Deep generative priors for biomolecular 3D heterogeneous reconstruction from cryo-EM projections.

Cryo-electron microscopy has become a powerful tool to determine three-dimensional (3D) structures of rigid biological macromolecules from noisy micrographs with single-particle reconstruction. Recently, deep neural networks, e.g., CryoDRGN, have demonstrated conformational and compositional heterogeneity of complexes. However, the lack of ground-truth conformations poses a challenge to assess the performance of heterogeneity analysis methods. In this work, variational autoencoders (VAE) with three types of deep generative priors were learned for latent variable inference and heterogeneous 3D reconstruction via Bayesian inference. More specifically, VAEs with "Variational Mixture of Posteriors" priors (VampPrior-SPR), non-parametric exemplar-based priors (ExemplarPrior-SPR) and priors from latent score-based generative models (LSGM-SPR) were quantitatively compared with CryoDRGN. We built four simulated datasets composed of hypothetical continuous conformation or discrete states of the hERG K + channel. Empirical and quantitative comparisons of inferred latent representations were performed with affine-transformation-based metrics. These models with more informative priors gave better regularized, interpretable factorized latent representations with better conserved pairwise distances, less deformed latent distributions and lower within-cluster variances. They were also tested on experimental datasets to resolve compositional and conformational heterogeneity (50S ribosome assembly, cowpea chlorotic mottle virus, and pre-catalytic spliceosome) with comparable high resolution. Codes and data are available: https://github.com/benjamin3344/DGP-SPR.

Defect-Induced Secondary Crystals Drive Two-Dimensional to Three-Dimensional Morphological Evolution in the Co-Self-Assembly of Polyferrocenylsilane Block Copolymer and Homopolymer.

Bottom-up fabrication protocols for uniform 3D hierarchical structures in solution are rare. We report two different approaches to fabricate uniform 3D spherulites and their precursors using mixtures of poly(ferrocenyldimethylsilane) (PFS) block copolymer (BCP) and PFS homopolymer (HP). Both protocols are designed to promote defects in 2D assemblies that serve as intermediate structures. In a multistep seeded growth protocol, we add the BCP/HP mixture to (1D) rod-like PFS micelles in a selective solvent as first-generation seeds. This leads to 2D platelet structures. If this step is conducted at a high supersaturation, secondary crystals form on the basal surface of these platelets. Co-crystallization and rapid crystallization of BCP/HP promote the formation of defects that act as nucleation sites for secondary crystals, resulting in multilayer platelets. This is the key step. The multilayer platelets serve as second-generation seeds upon subsequent addition of BCP/HP blends and, with increasing supersaturation, lead to the sequential formation of uniform (3D) hedrites, sheaves, and spherulites. Similar structures can also be obtained by a simple one-pot direct self-assembly (heating-cooling-aging) protocol of PFS BCP/HP blends. In this case, for a carefully chosen but narrow temperature range, PFS HPs nucleate formation of uniform structures, and the annealing temperature regulates the supersaturation level. In both protocols, the competitive crystallization kinetics of HP/BCP affects the morphology. Both protocols exhibit broad generality. We believe the morphological transformation from 2D to 3D structures, regulated by defect formation, co-crystallization, and supersaturation levels, could apply to various semicrystalline polymers. Moreover, the 3D structures are sufficiently robust to serve as recoverable carriers for nanoparticle catalysts, exhibiting valuable catalytic activity and opening new possibilities for applications requiring exquisite 3D structures.

Polyferrocenylsilane Block Copolymer Spherulites in Dilute Solution.

Self-assembly of block copolymers (BCP) into uniform 3D structures in solution is an extremely rare phenomenon. Furthermore, the investigation of general prerequisites for fabricating a specific uniform 3D structure remains unknown and challenging. Here, through a simple one-pot direct self-assembly (heating and cooling) protocol, we show that uniform spherulite-like structures and their precursors can be prepared with various poly(ferrocenyldimethylsilane) (PFS) BCPs in a variety of polar and non-polar solvents. These structures all evolve from elongated lamellae into hedrites, sheaf-like micelles, and finally spherulites as the annealing temperature and supersaturation degree are increased. The key feature leading to this growth trajectory is the formation of secondary crystals by self-nucleation on the surface of early-elongated lamellae. We identified general prerequisites for fabricating PFS BCP spherulites in solution. These include corona/PFS core block ratios in the range of 1-5.5 that favor the formation of 2D structures as well as the development of secondary crystals on the basal faces of platelets at early stages of the self-assembly. The one-pot direct self-assembly provides a general protocol to form uniform spherulites and their precursors consisting of PFS BCPs that match these prerequisites. In addition, we show that manipulation of various steps in the direct self-assembly protocol can regulate the size and shape of the structures formed. These general concepts show promise for the fabrication and optimization of spherulites and their precursors from semicrystalline BCPs with interesting optical, electronic, or biomedical properties using the one-pot direct self-assembly protocol.

Uniform 1D Micelles and Patchy & Block Comicelles via Scalable, One-Step Crystallization-Driven Block Copolymer Self-Assembly.

Fiber-like (1D) core-crystalline micelles of uniform length can be obtained in protocols involving multiple steps from block copolymers (BCPs) in which crystallization of the core-forming polymer drives the self-assembly. Here we report a systematic study that shows that adding small amounts (<5 w/w%) of a homopolymer corresponding to the core-forming block of the BCP enables uniform 1D micelles (mean lengths Ln = 0.6 to 9.7 µm) to be obtained in a single step, simply by heating the mixture in a selective solvent followed by slow cooling. A series of poly(ferrocenyldimethylsilane) (PFS) BCPs with different corona-forming blocks and different compositions as well as PFS homopolymers of different lengths were examined. Dye labeling and confocal fluorescence microscopy showed that the homopolymer ends up in the center of the micelle, signaling that it served as the initial seed for epitaxial micelle growth. The rate of unimer addition was strongly enhanced by the length of the PFS block, and this enabled more complex structures to be formed in one-pot self-assembly experiments from mixtures of two or three BCPs with different PFS block lengths. Furthermore, BCP mixtures that included PFS-b-PI (PI = polyisoprene) and PFS-b-PDMS with similar PFS block lengths resulted in simultaneous addition to growing micelles, resulting in a patchy block that could be visualized by staining the vinyl groups of the PI with Pt nanoparticles. This approach also enabled scale up, so that uniform 1D micelles of controlled architecture can be obtained at concentrations of 10 w/w % solids or more.

Spherulite-Like Micelles.

One-dimensional (1D) and 2D structures by crystallization-driven self-assembly of block copolymers (BCPs) can form fascinating hierarchical structures through secondary self-assembly. But examples of 3D structures formed via hierarchical self-assembly are rare. Here we report seeded growth experiments in decane of a poly(ferrocenyldimethylsilane) BCP with an amphiphilic corona forming block in which lenticular platelets grow into classic spherulite-like uniform colloidally stable structures. These 3D objects are spherically symmetric on the exterior, but asymmetric near the core, where there is a more open structure consisting of sheaf-like leaves. The most remarkable aspect of these experiments is that growth stops at different stages of growth process, depending upon how much unimer is added in the seeded growth step. The system provides a model for studying spherulitic growth where real-time observations on their growth at different stages remains challenging.

Conversion of Metal-Organic Cage to Ligand-Free Ultrasmall Noble Metal Nanocluster Catalysts Confined within Mesoporous Silica Nanoparticle Supports.

Supported ultrasmall noble metal nanocluster-based (UNMN-based) catalysts are one of the most important classes of solid materials for heterogeneous catalysis. In this work, we present a novel strategy for the controlled synthesis of ligand-free UNMN nanocatalysts based on in situ reduction of a palladium-based (Pd-based) metal-organic cage (MOC) confined within monosized, thiol-modified mesoporous silica nanoparticle (MSN) supports. By taking advantage of the high mutual solubility of MOCs and MSNs in DMSO and the strong interactions between the thiol-modified MSN pore wall and MOC surface, a good dispersion of MOC molecules was achieved throughout the MSN support. The close correspondence of the MSN pore diameter (ca. 5.0 nm) with the diameter of the MOC (ca. 4.0 nm) confines MOC packing to approximately a monolayer. Based on this spatial constraint and electrostatic binding of the MOC to the thiol-modified MSN pore surface, in situ MOC reduction followed by metal atom diffusion, coalescence, and anchoring on the active sites resulted in ligand-free Pd-based UNMNs of approximately 0.9 ± 0.2 nm in diameter decorating the MSN pore surfaces. Control experiments of the reduction of a conventional palladium source or the reduction of free, unconstrained cages in solution under the same conditions only produced large metal nanocrystals (NP, >2 nm), confirming the importance of confined reduction to achieve a highly catalytically active surface. In light of this strategy, two catalytic experiments including the reaction of 4-nitrophenol to 4-aminophenol and the Suzuki C-C coupling reaction show superior catalytic activity of the engineered MSN-supported UNMN nanocatalysts compared to their free form and state of the art commercial catalysts. We believe that our new strategy will open new avenues for artificially designed UNMN-inspired nanoarchitectures for wide applications.

Patchy Nanofibers from the Thin Film Self-Assembly of a Conjugated Diblock Copolymer.

An unexpected morphology comprising patchy nanofibers can be accessed from the self-assembly of an all-conjugated, polyselenophene-block-polythiophene copolymer. This morphology consists of very small (<10 nm), polythiophene- and polyselenophene-rich domains and is unprecedented for both conjugated polymers and diblock copolymers in general. We propose that the patchy morphology occurs from the enhanced miscibility of the blocks arising from the longer alkyl chains in comparison to similar block copolymers with shorter alkyl chains, which fully phase separate, as well as the difference in rigidity between the polythiophene and polyselenophene blocks. This work demonstrates a facile way to tune the self-assembly behavior of conjugated block copolymers by modification of the side chain substituents.

In situ TEM tensile testing of carbon-linked graphene oxide nanosheets using a MEMS device.

This paper reports in situ transmission electron microscopy (TEM) tensile testing of carbon-linked graphene oxide nanosheets using a monolithic TEM compatible microelectromechanical system device. The set-up allows direct on-chip nanosheet thickness mapping, high resolution electron beam linking of a pre-fractured nanosheet, and mechanical tensile testing of the nanosheet. This technique enables simultaneous mechanical and high energy electron beam characterization of 2D nanomaterials.

Strengthening in Graphene Oxide Nanosheets: Bridging the Gap between Interplanar and Intraplanar Fracture.

Graphene oxide (GO) is a layered material comprised of hierarchical features which possess vastly differing characteristic dimensions. GO nanosheets represent the critical hierarchical structure which bridges the length-scale of monolayer and bulk material architectures. In this study, the strength and fracture behavior of GO nanosheets were examined. Under uniaxial loading, the tensile strength of the nanosheets was measured to be as high as 12 ± 4 GPa, which approaches the intrinsic strength of monolayer GO and is orders of magnitude higher than that of bulk GO materials. During mechanical failure, brittle fracture was observed in a highly localized region through the cross-section of the nanosheets without interlayer pull-out. This transition in the failure behavior from interplanar fracture, common for bulk GO, to intraplanar fracture, which dominates failure in monolayer GO, is responsible for the high strength measured in the nanosheets. Molecular dynamics simulations indicate that the elastic release from the propagation of intraplanar cracks initiates global fracture due to interlayer load transmission through hydrogen bond networks within the gallery space of the GO nanosheets. Furthermore, the GO nanosheet strength and stiffness were found to be strongly correlated to the effective volume and thickness of the samples, respectively. These findings help to bridge the understanding of the mechanical behavior of hierarchical GO materials and will ultimately guide the application of this intermediate scale material.

Formation of Soluble Mercury Oxide Coatings: Transformation of Elemental Mercury in Soils.

The impact of mercury (Hg) on human and ecological health has been known for decades. Although a treaty signed in 2013 by 147 nations regulates future large-scale mercury emissions, legacy Hg contamination exists worldwide and small-scale releases will continue. The fate of elemental mercury, Hg(0), lost to the subsurface and its potential chemical transformation that can lead to changes in speciation and mobility are poorly understood. Here, we show that Hg(0) beads interact with soil or manganese oxide solids and X-ray spectroscopic analysis indicates that the soluble mercury coatings are HgO. Dissolution studies show that, after reacting with a composite soil, >20 times more Hg is released into water from the coated beads than from a pure liquid mercury bead. An even larger, >700 times, release occurs from coated Hg(0) beads that have been reacted with manganese oxide, suggesting that manganese oxides are involved in the transformation of the Hg(0) beads and creation of the soluble mercury coatings. Although the coatings may inhibit Hg(0) evaporation, the high solubility of the coatings can enhance Hg(II) migration away from the Hg(0)-spill site and result in potential changes in mercury speciation in the soil and increased mercury mobility.

Rational defect introduction in silicon nanowires.

The controlled introduction of planar defects, particularly twin boundaries and stacking faults, in group IV nanowires remains challenging despite the prevalence of these structural features in other nanowire systems (e.g., II-VI and III-V). Here we demonstrate how user-programmable changes to precursor pressure and growth temperature can rationally generate both transverse twin boundaries and angled stacking faults during the growth of <111> oriented Si nanowires. We leverage this new capability to demonstrate prototype defect superstructures. These findings yield important insight into the mechanism of defect generation in semiconductor nanowires and suggest new routes to engineer the properties of this ubiquitous semiconductor.

In-situ TEM study from the perspective of holders.

During the in situ transmission electron microscopy (TEM) observations, the diverse functionalities of different specimen holders play a crucial role. We hereby provide a comprehensive overview of the main types of holders, associated technologies and case studies pertaining to the widely employed heating and gas heating methods, from their initial developments to the latest advancement. In addition to the conventional approaches, we also discuss the emergence of holders that incorporate a micro-electro-mechanical system (MEMS) chip for in situ observations. The MEMS technology offers a multitude of functions within a single chip, thereby enhancing the capabilities and versatility of the holders. MEMS chips have been utilized in environmental-cell designs, enabling customized fabrication of diverse shapes. This innovation has facilitated their application in conducting in situ observations within gas and liquid environments, particularly in the investigation of catalytic and battery reactions. We summarize recent noteworthy studies conducted using in situ liquid TEM. These studies highlight significant advancements and provide valuable insights into the utilization of MEMS chips in environmental-cells, as well as the expanding capabilities of in situ liquid TEM in various research domains.

Simultaneous secondary electron microscopy in the scanning transmission electron microscope with applications for in situ studies.

Scanning/transmission electron microscopy (STEM) is a powerful characterization tool for a wide range of materials. Over the years, STEMs have been extensively used for in situ studies of structural evolution and dynamic processes. A limited number of STEM instruments are equipped with a secondary electron (SE) detector in addition to the conventional transmitted electron detectors, i.e. the bright-field (BF) and annular dark-field (ADF) detectors. Such instruments are capable of simultaneous BF-STEM, ADF-STEM and SE-STEM imaging. These methods can reveal the 'bulk' information from BF and ADF signals and the surface information from SE signals for materials <200 nm thick. This review first summarizes the field of in situ STEM research, followed by the generation of SE signals, SE-STEM instrumentation and applications of SE-STEM analysis. Combining with various in situ heating, gas reaction and mechanical testing stages based on microelectromechanical systems (MEMS), we show that simultaneous SE-STEM imaging has found applications in studying the dynamics and transient phenomena of surface reconstructions, exsolution of catalysts, lunar and planetary materials and mechanical properties of 2D thin films. Finally, we provide an outlook on the potential advancements in SE-STEM from the perspective of sample-related factors, instrument-related factors and data acquisition and processing.

Peltier cooling for the reduction of carbon contamination in scanning electron microscopy.

We used a novel Peltier anticontamination device (PAC) to reduce carbon contamination upon electron beam irradiation in scanning electron microscopy through a reduction of hydrocarbon molecules in the specimen chamber. Unlike liquid-nitrogen based cold traps, the PAC operates free of user maintenance and is suitable for lengthy imaging sessions without degradation of the anticontamination performance. Its performance as an alternative cold trap method provides considerable reduction of electron beam-assisted carbon build-up. We compared the thickness of carbon contamination deposited upon prolonged electron beam scans with the PAC system on and off. Topographical structures of the carbon build-up were characterized using atomic force microscopy. We report that under identical beam parameters, thickness of the carbon contamination was reduced by over 79 % for area scans (1.2 × 1.2 µm2), and by two orders of magnitude for stationary point scans when the PAC cooling mode is engaged.

Carbohydrate-derived hydrothermal carbons: a thorough characterization study.

Hydrothermal carbonization (HTC) is an aqueous-phase route to produce carbon materials using biomass or biomass-derived precursors. In this paper, a comprehensive physicochemical and textural characterization of HTC materials obtained using four different precursors, namely, xylose, glucose, sucrose, and starch, is presented. The development of porosity in the prepared HTC materials as a function of thermal treatment (under an inert atmosphere) was specifically monitored using N(2) and CO(2) sorption analysis. The events taking place during the thermal treatment process were studied by a combined thermogravimetric/infrared (TGA-IR) measurement. Interestingly, these inexpensive biomass-derived carbon materials show good selectivity for CO(2) adsorption over N(2) (CO(2)/N(2) selectivity of 20 at 273 K, 1 bar and 1:1 gas composition). Furthermore, the elemental composition, morphologies, degree of structural order, surface charge, and functional groups are also investigated.

Direct assembly of hydrophobic nanoparticles to multifunctional structures.

We present a general process that allows convenient production of multifunctional composite particles by direct self-assembly of hydrophobic nanoparticles on host nanostructures containing high-density surface thiol groups. Hydrophobic nanoparticles of various compositions and combinations can be directly assembled onto the host surface through the strong coordination interactions between metal cations and thiol groups. The resulting structures can be further conveniently overcoated with a layer of normal silica to stabilize the assemblies and render them highly dispersible in water for biomedical applications. As the entire fabrication process does not involve complicated surface modification procedures, the hydrophobic ligands on the nanoparticles are not disturbed significantly so that they retain their original properties such as highly efficient luminescence. Many complex composite nanostructures with tailored functions can be efficiently produced by using this versatile approach. For example, multifunctional nonspherical nanostructures can be efficiently produced by using mercapto-silica coated nano-objects of arbitrary shapes as hosts for immobilizing functional nanoparticles. Multilayer structures can also be achieved by repeating the mercapto-silica coating and nanoparticle immobilization processes. Such assembly approach will provide the research community a highly versatile, configurable, scalable, and reproducible process for the preparation of various multifunctional structures.

The role of cooling rate in crystallization-driven block copolymer self-assembly.

Self-assembly of crystalline-coil block copolymers (BCPs) in selective solvents is often carried out by heating the mixture until the sample appears to dissolve and then allowing the solution to cool back to room temperature. In self-seeding experiments, some crystallites persist during sample annealing and nucleate the growth of core-crystalline micelles upon cooling. There is evidence in the literature that the nature of the self-assembled structures formed is independent of the annealing time at a particular temperature. There are, however, no systematic studies of how the rate of cooling affects self-assembly. We examine three systems based upon poly(ferrocenyldimethylsilane) BCPs that generated uniform micelles under typical conditions where cooling took pace on the 1-2 h time scale. For example, several of the systems generated elongated 1D micelles of uniform length under these slow cooling conditions. When subjected to rapid cooling (on the time scale of a few minutes or faster), branched structures were obtained. Variation of the cooling rate led to a variation in the size and degree of branching of some of the structures examined. These changes can be explained in terms of the high degree of supersaturation that occurs when unimer solutions at high temperature are suddenly cooled. Enhanced nucleation, seed aggregation, and selective growth of the species of lowest solubility contribute to branching. Cooling rate becomes another tool for manipulating crystallization-driven self-assembly and controlling micelle morphologies.

Microstructure-Dependent Thermal Stability of Super-Tetragonal Nanocomposite Films through In Situ TEM/EELS Study.

Smart microstructure design in nanocomposite films allows us to tailor physical properties such as ferroelectricity and thermal stability to broaden applications of next-generation electronic devices. Here, we study the thermal stability of self-assembled PbTiO3 (PTO)/PbO nanocomposite films with nano-spherical and nanocolumnar microstructures by utilizing an environmental transmission electron microscopy (TEM) combined with electron energy loss spectroscopy (EELS). The real-time study reveals that the microstructure-dependent interphase strain has an effect on the stabilization of the tetragonal phase. Compared to the nano-spherical configuration, the nanocomposite film with the nanocolumnar microstructure can maintain the giant tetragonality of ∼1.20 up to 450 °C, and the tetragonal phase is predicted to be stable at elevated temperatures > 600 °C. Moreover, the temperature-dependent EELS further demonstrates the sensitivity of the chemical bonding of Pb and Ti with O to the PTO lattice distortion, correlating the structural variation and electronic properties at different temperatures. Such in situ heating TEM study provides insights into the thermal stability of nanocomposites with different microstructures and facilitates the advancement of power electronics applications in harsh environments.

Automatic quantification and classification of microplastics in scanning electron micrographs via deep learning.

Microplastics quantification and classification are demanding jobs to monitor microplastic pollution and evaluate the potential health risks. In this paper, microplastics from daily supplies in diverse chemical compositions and shapes are imaged by scanning electron microscopy. It offers a greater depth and finer details of microplastics at a wider range of magnification than visible light microscopy or a digital camera, and permits further chemical composition analysis. However, it is labour-intensive to manually extract microplastics from micrographs, especially for small particles and thin fibres. A deep learning approach facilitates microplastics quantification and classification with a manually annotated dataset including 237 micrographs of microplastic particles (fragments or beads) in the range of 50 µm-1 mm and fibres with diameters around 10 µm. For microplastics quantification, two deep learning models (U-Net and MultiResUNet) were implemented for semantic segmentation. Both significantly outmatched conventional computer vision techniques and achieved a high average Jaccard index over 0.75. Especially, U-Net was combined with object-aware pixel embedding to perform instance segmentation on densely packed and tangled fibres for further quantification. For shape classification, a fine-tuned VGG16 neural network classifies microplastics based on their shapes with high accuracy of 98.33%. With trained models, it takes only seconds to segment and classify a new micrograph in high accuracy, which is remarkably cheaper and faster than manual labour. The growing datasets may benefit the identification and quantification of microplastics in environmental samples in future work.

New black indium oxide-tandem photothermal CO2-H2 methanol selective catalyst.

It has long been known that the thermal catalyst Cu/ZnO/Al2O3(CZA) can enable remarkable catalytic performance towards CO2 hydrogenation for the reverse water-gas shift (RWGS) and methanol synthesis reactions. However, owing to the direct competition between these reactions, high pressure and high hydrogen concentration (≥75%) are required to shift the thermodynamic equilibrium towards methanol synthesis. Herein, a new black indium oxide with photothermal catalytic activity is successfully prepared, and it facilitates a tandem synthesis of methanol at a low hydrogen concentration (50%) and ambient pressure by directly using by-product CO as feedstock. The methanol selectivities achieve 33.24% and 49.23% at low and high hydrogen concentrations, respectively.