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Short overview of operando electron microscopy for energy generation, use, and storage. Highlights research in energy storage, with connections to analytical and cryogenic electron microscopy.
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Asteroids are made up of rocky material that is left over from the formation of our solar system [1]. This material holds the key to unlocking information about the history of the planets and our sun. Hayabusa2 and OSIRIS-REx are two sample return missions that have collected rocky material from carbonaceous asteroids which are rich in water, carbon, and organic compounds [1]. Hayabusa2 is a spacecraft that collected samples from asteroid Ryugu launched by the Japan Aerospace Exploration Agency (JAXA). Samples were returned to Earth in December 2020 and 10 percent of the collected material is currently housed in a state-of-the-art laboratory at NASA Johnson Space Center (JSC) dedicated to Ryugu material [1]. OSIRIS-REx is a spacecraft launched by NASA that collected samples from asteroid Bennu. The samples are on their journey to Earth and will arrive in September 2023. They will be stored in a separate cleanroom dedicated to Bennu material. From these two missions, scientists will examine dust particles and rocky material to research clues on how the early solar system formed and how life began [2]. Because these samples are so precious and rare, it is important to keep them free from Earth contamination. In September of 2020, a massive construction project began to provide curation facilities to meet the needs of two incoming asteroid collections, Hayabusa2 and OSIRIS-REx. Despite numerous setbacks caused by the coronavirus pandemic, these facilities have been completed in the fall of 2021. Some of these setbacks included strict PPE requirements, keeping safe working distances as well as access to the facilities as NASA was operating under a Stage 3 condition in response to the pandemic. These facilities will provide curation laboratories to safely house the precious asteroid samples as well as facilities to manipulate samples, do sample preparation and process samples into and out of the labs for future studies by the scientific community. The lab suite consists of several different rooms including an initial anteroom (ISO 7), a staging area (ISO 7), two gowning areas (one for OSIRIS-REx and one for Hayabusa2; both ISO 6)), the Haybusa2 Curation Laboratory (ISO 5), the OSIRIS-REx Curation Laboratory (ISO 5), an ultramicrotomy laboratory (ISO 7) and a thin section laboratory (not a cleanroom). To maintain cleanliness levels necessary for these lab spaces quarterly to monthly Balazs organic and inorganic sampling has been conducted on a regular basis before, during and after the construction project to monitor the cleanliness levels in the new labs that will house the asteroid samples. These new facilities feature a Ruuvi (Grafana) monitoring system that measures temperature, humidity, and pressure of the lab suite. This is a small puck shaped sensor that sends signals to the Grafana cloud where the data can be plotted and set up on dashboards within the lab suite. These sensors are easy to set up and install and they run off a cell battery that is good for 3-5 years before needing replacement. This system can deliver text messages and emails to the appropriate personnel when values fall outside of set limits. In addition to the monitoring system, this new lab suite features viewing windows from several different locations and sliding motion censored doors. We have obtained some state-of-the-art equipment to outfit the lab spaces (desiccators, microscopes, ultramicrotome) and are in the process of outfitting the suite with additional items such as: nitrogen gloveboxes, additional desiccators, micromanipulators, etc. The Hayabusa2 samples provided to NASA by JAXA included 23 millimeter sized grains and 4 containers with finer material. OSIRIS-REx is expected to return as much as 400 grams of asteroid Bennu. Samples will be stored in a nitrogen glove box and separated into nitrogen sealed transfer containers. Small Bennu and Ryugu particles will be handled and prepared (e.g., ultra-thin sections for scanning electron microscopy (SEM) or transmission electron microscopy (TEM)) using an Axis Pro micromanipulator and Leica EM UC7 ultramicrotome. Analyzing samples at the atomic scale will provide greater insight to the origins of the solar system and life formation on Earth. All the curation preparation for these sample collections is essential to ongoing research and efforts to understand our solar system, both nowand for future generations.
Light and electron microscopy studies were carried out on decalcified tibial epiphyseal plates of rats flown aboard Cosmos 1887 (12.5d flight plus 53.5h recovery). Analysis of variance showed that the proliferative zone of flight animals was significantly higher than that of synchronous controls, while the hypertrophic/calcification zone was significantly reduced. Flight animals had more cells than synchronous controls in the proliferative zone, and less in the hypertrophic/calcification region. The total number of cells, however, was significantly higher in flight animals. No differences were found for perimeter or shape factor of growth plates, but area was significantly lower in flight animals in comparison to synchronous controls. Collagen fibrils in flight animals were shorter and wider than in synchronous controls. The time required for a cell to cycle through the growth plate is 2 to 3 days, so most of the cells and matrix present were formed after the animals had returned to 1 g, and probably represent stages of recovery from microgravity exposure, which in itself is an interesting question.
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Data and experimental evidence which support the contention that the C delta diamonds may result from grain-grain collisions in supernova shocks in the interstellar medium are presented. Fragments of the Allende CV3 chondrite were acid-treated. A whitish powder was obtained. For the Analytical Electron Microscopy (AEM) a small drop of ethanol suspension was transferred onto holey carbon support films on 3 mm EM grids. The AEM was performed on transmission-thin fragments of the material which overlay holes in the film, to eliminate interference from the substrate. Electron Spectroscopy for Chemical Analysis (ESCA) was performed on a large aliquot of C. Diamond was identified by selected area electron diffraction. Scanning Transmission Electron Microscope / Energy Dispersive X-ray (STEM-EDS) microanalyses of the C delta diamond, using a light-element detector, show that oxygen and possibly nitrogen are the only impurities consistently present. ESCA spectra from bulk C delta material confirm the presence of N at a level of 0.35 percent or less. Under UV irradiation a yellow-red fluorescence is observed, consistent with that of natural diamonds containing substitutional N. Electron Energy Loss Spectra (EELS) were recorded at 2 eV resolution from the C delta diamond, high pressure synthetic diamond, a diamond film produced in a low pressure plasma by chemical vapor deposition (CVD) on a heated silicon substrate (Roy, 1987), graphite, and amorphous arc sputtered carbon. Comparison of the carbon K edge shape and fine structure shows the Allende C delta phase to be largely diamond, but with a significant pre-edge absorption feature indicative of transitions of C 1s electrons into pi asterisk orbitals which are absent in the purely sp(3)-bonded diamond but present in graphite and amorphous carbon.
Analysis of the three-dimensional (3D) structures of nanocrystals with solution-phase transmission electron microscopy is beginning to reveal their unique physiochemical properties. We developed a “one-particle Brownian 3D reconstruction method” based on imaging of ensembles of colloidal nanocrystals using graphene liquid cell electron microscopy. Projection images of differently rotated nanocrystals are acquired using a direct electron detector with high temporal (<2.5 ms) resolution and analyzed to obtain an ensemble of 3D reconstructions. Here, we introduce computational methods required for successful atomic-resolution 3D reconstruction: (i) tracking of the individual particles throughout the time series, (ii) subtraction of the interfering background of the graphene liquid cell, (iii) identification and rejection of low-quality images, and (iv) tailored strategies for 2D/3D alignment and averaging that differ from those used in biological cryo–electron microscopy. Our developments are made available through the open-source software package SINGLE.
Abstract The accurate assignment of cofactors in cryo-electron microscopy maps is crucial in determining protein function. This is particularly true for chlorophylls (Chls), for which small structural differences lead to important functional differences. Recent cryo-electron microscopy structures of Chl-containing protein complexes exemplify the difficulties in distinguishing Chl b and Chl f from Chl a . We use these structures as examples to discuss general issues arising from local resolution differences, properties of electrostatic potential maps, and the chemical environment which must be considered to make accurate assignments. We offer suggestions for how to improve the reliability of such assignments.
Mitochondrial division is a fundamental biological process essensial for cellular functionality and vitality. The prevailing hypothesis that dynamin related protein 1 (Drp1) provides principal control in mitochondrial division, in which it also involves the endoplasmic reticulum (ER) and the cytoskeleton, does not account for all the observations. Therefore. the hypothesis may be incomplete. Our previous study in HeLa cells led to a new hypothesis of mitochondrial division by budding. To follow-up our previous study, we employed in situ cryo-electron tomography to visualize mitochondrial budding in the intact healthy monkey kidney cells (BS-C-1 cells). Our findings reaffirm single and multiple mitochondrial budding, consistent with our observations in HeLa cells. Notably, the budding regions vary significantly in diameter and length, which may represent different stages of budding. More interestingly, neither rings nor ring-like structures, nor the wrapping of ER tubes was observed in the budding regions, suggesting mitochondrial budding is independent from Drp1 and ER. Meanwhile, we uncovered direct interactions between mitochondria and large vesicles that are distinct from small mitochondrial-derived vesicles and extracellular mitovesicles. In conclusion, we propose that these interacting vesicles may have mitochondrial origins.
Viruses are numerically the most abundant forms on Earth, and most are present in soil. Even though viruses are highly abundant in soil and critical to rhizosphere function, visualizing the diverse morphotypes within soil has been challenging. The difficulty is primarily due to the heterogenous nature of isolated suspensions that typically contain nanometer to micron scale debris which renders protein crystallography for structural studies unfeasible and hinders cryo-electron microscopy due to ice thickness and contrast issues. Here we employed and compared a simple spin filtration method to cleanup solutions of extracted viruses for direct observation with cryo-electron microscopy. The method employs common physical biochemical separation steps to remove large and small debris which dramatically improves image quality and preservation of structural features to permit visualizing morphotypes not typically seen with conventional negative stain approaches. In addition to tailed and non-tailed polyhedral phages, several under reported or novel morphotypes of soil viruses are directly visualized as a particle library with both 2D and 3D information.
Atmosphere- and/or moisture-sensitive materials can be challenging to characterize using electron microscopy techniques due to sample preparation workflows that generally require exposure to ambient conditions. Here, we describe a novel preparation method that uses aluminum foil in combination with a commercial cryo-EM transfer system to circumvent undesired exposure to the atmosphere. First, hygroscopic MgCl 2 was used as a model material, and prepared samples (both protected and unprotected) were placed in a controlled-humidity environment (> 80% relative humidity) for various exposure lengths (circa seconds to hours). Following this, the effectiveness of the sample preparation method was determined by comparing qualitative photos and quantitative X-ray diffraction patterns between the two sample subsets. The combined results of these experiments suggest that the outlined preparation method effectively protects MgCl 2 from atmospheric contamination compared to MgCl 2 samples that had no protective measures taken. Finally, the preparation method was utilized to protect a highly hygroscopic crystalline BaO thin film for characterization via scanning electron microscopy, thereby demonstrating a functional application of the outlined preparation technique and an additional use for the commercial cryo-EM transfer system beyond its intended application.
Bacteria rapidly adapt to environmental cues through morphological and ultrastructural changes that correlate with physiology and behavior. Cryogenic transmission electron microscopy (cryo‐TEM) can capture these phenotypic changes in near‐native, vitrified states, but manual analysis of low‐dose micrographs is labor intensive and limits throughput. Here, we present an end‐to‐end workflow that combines low‐dose cryo‐TEM imaging with a YOLOv11‐based instance‐segmentation model to automatically identify bacteria and quantify key structural features directly from the micrographs. This workflow enables (i) robust bacterial localization and counting from low‐magnification atlas/montage images, (ii) automated measurements of cell‐envelope (outer–inner membrane) thickness and anisotropy from higher‐magnification views, and (iii) detection and quantification of bacteria–flagella interactions, including overlap length and curvature metrics for interacting versus noninteracting flagella. Using Pantoea sp. YR343 grown under distinct media conditions, we show that the automated measurements agree with manual annotations while substantially reducing analysis time. Together, these tools provide a practical framework for scalable bacterial identification and quantitative phenotyping in low‐dose cryo‐TEM datasets and establish a foundation for extending cryo‐TEM image analysis toward higher‐throughput studies of microbial heterogeneity and biointerfaces.
Neutralizing antibody responses to coronaviruses mainly target the receptor-binding domain (RBD) of the trimeric spike. Here, we characterized polyclonal immunoglobulin Gs (IgGs) and Fabs from COVID-19 convalescent individuals for recognition of coronavirus spikes. Plasma IgGs differed in their focus on RBD epitopes, recognition of alpha- and beta-coronaviruses, and contributions of avidity to increased binding/ neutralization of IgGs over Fabs. Using electron microscopy, we examined specificities of polyclonal plasma Fabs, revealing recognition of both S1 A and RBD epitopes on SARS-CoV-2 spike. Moreover, a 3.4 Å cryoelectron microscopy (cryo-EM) structure of a neutralizing monoclonal Fab-spike complex revealed an epitope that blocks ACE2 receptor binding. Modeling based on these structures suggested different potentials for inter-spike crosslinking by IgGs on viruses, and characterized IgGs would not be affected by identified SARS-CoV-2 spike mutations. Overall, our studies structurally define a recurrent anti-SARS-CoV-2 antibody class derived from VH3-53/VH3-66 and similarity to a SARS-CoV VH3-30 antibody, providing criteria for evaluating vaccine-elicited antibodies.
We derive an asymptotic expansion for the log-likelihood of Gaussian mixture models (GMMs) with equal covariance matrices in the low signal-to-noise regime. The expansion reveals an intimate connection between two types of algorithms for parameter estimation: the method of moments and likelihood optimizing algorithms such as Expectation-Maximization (EM). We show that likelihood optimization in the low SNR regime reduces to a sequence of least squares optimization problems that match the moments of the estimate to the ground truth moments one by one. This connection is a stepping stone towards the analysis of EM and maximum likelihood estimation in a wide range of models. A motivating application for the study of low SNR mixture models is cryo-electron microscopy data, which can be modeled as a GMM with algebraic constraints imposed on the mixture centers. We discuss the application of our expansion to algebraically constrained GMMs, among other example models of interest. © 2022 The Authors. Communications on Pure and Applied Mathematics published by Wiley Periodicals LLC.
The discovery and design of biologically important RNA molecules is outpacing three-dimensional structural characterization. In this work, we demonstrate that cryo-electron microscopy can routinely resolve maps of RNA-only systems and that these maps enable subnanometer-resolution coordinate estimation when complemented with multidimensional chemical mapping and Rosetta DRRAFTER computational modeling. Furthermore ,this hybrid ‘Ribosolve’ pipeline detects and falsifies homologies and conformational rearrangements in 11 previously unknown 119- to 338-nucleotide protein-free RNA structures: full-length Tetrahymena ribozyme, hc16 ligase with and without substrate, full-length Vibrio cholerae and Fusobacterium nucleatum glycine riboswitch aptamers with and without glycine, Mycobacterium SAM-IV riboswitch with and without S-adenosylmethionine, and the computer-designed ATP-TTR-3 aptamer with and without AMP. Simulation benchmarks, blind challenges, compensatory mutagenesis, cross-RNA homologies and internal controls demonstrate that Ribosolve can accurately resolve the global architectures of RNA molecules but does not resolve atomic details. These tests offer guidelines for making inferences in future RNA structural studies with similarly accelerated throughput.
Organic semiconducting polymers are a powerful platform for the design of next-generation technologies due to their excellent optoelectronic properties and solution processability, allowing access to low-cost and scalable manufacturing techniques such as spin-coating, slot-die coating and roll-to-roll printing. However, their extended π-conjugation results in low solubility, requiring the use of toxic halogenated solvents to generate thin films and devices. Furthermore, accessible post-functionalization of semiconductors toward the development of multifunctional devices and sensors remains a challenge due to limited solid-state chemistry for alkyl side chains. In this work, an asymmetric side-chain engineering approach was used to introduce terminal hydroxyl moieties alongside traditional solubilizing branched alkyl chains into an isoindigo-based polymer. The hydroxyl moieties led to significantly improved processability in alcohol-based solvents without sacrificing electronic performance in thin film organic field-effect transistors. Solid state morphologies of the thin films processed from both alcohol-based and traditional halogenated solvents were further characterized using atomic force microscopy and grazing incidence wide angle X-ray scattering. Additionally, Cryo-EM was utilized in order to characterize the role of asymmetric side-chain functionality in solution state aggregation. The versatility of this design was further probed using fluorescein isothiocyanate to directly functionalize the asymmetric polymer in thin film. Furthermore, this facile solid-state post-functionalization further demonstrates asymmetric side-chain engineering to be a viable approach toward the development of sustainably manufactured multifunctional electronics.
Small-conductance Ca 2+ -activated K + (K Ca 2.1-K Ca 2.3) channels modulate neuronal and cardiac excitability. We report cryo-electron microscopy structures of the K Ca 2.2 channel in complex with calmodulin and Ca 2+ , alone or bound to two small molecule inhibitors, at 3.18, 3.50, 2.99 and 2.97 angstrom resolution, respectively. Extracellular S3-S4 loops in β-hairpin configuration form an outer canopy over the pore with an aromatic box at the canopy’s center. Each S3-S4 β-hairpin is tethered to the selectivity filter in the neighboring subunit by inter-subunit hydrogen bonds. This hydrogen bond network flips the aromatic residue (Tyr362) in the filter’s GYG signature by 180°, causing the outer selectivity filter to widen and water to enter the filter. Disruption of the tether by a mutation narrows the outer selectivity filter, realigns Tyr362 to the position seen in other K + channels, and significantly increases unitary conductance. UCL1684, a mimetic of the bee venom peptide apamin, sits atop the canopy and occludes the opening in the aromatic box. AP14145, an analogue of a therapeutic for atrial fibrillation, binds in the central cavity below the selectivity filter and induces closure of the inner gate. These structures provide a basis for understanding the small unitary conductance and pharmacology of K Ca 2.x channels.