Liquid Transmission Electron Microscopy for Probing Collagen Biomineralization.

Collagen biomineralization is fundamental to hard tissue assembly. While studied extensively, collagen mineralization processes are not fully understood, with the majority of theories derived from electron microscopy (EM) under static, dehydrated, or frozen conditions, unlike the liquid phase environment where mineralization occurs. Herein, novel liquid transmission EM (TEM) strategies are presented, in which collagen mineralization was explored in liquid for the first time via TEM. Custom thin-film enclosures were employed to visualize the mineralization of reconstituted collagen fibrils in a calcium phosphate and polyaspartic acid solution to promote intrafibrillar mineralization. TEM highlighted that at early time points precursor mineral particles attached to collagen and progressed to crystalline mineral platelets aligned with fibrils at later time points. This aligns with observations from other techniques and validates the liquid TEM approach. This work provides a new liquid imaging approach for exploring collagen biomineralization, advancing toward understanding disease pathogenesis and remineralization strategies for hard tissues.

Stabilization of Dinuclear Rhodium and Iridium Clusters on Layered Titanate and Niobate Supports.

Atomically dispersed organometallic clusters can provide well-defined nuclearity of active sites for both fundamental studies as well as new regimes of activity and selectivity in chemical transformations. More recently, dinuclear clusters adsorbed onto solid surfaces have shown novel catalytic properties resulting from the synergistic effect of two metal centers to anchor different reactant species. Difficulty in synthesizing, stabilizing, and characterizing isolated atoms and clusters without agglomeration challenges allocating catalytic performance to atomic structure. Here, we explore the stability of dinuclear rhodium and iridium clusters adsorbed onto layered titanate and niobate supports using molecular precursors. Both systems maintain their nuclearity when characterized using aberration-corrected high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM). Statistical analysis of HAADF-STEM images revealed that rhodium and iridium dimers had mean cluster-to-cluster distances very similar to what is expected from a random distribution of atoms over a large area, indicating that they are dispersed without aggregation. The stability of dinuclear rhodium clusters supported on titanate nanosheets was also investigated by X-ray absorption fine structure (EXAFS), DRIFTS, and first-principles calculations. Both X-ray absorption spectroscopy and HAADF-STEM simulations, guided by density functional theory (DFT)-optimized structure models, suggested that rhodium dimers adsorb onto the nanosheets in an end-on binding mode that is stable up to 100 °C under reducing conditions. This study highlights that crystalline nanosheets derived from layered metal oxides can be used as model supports to selectively stabilize dinuclear clusters, which could have implications for heterogeneous catalysis.

Automated Tools to Advance High-Resolution Imaging in Liquid.

Liquid-electron microscopy (EM), the room-temperature correlate to cryo-EM, is a rapidly growing field providing high-resolution insights of macromolecules in solution. Here, we describe how liquid-EM experiments can incorporate automated tools to propel the field to new heights. We demonstrate fresh workflows for specimen preparation, data collection, and computing processes to assess biological structures in liquid. Adeno-associated virus (AAV) and the SARS-CoV-2 nucleocapsid (N) were used as model systems to highlight the technical advances. These complexes were selected based on their major differences in size and natural symmetry. AAV is a highly symmetric, icosahedral assembly with a particle diameter of ~25 nm. At the other end of the spectrum, N protein is an asymmetric monomer or dimer with dimensions of approximately 5­7 nm, depending upon its oligomerization state. Equally important, both AAV and N protein are popular subjects in biomedical research due to their high value in vaccine development and therapeutic efforts against COVID-19. Overall, we demonstrate how automated practices in liquid-EM can be used to decode molecules of interest for human health and disease.

Nanoarchitecture through Strained Molecules: Cubane-Derived Scaffolds and the Smallest Carbon Nanothreads.

Relative to the rich library of small-molecule organics, few examples of ordered extended (i.e., nonmolecular) hydrocarbon networks are known. In particular, sp3 bonded, diamond-like materials represent appealing targets because of their desirable mechanical, thermal, and optical properties. While many covalent organic frameworks (COFs)-extended, covalently bonded, and porous structures-have been realized through molecular architecture with exceptional control, the design and synthesis of dense, covalent extended solids has been a longstanding challenge. Here we report the preparation of a sp3-bonded, low-dimensional hydrocarbon synthesized via high-pressure, solid-state diradical polymerization of cubane (C8H8), which is a saturated, but immensely strained, cage-like molecule. Experimental measurements show that the obtained product is crystalline with three-dimensional order that appears to largely preserve the basic structural topology of the cubane molecular precursor and exhibits high hardness (comparable to fused quartz) and thermal stability up to 300 °C. Among the plausible theoretical candidate structures, one-dimensional carbon scaffolds comprising six- and four-membered rings that pack within a pseudosquare lattice provide the best agreement with experimental data. These diamond-like molecular rods with extraordinarily small thickness are among the smallest members in the carbon nanothread family, and calculations indicate one of the stiffest one-dimensional systems known. These results present opportunities for the synthesis of purely sp3-bonded extended solids formed through the strain release of saturated molecules, as opposed to only unsaturated precursors.

Polymer-inorganic solid-electrolyte interphase for stable lithium metal batteries under lean electrolyte conditions.

The solid-electrolyte interphase (SEI) is pivotal in stabilizing lithium metal anodes for rechargeable batteries. However, the SEI is constantly reforming and consuming electrolyte with cycling. The rational design of a stable SEI is plagued by the failure to control its structure and stability. Here we report a molecular-level SEI design using a reactive polymer composite, which effectively suppresses electrolyte consumption in the formation and maintenance of the SEI. The SEI layer consists of a polymeric lithium salt, lithium fluoride nanoparticles and graphene oxide sheets, as evidenced by cryo-transmission electron microscopy, atomic force microscopy and surface-sensitive spectroscopies. This structure is different from that of a conventional electrolyte-derived SEI and has excellent passivation properties, homogeneity and mechanical strength. The use of the polymer-inorganic SEI enables high-efficiency Li deposition and stable cycling of 4 V Li|LiNi0.5Co0.2Mn0.3O2 cells under lean electrolyte, limited Li excess and high capacity conditions. The same approach was also applied to design stable SEI layers for sodium and zinc anodes.

CCK Receptor Inhibition Reduces Pancreatic Tumor Fibrosis and Promotes Nanoparticle Delivery.

The poor prognosis for pancreatic ductal adenocarcinoma (PDAC) patients is due in part to the highly fibrotic nature of the tumors that impedes delivery of therapeutics, including nanoparticles (NPs). Our prior studies demonstrated that proglumide, a cholecystokinin receptor (CCKR) antagonist, reduced fibrosis pervading PanIN lesions in mice. Here, we further detail how the reduced fibrosis elicited by proglumide achieves the normalization of the desmoplastic tumor microenvironment (TME) and improves nanoparticle uptake. One week following the orthotopic injection of PDAC cells, mice were randomized to normal or proglumide-treated water for 3-6 weeks. Tumors were analyzed ex vivo for fibrosis, vascularity, stellate cell activation, vascular patency, and nanoparticle distribution. The histological staining and three-dimensional imaging of tumors each indicated a reduction in stromal collagen in proglumide-treated mice. Proglumide treatment increased tumor vascularity and decreased the activation of cancer-associated fibroblasts (CAFs). Additionally, PANC-1 cells with the shRNA-mediated knockdown of the CCK2 receptor showed an even greater reduction in collagen, indicating the CCK2 receptors on tumor cells contribute to the desmoplastic TME. Proglumide-mediated reduction in fibrosis also led to functional changes in the TME as evidenced by the enhanced intra-tumoral distribution of small (<12 nm) Rhodamine-loaded nanoparticles. The documented in vivo, tumor cell-intrinsic anti-fibrotic effects of CCK2R blockade in both an immunocompetent syngeneic murine PDAC model as well as a human PDAC xenograft model demonstrates that CCK2R antagonists, such as proglumide, can improve the delivery of nano-encapsulated therapeutics or imaging agents to pancreatic tumors.

Non-lytic spread of poliovirus requires the nonstructural protein 3CD.

Non-enveloped viruses like poliovirus (PV) have evolved the capacity to spread by non-lytic mechanisms. For PV, this mechanism exploits the host secretory autophagy pathway. Virions are selectively incorporated into autophagosomes, double-membrane vesicles that travel to the plasma membrane, fuse, and release single-membrane vesicles containing virions. Loading of cellular cargo into autophagosomes relies on direct or indirect interactions with microtubule-associated protein 1B-light chain 3 (LC3) that are mediated by motifs referred to as LC3-interaction regions (LIRs). We have identified a PV mutant with a severe defect in non-lytic spread. An F-to-Y substitution in a putative LIR of the nonstructural protein 3CD prevented virion incorporation into LC3-positive autophagosomes and virion trafficking to the plasma membrane for release. Using high-angle annular dark-field scanning transmission electron microscopy to monitor PV-induced autophagosome biogenesis, for the first time, we show that virus-induced autophagic signals yield normal autophagosomes, even in the absence of virions. The F-to-Y derivative of PV 3CD was unable to support normal autophagosome biogenesis. Together, these studies make a compelling case for a direct role of a viral nonstructural protein in the formation and loading of the vesicular carriers used for non-lytic spread that may depend on the proper structure, accessibility, and/or dynamics of its LIR. The studies of PV 3CD protein reported here will hopefully provoke a more deliberate look at the presence and function of LIR motifs in viral proteins of viruses known to use autophagy as the basis for non-lytic spread.

3D structure determination of native mammalian cells using cryo-FIB and cryo-electron tomography.

Cryo-electron tomography (cryo-ET) has enabled high resolution three-dimensional(3D) structural analysis of virus and host cell interactions and many cell signaling events; these studies, however, have largely been limited to very thin, peripheral regions of eukaryotic cells or to small prokaryotic cells. Recent efforts to make thin, vitreous sections using cryo-ultramicrotomy have been successful, however,this method is technically very challenging and with many artifacts. Here, we report a simple and robust method for creating in situ, frozen-hydrated cell lamellas using a focused ion beam at cryogenic temperature (cryo-FIB), allowing access to any interior cellular regions of interest. We demonstrate the utility of cryo-FIB with high resolution 3D cellular structures from both bacterial cells and large mammalian cells. The method will not only facilitate high-throughput 3D structural analysis of biological specimens, but is also broadly applicable to sample preparation of thin films and surface materials without the need for FIB "lift-out".

Heterostructures of Cu2-xS/Cu2-xTe plasmonic semiconductors: disappearing and reappearing LSPR with anion exchange.

Localized surface plasmon resonance (LSPR) of Cu2-xS nanorods is quenched during the initial Cu2-xS/Cu2-xTe core/shell stage of anion exchange then returns as Cu2-xTe progresses into the nanorod. Phase change within the core accounts for this behaviour illustrating the complexity emergent from anion exchange.

Liquid-EM goes viral - visualizing structure and dynamics.

Liquid-electron microscopy (EM), the room temperature correlate to cryo-EM, is an exciting new technique delivering real-time data of dynamic reactions in solution. Here, we explain how liquid-EM gained popularity in recent years by examining key experiments conducted on viral assemblies and host-pathogen interactions. We describe developing workflows for specimen preparation, data collection, and computing processes that led to the first high-resolution virus structures in a liquid environment. Equally important, we review why liquid-electron tomography may become the next big thing in biomedical research due to its ability to monitor live viruses entering cells within seconds. Taken together, we pose the idea that liquid-EM can serve as a dynamic complement to current cryo-EM methods, inspiring the "real-time revolution" in nanoscale imaging.

Shear Modes in a 2D Polar Metal.

Low-frequency shear and breathing modes are important Raman signatures of two-dimensional (2D) materials, providing information on the number of layers and insights into interlayer interactions. We elucidate the nature of low-frequency modes in a 2D polar metal-2D Ga covalently bonded to a SiC substrate, using a first-principles Green's function-based approach. The low-frequency Raman modes are dominated by surface resonance modes, consisting primarily of out-of-phase shear modes in Ga, coupled to SiC phonons. Breathing modes are strongly coupled to the substrate and do not give rise to peaks in the phonon spectra. The highest-frequency shear mode blue-shifts significantly with increasing thickness, reflecting both an increase in the number of Ga layers and an increase in the effective interlayer force constant. The surface resonance modes evolve into localized 2D Ga modes as the phonon momentum increases. The predicted low-frequency modes are consistent with Raman measurements on 2D polar Ga.

Advancing High-Resolution Imaging of Virus Assemblies in Liquid and Ice.

Interest in liquid-electron microscopy (liquid-EM) has skyrocketed in recent years as scientists can now observe real-time processes at the nanoscale. It is extremely desirable to pair high-resolution cryo-EM information with dynamic observations as many events occur at rapid timescales - in the millisecond range or faster. Improved knowledge of flexible structures can also assist in the design of novel reagents to combat emerging pathogens, such as SARS-CoV-2. More importantly, viewing biological materials in a fluid environment provides a unique glimpse of their performance in the human body. Presented here are newly developed methods to investigate the nanoscale properties of virus assemblies in liquid and vitreous ice. To accomplish this goal, well-defined samples were used as model systems. Side-by-side comparisons of sample preparation methods and representative structural information are presented. Sub-nanometer features are shown for structures resolved in the range of ~3.5-Å-10 Å. Other recent results that support this complementary framework include dynamic insights of vaccine candidates and antibody-based therapies imaged in liquid. Overall, these correlative applications advance our ability to visualize molecular dynamics, providing a unique context for their use in human health and disease.

Fabrication of discrete gallium nanoislands on the surface of a Si(001) substrate using a focused ion beam.

A gallium focused ion beam (FIB) has been used to implant Ga at specific sites on the surface of undoped Si(001) substrates. Upon annealing at 600 °C, discrete nanoscale surface islands form within the FIB patterned regions when the total Ga ion dose, or fluence, is greater than 1.0 × 10(16) ions cm( - 2). The number of islands depends on the size of the irradiated region and a single island can be achieved for a FIB milled region that is 100 nm × 100 nm. The average sizes of the islands were found to range from 24.5 nm when exposed to a total ion dose of 1.2 × 10(16) ions cm( - 2) to 45 nm for a dose of 3.0 × 10(16) ions cm( - 2). We have confirmed that these surface islands are metallic Ga by performing a selective chemical etch that removes the islands and by transmission electron microscopy characterization. These patterned Ga surface templates could serve as nucleation sites for the lateral arrangement of discrete quantum dot structures.

High-Resolution Imaging of Human Viruses in Liquid Droplets.

Liquid-phase electron microscopy (LP-EM) is an exciting new area in the materials imaging field, providing unprecedented views of molecular processes. Time-resolved insights from LP-EM studies are a strong complement to the remarkable results achievable with other high-resolution techniques. Here, the opportunities to expand LP-EM technology beyond 2D temporal assessments and into the 3D regime are described. The results show new structures and dynamic insights of human viruses contained in minute volumes of liquid while acquired in a rapid timeframe. To develop this strategy, adeno-associated virus (AAV) is used as a model system. AAV is a well-known gene therapy vehicle with current applications involving drug delivery and vaccine development for COVID-19. Improving the understanding of the physical properties of biological entities in a liquid state, as maintained in the human body, has broad societal implications for human health and disease.

Pulsed-Laser Deposited 35 Bi(Mg1/2Ti1/2) O3-65 PbTiO3 Thin Films-Part I: Influence of Processing on Composition, Microstructure, and Ferroelectric Hysteresis.

35 Bi(Mg1/2Ti1/2)O3 - 65 PbTiO3 (35 BiMT-65 PT) is a potential candidate material for a high-temperature nonvolatile ferroelectric memory. For pulsed-laser deposited 35 BiMT-65 PT films with the perovskite structure, it was found that as the chamber pressure during deposition decreased, the Mg and Pb contents in as-deposited films drop, while the concentration of Bi increases. Concurrently with the change in composition, the remanent polarization $P_{r}$ increases 64% to $\approx 21~\mu \text{C}$ /cm2 and the polarization-electric field loops rotated counterclockwise as the deposition pressure increases. Decreasing the seed layer thickness from 36 to 16 nm led to a decrease in $P_{r}$ to $\approx 14~\mu \text{C}$ /cm2. Adjusting the target composition allowed the deposition of films which had near-stoichiometric Bi and Mg concentrations, but in all cases, the grown films were lead deficient. These films had remanent polarizations of 18- $20~\mu \text{C}$ /cm2. If the lead content of the target was increased too far, the remanent polarization decreased, possibly due to the need to evolve more PbO from defective growth layers. Finally, the deposition rate showed no substantial effect on the film composition, but did have a significant impact on the ferroelectric properties. As the deposition rate decreased, the $P_{r}$ increased to $\approx 22~\mu \text{C}$ /cm2 due to enhanced crystalline quality. At laser frequencies of 5 Hz, a Mg-rich pyrochlore phase begins to form and films showed a maximum $P_{r} \approx 22~\mu \text{C}$ /cm2. The processing-composition behavior is explained via preferential adsorption of Bi on the A-site, which results in lead vacancies.

High-Pressure Reactivity of Triptycene Probed by Raman Spectroscopy.

The high-pressure reactivity of caged olefinic carbons and polyatomic aromatic hydrocarbons (PAHs) are of interest because of their ability to produce unique C-H networks with varying geometries and bonding environments. Here, we have selected triptycene to explore the creation of pores via high-pressure polymerization. Triptycene has internal free volume on a molecular scale that arises due to its paddle wheel-like structure, formed via fusion of three benzene rings via sp3-hybridized bridgehead carbon sites. At 25 GPa and 298 K, triptycene polymerizes to yield an amorphous hydrogenated carbon, with FTIR indicating an sp3 C-H content of approximately 40%. Vibrational spectroscopy conclusively demonstrates that triptycene polymerizes via cycloaddition reactions at the aromatic sites via a ring opening mechanism. The bridgehead carbons remain intact after polymerization, indicating the rigid backbone of the triptycene precursor is retained in the polymer, as well as molecular-level (∼1-3 Å) internal free volume. High resolution transmission electron microscopy, combined with dark field imaging, indicates the presence of ∼10 nm voids in the polymer, which we attribute to either polymeric clustering or a hierarchical tertiary porous network. Creation of a polymerized network that retains internal voids via high-pressure polymerization is attributed to the presence and retention of the bridgehead carbons.

Colloidal ZnO and Zn(1-x)Co(x)O tetrapod nanocrystals with tunable arm lengths.

Tetrapod-shaped ZnO nanocrystals exhibit exceptional optoelectronic properties, including intense ultraviolet photoluminescence emission, that make them attractive for applications that include lasers, sensors, and photocatalysts. However, synthetic methods that produce ZnO tetrapods typically include high-temperature vapor-deposition approaches that do not readily achieve characteristic dimensions of less than 100 nm or colloidal methods that require added metal dopants, which modify the inherent properties of ZnO. Here, we report a robust, modified solution-phase synthetic protocol for generating colloidal ZnO tetrapods that does not require the use of metal dopants. The ZnO tetrapod arm lengths can be tuned from 10 to 25 nm by adjusting the amount of Zn reagent used in the reaction. Subsequent seeded-growth produced even larger colloidal ZnO tetrapods with 62 nm arms. Photoluminescence (PL) measurements confirm that the tetrapods are of high crystalline quality, and the ultraviolet PL emission wavelengths that are observed fall between those of previously reported metal-doped colloidal ZnO tetrapods, which exhibit dopant-induced red- or blue-shifts. Furthermore, the reaction strategy can be modified to produce cobalt-substituted ZnO, offering a chemical pathway to tetrapod-shaped Zn1-xCoxO nanocrystals.