Transmission Electron Microscopy Grids Research Articles

Liquid Crystal‐Based Detection of Antigens with ELISA Sensitivity

AbstractA highly sensitive label‐free liquid crystal (LC)‐based technique is presented for detecting Immunoglobulin G (IgG) antigens used to uncover viral infections. The effectiveness, sensitivity, and selectivity of this detection method is demonstrated with goat IgG antigen at concentrations as low as 100 pg ml−1, which is comparable to the sensitivity of the current enzyme‐linked immunosorbent assay (ELISA). The sensor is fabricated by decorating a transmission electron microscopy grid immobilized glass surfaces with antibodies; the target antigen is detected by a liquid crystal suspended onto the grid. This is different from previous methods where the antigen is detected either at the LC‐aqueous interface or in an LC sandwich cell with an antibody/antigen‐decorated substrate. This new approach has advantages such as easy sample preparation, higher sensitivity, and better storage capabilities. Binding the target antigen to the antibody results in a reorientation of the LC director that is detected optically. In addition to demonstrating the sensitivity, the physical principle of the detection is also discussed. This technique may apply to detect virtually any antigen of interest.

Blue hydrochars formed on hydrothermal carbonization of glucose using an iron catalyst

We hypothesized that the morphology of the hydrochar from hydrothermal carbonization of glucose would be affected by Fe2+; and indeed, with such ions, large pieces of hydrochar formed that comprised aggregated spherical particles and blue and thin films. Thin carbonized films formed at the bottom of the autoclave liners or on TeflonTM tape used as a template. Free-standing films could be prepared by stretching the TeflonTM tape after the synthesis. The carbonized films that formed at the bottom of the autoclave adhered to spherical hydrochar particles. The blueness was ascribed to thin-film interference under white-light irradiation and related to the film thickness, which was about 200 nm. Analysis of transmission electron microscopy (TEM) images showed that the films consisted of a layered amorphous carbon. The amorphous and thin films were more carbonized than the amorphous carbon of the TEM grid, as derived via electron energy loss spectroscopy (EELS). Additional analysis of one of the thin films by X-ray photoelectron spectral analysis showed a higher carbon fraction than for bulk hydrochar, supporting the EELS analysis. We believe that the synthesis of thin films of hydrochar can open up new colloidal processing pathways, which could be useful in the preparation of carbon-based materials and alike.

A non-destructive and efficient transfer method for preparing 2D materials samples for transmission electron microscopy study

Studying two-dimensional (2D) materials using transmission electron microscopy (TEM) is necessary and very important in many aspects. However, some 2D materials are not resistant to acids or alkalis, which are widely used in normal wet transfer techniques to transfer the exfoliated 2D nanosheets onto the TEM grids. On the other hand, dry stamping method can damage the holey carbon film on the TEM grids. In this article, we present a non-destructive, efficient, and widely applicable transfer method for preparing the TEM samples of the exfoliated 2D materials. Our method only uses the heat-release tape, PMMA, and blue Nitto tape. Neither acid nor alkali is involved in our method, therefore, impurities and damage can be avoided to the greatest extent. The method is also very efficient and can be accomplished in less than 30 min after the exfoliation of the 2D materials. This method is particularly useful for preparing the TEM samples of the 2D materials that are not resistant to acids and alkalis. The present method is also applicable to various 2D materials and various substrates.

Formation of styrene maleic acid lipid nanoparticles (SMALPs) using SMA thin film on a substrate

Despite the important role of membrane proteins in biological function and physiology, studying them remains challenging because of limited biomimetic systems for the protein to remain in its native membrane environment. Cryo electron microscopy (Cryo-EM) is emerging as a powerful tool for analyzing the structure of membrane proteins. However, Cryo-EM and other membrane protein analyses are better studied in a native lipid bilayer. Although traditional, mimetic systems have disadvantages that limit their use in the study of membrane proteins. As an alternative, styrene-maleic acid copolymers are used to form nanoparticles with POPC:POPG lipids. Traditional characterization of these styrene maleic acid lipid nanoparticles (SMALPs) includes dynamic light scattering (DLS), electron paramagnetic resonance (EPR), nuclear magnetic resonance (NMR), and transmission electron microscopy (TEM). In this study a new method was developed that utilizes SMALPs using a styrene-maleic acid copolymer (SMA) thin film on a TEM grid, acting as a substrate. By directly adding POPC:POPG lipid vesicles to the SMA coated grid SMALPs can be formed, visualized, and characterized by TEM without the need to make them in solution prior to imaging. We envision these functionalized grids could aid in single particle specimen preparation, increasing the efficiency of structural biology and biophysical techniques such as Cryo-EM.

Automation and Standardization-A Coupled Approach towards Reproducible Sample Preparation Protocols for Nanomaterial Analysis.

Whereas the characterization of nanomaterials using different analytical techniques is often highly automated and standardized, the sample preparation that precedes it causes a bottleneck in nanomaterial analysis as it is performed manually. Usually, this pretreatment depends on the skills and experience of the analysts. Furthermore, adequate reporting of the sample preparation is often missing. In this overview, some solutions for techniques widely used in nano-analytics to overcome this problem are discussed. Two examples of sample preparation optimization by automation are presented, which demonstrate that this approach is leading to increased analytical confidence. Our first example is motivated by the need to exclude human bias and focuses on the development of automation in sample introduction. To this end, a robotic system has been developed, which can prepare stable and homogeneous nanomaterial suspensions amenable to a variety of well-established analytical methods, such as dynamic light scattering (DLS), small-angle X-ray scattering (SAXS), field-flow fractionation (FFF) or single-particle inductively coupled mass spectrometry (sp-ICP-MS). Our second example addresses biological samples, such as cells exposed to nanomaterials, which are still challenging for reliable analysis. An air–liquid interface has been developed for the exposure of biological samples to nanomaterial-containing aerosols. The system exposes transmission electron microscopy (TEM) grids under reproducible conditions, whilst also allowing characterization of aerosol composition with mass spectrometry. Such an approach enables correlative measurements combining biological with physicochemical analysis. These case studies demonstrate that standardization and automation of sample preparation setups, combined with appropriate measurement processes and data reduction are crucial steps towards more reliable and reproducible data.

Wetting of surfaces decorated by gas-phase synthesized silver nanoparticles: Effects of Ag adatoms, nanoparticle aging, and surface mobility.

The wetting state of surfaces can be rendered to a highly hydrophobic state by the deposition of hydrophilic gas phase synthesized Ag nanoparticles (NPs). The aging of Ag NPs leads to an increase in their size, which is also associated with the presence of Ag adatoms on the surface between the NPs that have a strong effect on the wetting processes. Furthermore, surface airborne hydrocarbons were removed by UV-ozone treatment, providing deeper insight into the apparent mobility of the NPs on different surfaces and their subsequent ripening and aging. In addition, the UV-ozone treatment revealed the presence of adatoms during the magnetron sputtering process. This surface treatment lowers the initial contact angle of the substrates and facilitates the mobility of Ag NPs and adatoms on the surface of substrates. Adatoms co-deposited on clean high surface energy substrates will nucleate on Ag NPs that will remain closely spherical and preserve the pinning effect due to the water nanomeniscus. If the adatoms are co-deposited on a UV-ozone cleaned low surface energy substrate, their mobility is restricted, and they will nucleate in two-dimensional islands and/or nanoclusters on the surface instead of connecting to existing Ag NPs. This growth results in a rough surface without overhangs, where the wetting state is reversed from hydrophobic to hydrophilic. Finally, different material surfaces of transmission electron microscopy grids revealed strong differences in the sticking coefficient for the Ag NPs, suggesting another factor that can strongly affect their wetting properties.

Optimization of the In Situ Biasing FIB Sample Preparation for Hafnia-Based Ferroelectric Capacitor.

Hafnia-based ferroelectric (FE) thin films have received extensive attention in both academia and industry, benefitting from their outstanding scalability and excellent CMOS compatibility. Hafnia-based FE capacitors in particular have the potential to be used in dynamic random-access memory (DRAM) applications. Obtaining fine structure characterization at ultra-high spatial resolution is helpful for device performance optimization. Hence, sample preparation by the focused ion beam (FIB) system is an essential step, especially for in situ biasing experiments in a transmission electron microscope (TEM). In this work, we put forward three tips to improve the success rate of in situ biasing experiments: depositing a carbon protective layer to position the interface, welding the sample on the top of the Cu column of the TEM grid, and cutting the sample into a comb-like shape. By these means, in situ biasing of the FE capacitor was realized in TEM, and electric-field-induced tetragonal (t-) to monoclinic (m-) structure transitions in Hf0.5Zr0.5O2 FE film were observed. The improvement of FIB sample preparation technology can greatly enhance the quality of in situ biasing TEM samples, improve the success rate, and extend from capacitor sample preparation to other types.

Nonclassical Crystallization of an Al2O3 Film by Positively Charged Secondary Nanoparticles during Aerosol Deposition

Thin film growth through nonclassical crystallization with charged nanoparticles as building blocks has been studied in chemical vapor deposition and physical vapor deposition. A similar mechanism might be applied to aerosol deposition (AD), with which the mechanism for the evolution of dense ceramic films at room temperature has not been clearly understood. Aerosol particles fracture into fragments of secondary nanoparticles during AD. In this work, secondary particles passing between the two parallel biased and grounded electrodes were captured by the transmission electron microscopy (TEM) grid membrane on each electrode, and it was revealed that they were mainly positively charged. Neutral secondary nanoparticles, not deflected by the electric field, produced a porous film on the silicon substrate, whereas charged secondary nanoparticles produced a dense film. The positive charge of the secondary nanoparticles appeared to enhance the plastic deformation required for the evolution of dense films during AD, which was supported by ab initio calculations.

Quantification of the Nano-Voids in Electroless Deposited Copper Layers Using SiNx TEM Grids

A plan-view analytical method to detect nano-voids of deposited copper layers, especially for the fabrication of printed circuit boards, was investigated using transmission electron microscopy (TEM). Through electroless plating, thin copper layers were directly deposited on small silicon nitride window grids, and the presence of nano-voids within the deposited copper layers were confirmed with various TEM analytical techniques. The amount and size of the nano-voids were further quantified from the plan-view TEM images, making it possible to compare quickly multiple samples prepared under various plating conditions. This analytical method is practical as it is faster in detecting and measuring nano-scale defects when compared to typical cross-sectional analytical methods.

Major Factors Influencing the Size Distribution Analysis of Cellulose Nanocrystals Imaged in Transmission Electron Microscopy.

Size distributions of cellulose nanocrystals (CNCs), extracted from softwood pulp via strong sulfuric acid hydrolysis, exhibit large variability when analyzed from transmission electron microscopy (TEM) images. In this article, the causes of this variability are studied and discussed. In order to obtain results comparable with those reported, a reference material of CNCs (CNCD-1) was used to evaluate size distribution. CNC TEM specimens were prepared as-stained and dried with a rapid-flushing staining method or hydrated and embedded in vitreous ice with the plunge-freezing method. Several sets of bright-field TEM (BF-TEM), annular dark-field scanning TEM (ADF-STEM) and cryogenic-TEM (cryo-TEM) images were acquired for size distribution analysis to study the contributing factors. The rapid-flushing staining method was found to be the most effective for contrast enhancement of CNCs, not only revealing the helical structure of single CNCs but also resolving the laterally jointed CNCs. During TEM specimen preparation, CNCs were fractionated on TEM grids driven by the coffee-ring effect, as observed from contrast variation of CNCs with a stain-depth gradient. From the edge to the center of the TEM grids, the width of CNCs increases, while the aspect ratio (length to width) decreases. This fractionated dispersion of CNCs suggests that images taken near the center of a droplet would give a larger mean width. In addition to particle fractionation driven by the coffee-ring effect, the arrangement and orientation of CNC particles on the substrate significantly affect the size measurement when CNC aggregation cannot be resolved in images. The coexistence of asymmetric cross-section CNC particles introduces a large variation in size measurement, as TEM images of CNCs are mixed projections of the width and height of particles. As a demonstration of how this contributes to inflated size measurement, twisted CNC particles, rectangular cross-section particles and end-to-end jointed CNCs were revealed in reconstructed three-dimensional (3D) micrographs by electron tomography (ET).

Nanodissection of Selected Viral Particles by Scanning Transmission Electron Microscopy/Focused Ion Beam for Genetic Identification.

This study presents the development of a new correlative workflow to bridge the gap between electron microscopy imaging and genetic analysis of viruses. The workflow enables the assignment of genetic information to a specific biological entity by harnessing the nanodissection capability of focused ion beam (FIB). This correlative workflow is based on scanning transmission electron microscopy (STEM) and FIB followed by a polymerase chain reaction (PCR). For this purpose, we studied the tomato brown rugose fruit virus (ToBRFV) and the adenovirus that have significant impacts on plant integrity and human health, respectively. STEM imaging was used for the identification and localization of virus particles on a transmission electron microscopy (TEM) grid followed by FIB milling of the desired region of interest. The final-milled product was subjected to genetic analysis by the PCR. The results prove that the FIB-milling process maintains the integrity of the genetic material as confirmed by the PCR. We demonstrate the identification of RNA and DNA viruses extracted from a few micrometers of an FIB-milled TEM grid. This workflow enables the genetic analysis of specifically imaged viral particles directly from heterogeneous clinical samples. In addition to viral diagnostics, the ability to isolate and to genetically identify specific submicrometer structures may prove valuable in additional fields, including subcellular organelle and granule research.

Etching-Free Transfer and Nanoimaging of CVD-Grown MoS2 Monolayers

The transfer of two-dimensional materials from grown substrates onto target substrates is critical for device applications and postgrown analysis. The traditional transfer method for as-grown two-dimensional materials requires a wet chemical etching process that damages the crystals and the substrates. These issues deteriorate the electrical and optical performances of two-dimensional-material-based devices fabricated afterward. Herein, we developed an etching-free method and nanoimaging for transferring and analyzing monolayers of MoS2 onto arbitrary substrates using polyurethane as a sacrifice polymer. The polymer layer and the MoS2 crystals can be peeled off from the substrate by tweezers and transferred to any substrate. We analyzed the transferred samples to a glass coverslip substrate with optical microscopy, atomic force microscopy, tip-enhanced Raman spectroscopy, and tip-enhanced photoluminesce spectroscopy. Also, we transferred monolayers of MoS2 to a transmission electron microscopy grid and acquired scanning electron microscopy together with high-resolution scanning transmission electron microscopy images. The results of nanoimaging indicate that the method preserved the sample’s optical and structural properties and avoid undesirable cracks, wrinkles, and polymer residues. The etching-free transfer method of as-grown two-dimensional materials will improve the quality of transferred samples enhance the devices’ performance.

One-dimensional van der Waals heterostructures: Growth mechanism and handedness correlation revealed by nondestructive TEM

We recently synthesized one-dimensional (1D) van der Waals heterostructures in which different atomic layers (e.g., boron nitride or molybdenum disulfide) seamlessly wrap around a single-walled carbon nanotube (SWCNT) and form a coaxial, crystalized heteronanotube. The growth process of 1D heterostructure is unconventional-different crystals need to nucleate on a highly curved surface and extend nanotubes shell by shell-so understanding the formation mechanism is of fundamental research interest. In this work, we perform a follow-up and comprehensive study on the structural details and formation mechanism of chemical vapor deposition (CVD)-synthesized 1D heterostructures. Edge structures, nucleation sites, and crystal epitaxial relationships are clearly revealed using transmission electron microscopy (TEM). This is achieved by the direct synthesis of heteronanotubes on a CVD-compatible Si/SiO2 TEM grid, which enabled a transfer-free and nondestructive access to many intrinsic structural details. In particular, we have distinguished different-shaped boron nitride nanotube (BNNT) edges, which are confirmed by electron diffraction at the same location to be strictly associated with its own chiral angle and polarity. We also demonstrate the importance of surface cleanness and isolation for the formation of perfect 1D heterostructures. Furthermore, we elucidate the handedness correlation between the SWCNT template and BNNT crystals. This work not only provides an in-depth understanding of this 1D heterostructure material group but also, in a more general perspective, serves as an interesting investigation on crystal growth on highly curved (radius of a couple of nanometers) atomic substrates.

The effects of washing a collagen sample prior to TEM examination.

Transmission electron microscopy (TEM) is an important analysis technique to visualize (bio)macromolecules and their assemblies, including collagen fibers. Many protocols for TEM sample preparation of collagen involve one or more washing steps to remove excess salts from the dispersion that could hamper analysis when dried on a TEM grid. Such protocols are not standardized and washing times as well as washing solvents vary from procedure to procedure, with each research group typically having their own protocol. Here, we investigate the influence of washing with water, ethanol, but also methanol and 2‐propanol, for both mineralized and unmineralized collagen samples via a protocol based on centrifugation. Washing with water maintains the hydrated collagen structure and the characteristic banding pattern can be clearly observed. Conversely, washing with ethanol results in dehydration of the fibrils, often leading to aggregation of the fibers and a less obvious banding pattern, already within 1 min of ethanol exposure. As we show, this process is fully reversible. Similar observations were made for methanol and propanol. Based on these results, a standardized washing protocol for collagenous samples is proposed.

Multicomponent perovskite superlattices

Multicomponent perovskite superlattices

Direct Synthesis of ZIF-8 on Transmission Electron Microscopy Grids Allows Structure Analysis and 3D Reconstruction

The first example of layer‐by‐layer growth of a metal–organic framework (MOF) directly on transmission electron microscopy (TEM) grids is described. ZIF‐8 is deposited on thin amorphous carbon films and subjected to a structure analysis by (scanning) TEM ((S)TEM). This method serves as a two‐in‐one synthesis and TEM sample‐preparation technique and allows straightforward analysis of ZIF‐8 crystallites. Artifacts resulting from sample preparation are completely avoided by this approach. The morphological properties, crystal structure, and the chemical composition of the material are investigated with high spatial resolution by a variety of methods of (analytical) electron microscopy. Furthermore, the incorporation of metallic nanoparticles in ZIF‐8 by integrating a corresponding step into the layer‐by‐layer deposition process is examined. The formation of ZIF‐8 crystals on the film proceeds as under the absence of nanoparticle‐forming synthesis steps. However, the nanoparticles rather cover the supporting amorphous carbon film than being incorporated in the ZIF‐8 material. This information cannot be obtained from standard characterization techniques but requires the application of analytical (S)TEM techniques.

Nanoimprint Lithography Facilitated Plasmonic‐Photonic Coupling for Enhanced Photoconductivity and Photocatalysis

AbstractImprint lithography has emerged as a reliable, reproducible, and rapid method for patterning colloidal nanostructures. As a promising alternative to top‐down lithographic approaches, the fabrication of nanodevices has thus become effective and straightforward. In this study, a fusion of interference lithography (IL) and nanosphere imprint lithography on various target substrates ranging from carbon film on transmission electron microscope grid to inorganic and dopable polymer semiconductor is reported. 1D plasmonic photonic crystals are printed with 75% yield on the centimeter scale using colloidal ink and an IL‐produced polydimethylsiloxane stamp. Atomically smooth facet, single‐crystalline, and monodisperse colloidal building blocks of gold (Au) nanoparticles are used to print 1D plasmonic grating on top of a titanium dioxide (TiO2) slab waveguide, producing waveguide‐plasmon polariton modes with superior 10 nm spectral line‐width. Plasmon‐induced hot electrons are confirmed via two‐terminal current measurements with increased photoresponsivity under guiding conditions. The fabricated hybrid structure with Au/TiO2 heterojunction enhances photocatalytic processes like degradation of methyl orange (MO) dye molecules using the generated hot electrons. This simple colloidal printing technique demonstrated on silicon, glass, Au film, and naphthalenediimide polymer thus marks an important milestone for large‐scale implementation in optoelectronic devices.

Labile Fe(III) supersaturation controls nucleation and properties of product phases from Fe(II)-catalyzed ferrihydrite transformation

Fe(II)-catalyzed ferrihydrite (Fh) transformation to more crystalline iron (oxyhydr)oxide phases is a widely occurring geochemical process which has been extensively studied as a function of Fe(II)/Fh ratios at fixed Fh loadings. However, recent isolation of an intermediate Fe(III) species resulting from Fe(II)-Fh contact that facilitates transformation by dissolution/reprecipitation suggests that the kinetics and properties of product phases will instead depend mostly on its rate of accumulation to a critical concentration, consistent with principles in the classical nucleation theory (CNT). This suggests a dependence both on the loading of Fe(II) on the surface, which controls the rate of labile Fe(III) formation, as well as the available volume of solution, which also impacts how fast it can achieve its critical concentration to nucleate product phases. To specifically examine the latter effect, here we studied transformation of 15 mg Fh in 1 mM FeSO4 solutions at pH 7.2 in batch suspensions of 30 mL, 150 mL, and 450 mL volumes. Time-dependent concentrations of aqueous Fe(II), surface-associated Fe(II), and resulting labile Fe(III) were monitored along with bulk solids characterization as a function of time. Transmission electron microscopy (TEM) was used to visualize the evolution of phases at identical locations on TEM grids. The collective results show that the rates of Fh loss and emergence of product lepidocrocite (Lp) and goethite (Gt) as well as their phase proportions, nucleation mode and morphological properties depend directly on the rate of accumulation of the labile Fe(III) precursor to its critical concentration, which in our experiments was controlled simply by varying the available volume of solution into which it enters. Statistical analyses of TEM image data suggest that while both heterogeneous and homogeneous nucleation occurred in all experiments, the former was increasingly favored at lower Fh/solution ratio due to its lower nucleation barrier being more favorable at attendant lower supersaturations of Fe(III). Analysis of the collective results in the framework of CNT shows that the transformation process is fully consistent with dissolution/reprecipitation and that transformation kinetics, phase outcomes and their properties accordingly are directly related to effective supersaturation of the intermediate labile Fe(III) species.

Quantification of Nanoparticles in Dispersions Using Transmission Electron Microscopy.

The quantification of the particle size and the number concentration (PNC) of nanoparticles (NPs) is key for the characterization of nanomaterials. Transmission electron microscopy (TEM) is often considered as the gold standard for assessing the size of NPs; however, the TEM sample preparation suitable for estimating the PNC based on deposited NPs is challenging. Here, we use an ultrasonic nebulizer (USN) to transfer NPs from aqueous suspensions into dried aerosols which are deposited on TEM grids in an electrostatic precipitator of an aerosol monitor. The deposition efficiency of the electrostatic precipitator was ≈2%, and the transport efficiency of the USN was ≈7%. Experiments using SiO2 NPs (50–200 nm) confirmed an even deposition of the nebulized particles in the center of the TEM grids. PNCs of the SiO2 NPs derived from TEM images underestimated the expected PNCs of the suspensions by a factor of up to three, most likely resulting from droplet coagulation and NP aggregation in the USN. Nevertheless, single particles still dominated the PNC. Our approach results in reproducible and even deposition of particles on TEM grids suitable for morphological analysis and allows an estimation of the PNC in the suspensions based on the number of particles detected by TEM.

Understanding Substrate-Guided Assembly in van der Waals Epitaxy by in Situ Laser Crystallization within a Transmission Electron Microscope.

Understanding the bottom-up synthesis of atomically thin two-dimensional (2D) crystals and heterostructures is important for the development of new processing strategies to assemble 2D heterostructures with desired functional properties. Here, we utilize in situ laser-heating within a transmission electron microscope (TEM) to understand the stages of crystallization and coalescence of amorphous precursors deposited by pulsed laser deposition (PLD) as they are guided by 2D crystalline substrates into van der Waals (vdW) epitaxial heterostructures. Amorphous clusters of tungsten selenide were deposited by PLD at room temperature onto graphene or MoSe2 monolayer crystals that were suspended on TEM grids. The precursors were then stepwise evolved into 2D heterostructures with pulsed laser heating treatments within the TEM. The lattice-matching provided by the MoSe2 substrate is shown to guide the formation of large-domain, heteroepitaxial vdW WSe2/MoSe2 bilayers both during the crystallization process via direct templating and after crystallization by assisting the coalescence of nanosized domains through nonclassical particle attachment processes including domain rotation and grain boundary migration. The favorable energetics for domain rotation induced by lattice matching with the substrate were understood from first-principles calculations. These in situ TEM studies of pulsed laser-driven nonequilibrium crystallization phenomena represent a transformational tool for the rapid exploration of synthesis and processing pathways that may occur on extremely different length and time scales and lend insight into the growth of 2D crystals by PLD and laser crystallization.