of multiple scattering is studied using different numbers of slices. In the second part, different sample thicknesses of the ice-embedded TMV are considered in terms of additional ice layers. It is found that single-slice models yield full frequency transfer up to a resolution of 2.5 Å, followed by attenuation up to 1.4 Å. Three slices are sufficient to reach an information transfer up to 1.0 Å. In the third part, ptychographic reconstructions based on scanning transmission electron microscopy (STEM) and single-slice models are compared with conventional TEM simulations. The ptychographic reconstructions do not need the deliberate introduction of aberrations, are capable of post-acquisition aberration correction and promise benefits for information transfer, especially at resolutions beyond 1.8 Å.

A low-cost smartphone fluorescence microscope for research, life science education, and STEM outreach.

Much of our understanding of cell and tissue development, structure, and function stems from fluorescence microscopy. The acquisition of colorful and glowing images engages and excites users ranging from seasoned microscopists to STEM students. Fluorescence microscopes range in cost from several thousand to several hundred thousand US dollars. Therefore, the use of fluorescence microscopy is typically limited to well-funded institutions and biotechnology companies, research core facilities, and medical laboratories, but is financially impractical at many universities and colleges, primary and secondary schools (K-12), and in science outreach settings. In this study, we developed and characterized components that when used in combination with a smartphone or tablet, perform fluorescence microscopy at a cost of less than $50 US dollars per unit. We re-purposed recreational LED flashlights and theater stage lighting filters to enable viewing of green and red fluorophores including EGFP, DsRed, mRFP, and mCherry on a simple-to-build frame made of wood and plexiglass. These devices, which we refer to as glowscopes, were capable of 10 µm resolution, imaging fluorescence in live specimens, and were compatible with all smartphone and tablet models we tested. In comparison to scientific-grade fluorescence microscopes, glowscopes may have limitations to sensitivity needed to detect dim fluorescence and the inability to resolve subcellular structures. We demonstrate capability of viewing fluorescence within zebrafish embryos, including heart rate, rhythmicity, and regional anatomy of the central nervous system. Due to the low cost of individual glowscope units, we anticipate this device can help to equip K-12, undergraduate, and science outreach classrooms with fleets of fluorescence microscopes that can engage students with hands-on learning activities.

Low-energy scanning transmission electron microscopy applied to ice-embedded biological macromolecules.

In this report, we applied annular bright-field and annular dark-field low-energy (30 keV) scanning transmission electron microscopy imaging to a vitreous ice-embedded biological macromolecule, T4 phage, to investigate the applicability of these methods for morphological investigation and sample screening. Multiple camera lengths were examined to find the optimal acceptance angle for both modes. Image clarity differed substantially between the modes, with the presence of ice also strongly influencing the quality of acquired micrographs. In annular dark-field mode, the proper discrimination of electrons scattered by the specimen from those scattered by the background ice was found to be difficult due to the severe overlap of the scattered electrons. The resulting micrographs lacked clarity, and the ice-embedded phage particles could only be discerned after post-processing image adjustment. However, in annular bright-field mode, despite similar overlapping of the scattered electrons, it was possible to assess the morphology and intactness of the specimen in the embedding ice, suggesting that this mode may find utility in low-energy cryo-scanning transmission electron microscopy imaging methods.

Near-real-time diagnosis of electron optical phase aberrations in scanning transmission electron microscopy using an artificial neural network.

The key to optimizing spatial resolution in a state-of-the-art scanning transmission electron microscope is the ability to measure and correct for electron optical aberrations of the probe-forming lenses precisely. Several diagnostic methods for aberration measurement and correction have been proposed, albeit often at the cost of relatively long acquisition times. Here, we illustrate how artificial intelligence can be used to provide near-real-time diagnosis of aberrations from individual Ronchigrams. The demonstrated speed of aberration measurement is important because microscope conditions can change rapidly. It is also important for the operation of MEMS-based hardware correction elements, which have less intrinsic stability than conventional electromagnetic lenses.

Elemental compositions of sea urchin larval cell vesicles evaluated by cryo-STEM-EDS and cryo-SEM-EDS.

During spicule formation in sea urchin larvae, calcium ions translocate within the primary mesenchymal cells (PMCs) from endocytosed seawater vacuoles to various organelles and vesicles where they accumulate, and subsequently precipitate. During this process, calcium ions are concentrated by more than three orders of magnitude, while other abundant ions (Na, Mg) must be removed. To obtain information about the overall ion composition in the vesicles, we used quantitative cryo-SEM-EDS and cryo-STEM-EDS analyzes. For cryo-STEM-EDS, thin (500 nm) frozen hydrated lamellae of PMCs were fabricated using cryo-focused ion beam-SEM. The lamellae were then loaded into a cryo-TEM, imaged and the ion composition of electron dense bodies was measured. Analyzes performed on 18 Ca-rich particles/particle clusters from 6 cells contained Ca, Na, Mg, S and P in different ratios. Surprisingly, all the Ca-rich particles contained P in amounts up to almost 1:1 of Ca. These cryo-STEM-EDS results were qualitatively confirmed by cryo-SEM-EDS analyzes of 310 vesicles, performed on high pressure frozen and cryo-planed samples. We discuss the advantages and limitations of the two techniques, and their potential applicability, especially to study ion transport pathways and ion trafficking in cells involved in mineralization. STATEMENT OF SIGNIFICANCE: The 'inorganic side of life', encompassing ion trafficking and ion storage in soft tissues of organisms, is a generally overlooked problem. Addressing such a problem becomes possible through the application of innovative techniques, performed in cryogenic conditions, which preserve the tissues in quasi-physiological state. We developed here a set of analytical tools, cryo-SEM-EDS, and cryo-STEM-EDS, which allow reconstructing the ion composition inside vesicles in sea urchin larval cells, on their way to deposit mineral in the skeletons. The techniques are complex, and we evaluate here the advantages and disadvantages of each technique. The methodologies that we are developing here can be applied to other cells and other pathways as well, eventually leading to quantitative elemental analyzes of tissues under cryogenic conditions.

Ni Nanoparticles Stabilized by Hyperbranched Polymer: Does the Architecture of the Polymer Affect the Nanoparticle Characteristics and Their Performance in Catalysis?

Heat-up and hot-injection methods were employed to synthesize Ni nanoparticles (NPs) with narrow size distribution in the presence of hyperbranched pyridylphenylene polymer (PPP) as a stabilizing agent. It was shown that depending on the synthetic method, Ni NPs were formed either in a cross-linked polymer network or stabilized by a soluble hyperbranched polymer. Ni NPs were characterized by a combination of transmission electron microscopy (TEM), scanning TEM, thermogravimetric analysis, powder X-ray diffraction, X-ray photoelectron spectroscopy, energy dispersive X-ray analysis, and magnetic measurements. The architecture of polymer support was found to significantly effect Ni NPs characteristics and behavior. The Ni NPs demonstrated a high catalytic activity in a model Suzuki-Miyaura cross-coupling reaction. No significant drop in activity was observed upon repeated use after magnetic separation in five consecutive catalytic cycles. We believe that hyperbranched PPP can serve as universal platform for the controllable synthesis of Ni NPs, acting as highly active and stable catalysts.

Physics Discovery in Nanoplasmonic Systems via Autonomous Experiments in Scanning Transmission Electron Microscopy.

Physics-driven discovery in an autonomous experiment has emerged as a dream application of machine learning in physical sciences. Here, this work develops and experimentally implements a deep kernel learning (DKL) workflow combining the correlative prediction of the target functional response and its uncertainty from the structure, and physics-based selection of acquisition function, which autonomously guides the navigation of the image space. Compared to classical Bayesian optimization (BO) methods, this approach allows to capture the complex spatial features present in the images of realistic materials, and dynamically learn structure-property relationships. In combination with the flexible scalarizer function that allows to ascribe the degree of physical interest to predicted spectra, this enables physical discovery in automated experiment. Here, this approach is illustrated for nanoplasmonic studies of nanoparticles and experimentally implemented in a truly autonomous fashion for bulk- and edge plasmon discovery in MnPS3 , a lesser-known beam-sensitive layered 2D material. This approach is universal, can be directly used as-is with any specimen, and is expected to be applicable to any probe-based microscopic techniques including other STEM modalities, scanning probe microscopies, chemical, and optical imaging.

Identifying and manipulating single atoms with scanning transmission electron microscopy.

The manipulation of individual atoms has developed from visionary speculation into an established experimental science. Using focused electron irradiation in a scanning transmission electron microscope instead of a physical tip in a scanning probe microscope confers several benefits, including thermal stability of the manipulated structures, the ability to reach into bulk crystals, and the chemical identification of single atoms. However, energetic electron irradiation also presents unique challenges, with an inevitable possibility of irradiation damage. Understanding the underlying mechanisms will undoubtedly continue to play an important role to guide experiments. Great progress has been made in several materials including graphene, carbon nanotubes, and crystalline silicon in the eight years since the discovery of electron-beam manipulation, but the important challenges that remain will determine how far we can expect to progress in the near future.

Single-particle cryo-EM structures from iDPC-STEM at near-atomic resolution.

In electron cryomicroscopy (cryo-EM), molecular images of vitrified biological samples are obtained by conventional transmission microscopy (CTEM) using large underfocuses and subsequently computationally combined into a high-resolution three-dimensional structure. Here, we apply scanning transmission electron microscopy (STEM) using the integrated differential phase contrast mode also known as iDPC-STEM to two cryo-EM test specimens, keyhole limpet hemocyanin (KLH) and tobacco mosaic virus (TMV). The micrographs show complete contrast transfer to high resolution and enable the cryo-EM structure determination for KLH at 6.5 Å resolution, as well as for TMV at 3.5 Å resolution using single-particle reconstruction methods, which share identical features with maps obtained by CTEM of a previously acquired same-sized TMV data set. These data show that STEM imaging in general, and in particular the iDPC-STEM approach, can be applied to vitrified single-particle specimens to determine near-atomic resolution cryo-EM structures of biological macromolecules.

Deep learning-based noise filtering toward millisecond order imaging by using scanning transmission electron microscopy.

Application of scanning transmission electron microscopy (STEM) to in situ observation will be essential in the current and emerging data-driven materials science by taking STEM's high affinity with various analytical options into account. As is well known, STEM's image acquisition time needs to be further shortened to capture a targeted phenomenon in real-time as STEM's current temporal resolution is far below the conventional TEM's. However, rapid image acquisition in the millisecond per frame or faster generally causes image distortion, poor electron signals, and unidirectional blurring, which are obstacles for realizing video-rate STEM observation. Here we show an image correction framework integrating deep learning (DL)-based denoising and image distortion correction schemes optimized for STEM rapid image acquisition. By comparing a series of distortion corrected rapid scan images with corresponding regular scan speed images, the trained DL network is shown to remove not only the statistical noise but also the unidirectional blurring. This result demonstrates that rapid as well as high-quality image acquisition by STEM without hardware modification can be established by the DL. The DL-based noise filter could be applied to in-situ observation, such as dislocation activities under external stimuli, with high spatio-temporal resolution.

Scanning Transmission Electron Microscopy in a Scanning Electron Microscope for the High-Throughput Imaging of Biological Assemblies.

Electron microscopy of soft and biological materials, or "soft electron microscopy", is essential to the characterization of macromolecules. Soft microscopy is governed by enhancing contrast while maintaining low electron doses, and sample preparation and imaging methodologies are driven by the length scale of features of interest. While cryo-electron microscopy offers the highest resolution, larger structures can be characterized efficiently and with high contrast using low-voltage electron microscopy by performing scanning transmission electron microscopy in a scanning electron microscope (STEM-in-SEM). Here, STEM-in-SEM is demonstrated for a four-lobed protein assembly where the arrangement of the proteins in the construct must be examined. STEM image simulations show the theoretical contrast enhancement at SEM-level voltages for unstained structures, and experimental images with multiple STEM modes exhibit the resolution possible for negative-stained proteins. This technique can be extended to complex protein assemblies, larger structures such as cell sections, and hybrid materials, making STEM-in-SEM a valuable high-throughput imaging method.

Analysis of three-dimensional chromatin packing domains by chromatin scanning transmission electron microscopy (ChromSTEM).

Chromatin organization over multiple length scales plays a critical role in the regulation of transcription. Deciphering the interplay of these processes requires high-resolution, three-dimensional, quantitative imaging of chromatin structure in vitro. Herein, we introduce ChromSTEM, a method that utilizes high-angle annular dark-field imaging and tomography in scanning transmission electron microscopy combined with DNA-specific staining for electron microscopy. We utilized ChromSTEM for an in-depth quantification of 3D chromatin conformation with high spatial resolution and contrast, allowing for characterization of higher-order chromatin structure almost down to the level of the DNA base pair. Employing mass scaling analysis on ChromSTEM mass density tomograms, we observed that chromatin forms spatially well-defined higher-order domains, around 80 nm in radius. Within domains, chromatin exhibits a polymeric fractal-like behavior and a radially decreasing mass-density from the center to the periphery. Unlike other nanoimaging and analysis techniques, we demonstrate that our unique combination of this high-resolution imaging technique with polymer physics-based analysis enables us to (i) investigate the chromatin conformation within packing domains and (ii) quantify statistical descriptors of chromatin structure that are relevant to transcription. We observe that packing domains have heterogeneous morphological properties even within the same cell line, underlying the potential role of statistical chromatin packing in regulating gene expression within eukaryotic nuclei.

Three-dimensional SEM, TEM, and STEM for analysis of large-scale biological systems.

A major aim in structural cell biology is to analyze intact cells in three dimensions, visualize subcellular structures, and even localize proteins at the best possible resolution in three dimensions. Though recently developed electron microscopy tools such as electron tomography, or three-dimensional (3D) scanning electron microscopy, offer great resolution in three dimensions, the challenge is that, the better the resolution, usually the smaller the volume under investigation. Several different approaches to overcome this challenge were presented at the Microscopy Conference in Vienna in 2021. These tools include array tomography, batch tomography, or scanning transmission electron tomography, all of which can nowadays be extended toward correlative light and electron tomography, with greatly increased 3D information. Here, we review these tools, describe the underlying procedures, and discuss their advantages and limits.

Atomic layer deposited Al2O3 as a protective overlayer for focused ion beam preparation of plan-view STEM samples.

Preservation of analyte integrity during focused ion beam (FIB) sample preparation is a significant challenge in the scanning transmission electron microscopy (STEM) characterization of plan-view samples with sensitive surface chemistries. This can preclude the characterization of atomic arrangements, nanoscale surface coverages, and distributions and morphologies of functional molecular materials composed of surface-immobilized metal nanoparticles, clusters or coordination complexes. This work demonstrates effective protection of Pt nanoparticle (NP) morphology through a plan-view FIB lift-out and thinning procedure by encapsulating the sample surface in an Al2O3 overlayer grown by atomic layer deposition (ALD). High-angle annular dark field (HAADF)-STEM analysis was used in concert with energy dispersive X-ray spectroscopy (EDS) to identify and image sub-10 nm features attributed to Pt and to evaluate the distribution of implanted Ga+ (derived from the FIB milling beam). ALD is a mild chemical vapor deposition (CVD) technique that has the capability to generate dense, pinhole-free films with tunable compositions and properties, making this ALD-FIB procedure applicable to many sample architectures for plan-view lamella preparation and STEM analysis.