Electron Microscope Grids Research Articles

Electron cryo-tomography of vestibular hair-cell stereocilia

High-resolution imaging of hair-cell stereocilia of the inner ear has contributed substantially to our understanding of auditory and vestibular function. To provide three-dimensional views of the structure of stereocilia cytoskeleton and membranes, we developed a method for rapidly freezing unfixed stereocilia on electron microscopy grids, which allowed subsequent 3D imaging by electron cryo-tomography. Structures of stereocilia tips, shafts, and tapers were revealed, demonstrating that the actin paracrystal was not perfectly ordered. This sample-preparation and imaging procedure will allow for examination of structural features of stereocilia in a near-native state.

A Computer-Vision-Guided Robot Arm for Automatically Placing Grids in Pioloform Film Preparation

Preparing pioloform/formvar support films on transmission electron microscopy (TEM) grids is a routine laboratory procedure in practically all electron microscopy units. In current practice, these grids are manually placed on the support film one by one using special tweezers, a process requiring a steady hand. The work is often ergonomically awkward to continue for a longer period of time. In this article, we describe a low-cost, computer vision-guided robot arm that automatically places the grids on the film. The success rate of the prototype robot is 90%, which is comparable to an experienced laboratory technician.

Recombinant Expression, Purification, and Assembly of p62 Filaments.

This chapter describes the recombinant overexpression of the canonical selective autophagy receptor p62/SQSTM1 in E. coli and affinity purification. Also described is the method to induce p62 filament assembly and their visualization by negative stain electron microscopy (EM). In cells, p62 forms large structures termed p62 bodies and has been shown to be aggregation prone. This tendency to aggregate poses problems for expression and purification in vitro, which is a prerequisite for structural analysis. Here, we describe the method to express and purify soluble p62, using the solubility tag, MBP, in conjunction with autoinduction. Furthermore, we describe the protocol to assemble p62 into filaments by controlling the ionic strength of its buffer, as well as the preparation of negative stain EM grids to visualize the filaments. In vitro formed p62 filaments can be used to study receptor cargo interactions in minimal reconstituted autophagy model systems.

Tailoring surface acoustic wave atomisation for cryo-electron microscopy sample preparation.

Surface acoustic wave (SAW) atomisation has been widely explored for use in pharmacological delivery, hence performance is characterised predominately in terms of droplet size and maximum delivery of fluid, to ensure sufficient dosage is delivered to the right location. For the application of cryo electron microscopy grid preparation, however, what is required is the transfer of very little fluid onto the grid in a well-defined manner. To meet this requirement, the analysis of SAW atomisation needs to focus on very different characteristics. Specifically, we examine the aerosol jet geometry, in terms of width, cone angle, and elevation angle, and its stability at low power, and hence low flow rates. The variables used are the width and the location of the channel delivering the fluid to the site of atomization. From the experiments, it is observed that we can reach a flowrate as low as 0.55 μl s-1 with reasonable aerosol jet stability, a jet width of 0.5 mm wide and an elevation angle variation as low as 2°.

Structural Changes in Tubulin Sheets Caused by Immobilization on Solid Supports.

In the presence of zinc, the protein tubulin assembles into two-dimensional sheets that are a useful model system for the study of both tubulin and microtubule structure. Tubulin sheets present an ideal protein structure for study with atomic force microscopy because they contain a two-dimensional crystalline protein lattice and retain many of the structural features of tubulin and microtubules. However, high-resolution imaging requires nonperturbative immobilization onto an appropriate imaging substrate. In this report, several substrates commonly used for scanning probe microscopy are evaluated for their ability to effectively immobilize tubulin sheets: mica, gold, highly ordered pyrolytic graphite, and carbon-coated electron microscopy grids. We hypothesize that the different intermolecular interactions presented by these substrates will affect the morphology of adsorbed tubulin sheets as well as the amount of other contaminating adsorbates. Tubulin sheets were successfully imaged on all of these substrates and structural characterization is reported. The most consistent results were obtained on carbon-coated electron microscopy grids, which preserved fine structural features of the sheets and had the least amount of contamination from the adsorption of unpolymerized tubulin. Images of tubulin sheets obtained with atomic force microscopy also compare favorably with published electron micrographs of sheets produced using similar procedures. This work demonstrates the importance of assessing substrate effects when studying two-dimensional protein crystals and identifies suitable substrates for immobilizing tubulin sheets.

Hierarchically Patterned Elastomeric and Thermoplastic Polymer Films through Nanoimprinting and Ultraviolet Light Exposure.

The surface relief structure of polymer films over large areas can be controlled by combining nanoscale imprinting and microscale ultraviolet–ozone (UVO) radiation, resulting in hierarchical structured surfaces. First, nanoscale patterns were formed by nanoimprinting elastomer [poly(dimethylsiloxane) (PDMS)] films with a pattern on a digital video disk. Micron-scale patterns were then superimposed on the nanoimprinted PDMS films by exposing them to ultraviolet radiation in oxygen (UVO) through a transmission electron microscopy grid mask having variable microscale patterning. UVO exposure leads to conversion and densification of PDMS to SiOx, leading to micron height relief features that follow a linear scaling relation with pattern dimension. Further, the pattern scopes are shown to collapse into a master curve by normalized feature values. Interestingly, these relief structures preserve the nanoscale features. In this paper, the influence of the self-limiting PDMS densification, wall stress at the boundary of micro-depression, and UVO exposure energy is studied in control of the micro-depression scale. This simple two-step imprinting process involving both nanoimprinting and UV radiation allows for facile fabrication of the dimension adjustable micro–nano hierarchically structures not only on elastomer films but also on thermoplastic polymer films. Coarse-grained molecular dynamics simulations were performed to correlate the surface tension and elastic properties of polymeric materials to the deformation of the pattern structure.

Magnetic Field-Induced Assembly of Superparamagnetic Cobalt Nanoparticles on Substrates and at Liquid-Air Interface.

Superparamagnetic cobalt nanoparticles (Co NPs) are an interesting material for self-assembly processes because of their magnetic properties. We investigated the magnetic field-induced assembly of superparamagnetic cobalt nanoparticles and compared three different approaches, namely, the assembly on solid substrates, at water-air, and ethylene glycol-air interfaces. Oleic acid- and trioctylphosphine oxide-coated Co NPs were synthesized via a thermolysis of cobalt carbonyl and dispersed into either hexane or toluene. The Co NP dispersion was dropped onto different substrates (e.g., transmission electron microscopy (TEM) grid, silicon wafer) and onto liquid surfaces. Transmission electron microscopy (TEM), scanning force microscopy, optical microscopy, as well as scanning electron microscopy showed that superparamagnetic Co NPs assembled into one-dimensional chains in an external magnetic field. By varying the concentration of the Co NP dispersion (1-5 mg/mL) and the strength of the magnetic field (4-54 mT), the morphology of the chains changed. Short, thin, and flexible chain structures were obtained at low NP concentration and low strength of magnetic field, whereas they became long, thick and straight when the NP concentration and the magnetic field strength increased. In comparison, the assembly of Co NPs from hexane dispersion at ethylene glycol-air interface showed the most regular and homogeneous alignment, since a more efficient spreading could be achieved on ethylene glycol than on water and solid substrates.

Effects of radio frequency power and gas ratio on barrier properties of SiOxNy films deposited by inductively coupled plasma chemical vapor deposition

SiOxNy barrier films were deposited at low temperature of ~ 23 °C using inductively coupled plasma chemical vapor deposition (ICP-CVD). When radio frequency power increased at constant ratios of Ar/SiH4 and O2/(O2 + N2), respectively, of 4.5 and 0.1, the aggregation tendency of nanoparticles slightly decreased, whereas when the ratio of Ar/SiH4 was changed from 4.5 to 41, almost spherical nanoparticles, each of which was isolated without aggregation, were observed. Such a near spherical nanoparticle without aggregation produced highly dense films with enhanced barrier property. However, heavily aggregated nanoparticles produced very porous films with deteriorated barrier property. In other words, the film density and barrier property depended significantly on whether nanoparticles were isolated or aggregated. To investigate the formation mechanism of dense SiOxNy barrier film, a transmission electron microscope grid membrane was exposed for 5 s to capture nanoparticles generated in the gas phase using a shutter above the grid membrane during ICP-CVD and the grid membrane was observed by scanning transmission electron microscope. The formation mechanism of the barrier film could be best explained by non-classical crystallization of CVD films, where nanoparticles formed in the gas phase contribute to the film formation.

Comparison between a low-voltage benchtop electron microscope and conventional TEM for number size distribution of nearly spherical shape constituent particles of nanomaterial powders and colloids

Nanomaterial powders and colloids are already a large industry and are expected to continue to grow rapidly. In the context of risk assessment associated with nanomaterials, characterization of nanoparticle size and morphology is required. Until now, the best method giving direct access to these parameters has been electron microscopy (EM), in particular, transmission electron microscopy (TEM). Although this method is widely used, several issues are highlighted such as cost, maintenance, sample representativity and damage for sensitive materials. Low-voltage transmission electron microscopes (LVTEMs) could be an alternative approach to solve some of these issues. This paper presents a first comparison between a benchtop LVTEM and a conventional device to determine the number size distribution of the constitutent particles of two polydispersed industrial powders (TiO2 and SiO2) with particle sizes close to 100 nm and two colloids referenced for their particle size (ERM FD 304 and NM 300 K). The samples were prepared with an optimized deposition protocol involving glow discharging and Alcian blue solution pre-treatment on the EM grids. The benchtop LVTEM produced a rather good resolution and the relative differences obtained for the median diameters D50 are generally within ± 15 %. On the basis of these results, benchtop LVTEM could be promoted for identifying nanomaterials within the framework of risk assessment strategy.

Effect of Bias Applied to the Substrate on the Low Temperature Growth of Silicon Epitaxial Films during RF-PECVD

The deposition behavior of silicon films by radio frequency plasma-enhanced chemical vapor deposition (RF-PECVD) was studied by nonclassical crystallization, where the building block of deposition is charged nanoparticles generated in the gas phase of the reactor. To confirm the existence of nanoparticles in the RF-CVD reactor, nanoparticles were captured on the membrane of the transmission electron microscope (TEM) grid under the condition of the DC bias applied to the holder. The capturing behavior of nanoparticles depended on the bias and the conductivity of the membrane. Also, to examine the effect of the bias on the epitaxial growth, films were deposited on the silicon wafer substrate under the condition of the biases of 0, +1000, and −1000 V applied to the substrate holder. A fully epitaxial film could be grown on a silicon wafer at 550 °C under the bias of −1000 V.

The Morphology and Assembly of Respiratory Syncytial Virus Revealed by Cryo-Electron Tomography

Human respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract disease in young children. With repeat infections throughout life, it can also cause substantial disease in the elderly and in adults with compromised cardiac, pulmonary and immune systems. RSV is a pleomorphic enveloped RNA virus in the Pneumoviridae family. Recently, the three-dimensional (3D) structure of purified RSV particles has been elucidated, revealing three distinct morphological categories: spherical, asymmetric, and filamentous. However, the native 3D structure of RSV particles associated with or released from infected cells has yet to be investigated. In this study, we have established an optimized system for studying RSV structure by imaging RSV-infected cells on transmission electron microscopy (TEM) grids by cryo-electron tomography (cryo-ET). Our results demonstrate that RSV is filamentous across several virus strains and cell lines by cryo-ET, cryo-immuno EM, and thin section TEM techniques. The viral filament length varies from 0.5 to 12 μm and the average filament diameter is approximately 130 nm. Taking advantage of the whole cell tomography technique, we have resolved various stages of RSV assembly. Collectively, our results can facilitate the understanding of viral morphogenesis in RSV and other pleomorphic enveloped viruses.

Formation and evolution of tar balls from northwestern US wildfires

Abstract. Biomass burning is a major source of light-absorbing black and brown carbonaceous particles. Tar balls (TBs) are a type of brown carbonaceous particle apparently unique to biomass burning. Here we describe the first atmospheric observations of the formation and evolution of TBs from forest fires. Aerosol particles were collected on transmission electron microscopy (TEM) grids during aircraft transects at various downwind distances from the Colockum Tarps wildland fire. TB mass fractions, derived from TEM and in situ measurements, increased from <1 % near the fire to 31–45 % downwind, with little change in TB diameter. Given the observed evolution of TBs, it is recommended that these particles be labeled as processed primary particles, thereby distinguishing TB formation–evolution from secondary organic aerosols. Single-scattering albedo determined from scattering and absorption measurements increased slightly with downwind distance. Similar TEM and single-scattering albedo results were observed sampling multiple wildfires. Mie calculations are consistent with weak light absorbance by TBs (i.e., m similar to the literature values 1.56−0.02i or 1.80−0.007i) but not consistent with absorption 1 order of magnitude stronger observed in different settings. The field-derived TB mass fractions reported here indicate that this particle type should be accounted for in biomass burning emission inventories.

A sampler designed for nanoparticles and respirable particles with direct analysis feature

A sampler has been designed to collect particles in the nanometer and respirable sizes directly onto a membrane filter and transmission electron microscopy (TEM) grid. The novel design aspects of this sampler include the selection of the diameter of the inlet probe, geometry of the sampler, and the resulting air flow to the sampler. Together, they control the cutoff diameter, which was determined experimentally to be a mass median aerodynamic diameter (MMAD) of 3.8 μm. The maximum aerodynamic diameter entering the sampler is designed to be approximately 8 μm. Nanometer-sized particles are collected on both the filter and grid through diffusion, as confirmed by testing with aluminum oxide engineered nanoparticles collected on the filter which measured a count median diameter (CMD) of 500 nm and a geometric standard deviation (GSD) of 1.97. The primary particles and small agglomerates collected on the grid have a CMD of 100 nm and GSD of 2.3. This diffusion sampler collected close to, if not 100%, of the particles entering the sampler. The sampler is easily wearable for personal exposure and environmental sampling, operates at 0.3 L/min, and can collect particles in various settings at indoor and outdoor environments. Particles are analyzed directly by transmission electron microscope on the grid and by scanning electron microscope on the filter to assess the exposure through particle counts and elemental composition analysis.

Nanostructure characterization of soot particles from biodiesel and diesel spray flame in a constant volume combustion chamber

This study investigates the effect of biodiesel fuels on morphological characteristics of soot particles from spray flame in a constant volume combustion chamber. The experiments were carried out under simulated diesel engine condition. The auto-ignition of injected fuel was carried out at an ambient pressure of 5 MPa and ambient temperature of 978.15 K. The soot particles were captured with a transmission electron microscope (TEM) grid inside the flame by thermophoretic effect. They were characterized as primary particle diameter, graphene layer (fringe) length, fringe tortuosity, and fringe spacing based on the image processing from original TEM image. Three different kinds of biodiesel fuels, waste cooking oil biodiesel, Jatropha biodiesel and Karanja biodiesel were used in the test. Conventional diesel fuel was utilized as a baseline fuel for the comparison. The tested fuels were injected with injection pressures of 40, 80, and 120 MPa by means of common-rail injection system. The experimental result showed that all of the biodiesel fuels had smaller primary particle diameter than that of conventional diesel regardless of injection pressures. The soot particles from biodiesel fuels were also distinguished showing characteristically shorter fringe length and lower tortuosity. These experiments unveiled a correlation between the nano-structural parameters for the early stage of oxidation inside the flame.

Discovery of nano-sized gold particles in natural plant tissues

Biological effects of nanoparticles have attracted widespread attention. However, the interaction between plants and nanoparticles remains unclear. The purpose of this study was to investigate characteristics of nano-sized metal particles in two representative plant species, Erigeron canadensis and Boehmeria nivea, in the Guangdong Province, China. The stems of the plants were sliced and placed on Ni–C grids for field-emission transmission electron microscopy (TEM). The metal-bearing nanoparticles were further analysed for their size, shape, composition, content and other characteristics using X-ray energy spectrum analysis, scanning TEM and selected-area electron diffraction pattern. The results revealed that the plants contain nano-sized Au-bearing particles with a diameter of 5–50 nm, ellipsoid, spherical and bone-rod shapes or irregular morphology with smooth edges. These nanoparticles primarily consisted of Au, Cu, O and Cl. The discovery of Au-bearing nanoparticles in natural plant tissues is of great significance for biological nanoscience. Here, we discuss the function and absorption mechanism of Au-bearing nanoparticles in plants and present the influence of the discovery of Au-bearing nanoparticles in natural plants.

Mobility and Poisoning of Mass-Selected Platinum Nanoclusters during the Oxygen Reduction Reaction

A major challenge in electrocatalysis is to understand the effect of electrochemical processes on the physicochemical properties of nanoparticle or nanocluster (NC) ensembles, especially for complex processes, such as the oxygen reduction reaction (ORR) considered herein. We describe an approach whereby electrocatalysis at a small number of well-defined mass-selected Pt NCs (Pt923±37, diameter, d ≈ 3 nm), deposited from a cluster beam source on carbon-coated transmission electron microscopy (TEM) grids, can be measured by a scanning electrochemical cell microscopy (SECCM) setup, in tandem with a range of complementary microscopy and spectroscopy techniques. The SECCM setup delivers high mass transport rates and allows the effects of transient reactive intermediates to be elucidated for different Pt surface coverages (NC spacing). A major observation is that the ORR activity decreases during successive electrochemical (voltammetric) measurements. This is shown to be due to poisoning of the Pt NCs by carbon...

Label- and enzyme-free detection of glucose by boronic acid-coupled poly(styrene-b-acrylic acid) at liquid crystal/aqueous interfaces

A glucose biosensor was developed based on a 4-cyano-4′-pentylbiphenyl (5CB)-filled transmission electron microscopy (TEM) grid cell by coating poly (styrene-b-acrylic acid) (PS-b-PAA) at the 5CB/aqueous interface and immobilising 3-aminophenyl boronic acid (APBA) on the PAA block chains by N-(3-dimethylaminopropyl)-N-ethylcarbodiimide coupling. Binding events between APBA and glucose were translated into homeotropic-to-planar configurational changes of 5CB, which were observed by polarised optical microscopy (POM) under crossed polarisers. This liquid-crystal-based glucose biosensor exhibited high sensitivity (limit of detection: 0.1 mM), even in complex serum and urine samples, high selectivity (against cholesterol, haemoglobin, and urea), and good stability for 40 d. This new and sensitive glucose biosensor has the advantages of low production cost, a simple immobilisation technique, and easy detection with POM, and may be useful for pre-screening glucose levels in human samples (serum and urine).

Probing the interaction of noble gases with pristine and nitrogen-doped graphene through Raman spectroscopy

The interactions of adsorbates with graphene have received increasing attention due to its importance in the development of applications involving graphene-based coatings. Here, we present a study of the adsorption of noble gases on pristine and nitrogen-doped graphene. Single-layer graphene samples were synthesized by chemical vapor deposition (CVD) and transferred to transmission electron microscopy (TEM) grids. Several noble gases were allowed to adsorb on the suspended graphene substrate at very low temperatures. Raman spectra show distinct frequency blue shifts in both the 2D and G bands, which are induced by gas adsorption onto high quality single layer graphene (1LG). These shifts, which we associate with compressive biaxial strain in the graphene layers induced by the noble gases, are negligible for nitrogen-doped graphene. Additionally, a thermal depinning transition, which is related to the desorption of a noble gas layer from the graphene surface at low temperatures (ranging from 20 to 35 K), was also observed at different transition temperatures for different noble gases. These transition temperatures were found to be 25 K for argon and 35 K for xenon. Moreover, we were able to obtain values for the compressive biaxial strain in graphene induced by the adsorbed layer of noble gases, using Raman spectroscopy. Ab initio calculations confirmed the correlation between the noble gas-induced strain and the changes in the Raman features observed.

Disc Antenna Enhanced Infrared Spectroscopy: From Self-Assembled Monolayers to Membrane Proteins.

Plasmonic surfaces have emerged as a powerful platform for biomolecular sensing applications and can be designed to optimize the plasmonic resonance for probing molecular vibrations at utmost sensitivity. Here, we present a facile procedure to generate metallic microdisc antenna arrays that are employed in surface-enhanced infrared absorption (SEIRA) spectroscopy of biomolecules. Transmission electron microscopy (TEM) grids are used as shadow mask deployed during physical vapor deposition of gold. The resulting disc-shaped antennas exhibit enhancement factors of the vibrational bands of 4 × 104 giving rise to a detection limit <1 femtomol (10-15 mol) of molecules. Surface-bound monolayers of 4-mercaptobenzoic acid show polyelectrolyte behavior when titrated with cations in the aqueous medium. Conformational rigidity of the self-assembled monolayer is validated by density functional theory calculations. The membrane protein sensory rhodopsin II is tethered to the disc antenna arrays and is fully functional as inferred from the light-induced SEIRA difference spectra. As an advance to previous studies, the accessible frequency range is improved and extended into the fingerprint region.

Engineering Ni-Mo-S Nanoparticles for Hydrodesulfurization.

Nanoparticle engineering for catalytic applications requires both a synthesis technique for the production of well-defined nanoparticles and measurements of their catalytic performance. In this paper, we present a new approach to rationally engineering highly active Ni-Mo-S nanoparticle catalysts for hydrodesulfurization (HDS), i.e., the removal of sulfur from fossil fuels. Nanoparticle catalysts are synthesized by the sputtering of a Mo75Ni25 metal target in a reactive atmosphere of Ar and H2S followed by the gas aggregation of the sputtered material into nanoparticles. The nanoparticles are filtered by a quadrupole mass filter and subsequently deposited on a planar substrate, such as a grid for electron microscopy or a microreactor. By varying the mass of the deposited nanoparticles, it is demonstrated that the Ni-Mo-S nanoparticles can be tuned into fullerene-like particles, flat-lying platelets, and upright-oriented platelets. The nanoparticle morphologies provide different abundances of Ni-Mo-S edge sites, which are commonly considered the catalytically important sites. Using a microreactor system, we assess the catalytic activity of the Ni-Mo-S nanoparticles for the HDS of dibenzothiophene. The measurements show that platelets are twice as active as the fullerene-like particles, demonstrating that the Ni-Mo-S edges are more active than basal planes for the HDS. Furthermore, the upright-standing orientation of platelets show an activity that is six times higher than the fullerene-like particles, demonstrating the importance of the edge site number and accessibility to reducing, e.g., sterical hindrance for the reacting molecules.