Carbon Support Film Research Articles

Cryo-electron microscopy at the Faculty of Biology of Lomonosov Moscow State University

This paper demonstrates an example of a successful upgrade of a JEOL JEM-2100 analytical transmission electron microscope to a low-resolution cryo-electron microscope designed for routine tasks of sample preparation and quality evaluation. As a result of the upgrade, the instrument allows the subnanometer resolution of protein molecule reconstructions (within 8 Å). The influence of graphene and amorphous carbon support films to prevent the effect of preferred orientation of protein particles in the frozen sample is discussed.

Crystal structure of 9,10-bis-((perchloro-phenyl)-ethynyl)anthracene determined from three-dimensional electron diffraction data

Abstract The crystal structure of the title compound was determined using electron diffraction data collected in continuous rotation mode. The structure was successfully solved and refined kinematically in the monoclinic space group P21/c, with a Z value of 2 and Z′ value of 0.5. Within the crystal structure, the entire molecule is predominantly flat. The molecular packing exhibits a herringbone pattern, distinct from that of the unchlorinated analogue molecule. The largest facet of the crystals, which faces the supporting carbon film, is designated as (0 1 ‾ 1 ‾ $\bar{1}\bar{1}$ ).

Atomic-scale in situ observation of electron beam and heat induced crystallization of Ge nanoparticles and transformation of Ag@Ge core-shell nanocrystals.

Crystallization of amorphous materials by thermal annealing has been investigated for numerous applications in the fields of nanotechnology, such as thin-film transistors and thermoelectric devices. The phase transition and shape evolution of amorphous germanium (Ge) and Ag@Ge core-shell nanoparticles with average diameters of 10 and 12nm, respectively, were investigated by high-energy electron beam irradiation and in situ heating within a transmission electron microscope. The transition of a single Ge amorphous nanoparticle to the crystalline diamond cubic structure at the atomic scale was clearly demonstrated. Depending on the heating temperature, a hollow Ge structure can be maintained or transformed into a solid Ge nanocrystal through a diffusive process during the amorphous to crystalline phase transition. Selected area diffraction patterns were obtained to confirm the crystallization process. In addition, the thermal stability of Ag@Ge core-shell nanoparticles with an average core of 7.4 and a 2.1nm Ge shell was studied by applying the same beam conditions and temperatures. The results show that at a moderate temperature (e.g., 385°C), the amorphous Ge shell can completely crystallize while maintaining the well-defined core-shell structure, while at a high temperature (e.g., 545°C), the high thermal energy enables a freely diffusive process of both Ag and Ge atoms on the carbon support film and leads to transformation into a phase segregated Ag-Ge Janus nanoparticle with a clear interface between the Ag and Ge domains. This study provides a protocol as well as insight into the thermal stability and strain relief mechanism of complex nanostructures at the single nanoparticle level with atomic resolution.

C-shaped dipper: A novel useful auxiliary tool for preparation of specimen grids for transmission electron microscopy.

The transmission electron microscopy (TEM) has played a pivotal role in visualizing and detailing the morphology of nanoscale objects of observation, but care must be taken to ensure that the object is in its original, unperturbed state. This is because not all objects under the observation are necessarily robust and structurally stable, and if the object being observed is so delicate and easily changeable in shape, one may be observing structural artifacts leading to wrong interpretations. I here present a novel prototype of an easy-to-use and reliable auxiliary tool, the C-shaped dipper, developed for the preparation of specimen grids for TEM, along with its handling instructions for diverse applications. Some nanostructures are so delicate and fragile that their nanocomplexes are easily destroyed when aspirated by a pipette with a narrow tip diameter. Using the C-shaped dipper, the observation objects can be scooped up in a droplet formed by the surface tension of the liquid, keeping their intact shapes, and placed on a grid precovered with a support film. The C-shaped dipper has proven to be excellent for handling soft assemblies having delicate structures, such as nano-scale composites, for facilitating thin layered formations with molecular constituent blocks, and for preparing a grid individually covered with a collodion film or an amorphous carbon support film.

Preparation of RNA Polymerase Complexes for Their Analysis by Single-Particle Cryo-Electron Microscopy.

Recent technological progress revealed new prospects of high-resolution structure determination of macromolecular complexes using cryo-electron microscopy (cryo-EM) . In the field of RNA polymerase (Pol) I research, a number of cryo-EM studies contributed to understanding the highly specialized mechanisms underlying the transcription of ribosomal RNA genes . Despite a broad applicability of the cryo-EM method itself, preparation of samples for high-resolution data collection can be challenging. Here, we describe strategies for the purification and stabilization of Pol I complexes, exemplarily considering advantages and disadvantages of the methodology. We further provide an easy-to-implement protocol for the coating of EM-grids with self-made carbon support films. In sum, we present an efficient workflow for cryo-grid preparation and optimization, including early stage cryo-EM screening that can be adapted to a wide range of soluble samples for high-resolution structure determination .

Binder‐Loaded Amorphous Nanometer Calcium Phosphate in Preventing Enamel Demineralization in Orthodontic Patients

With the popularization of oral health knowledge, people have gradually realized the importance of orthodontic treatment in oral health, so the number of patients undergoing orthodontic treatment has increased significantly. To improve the effect of orthodontic treatment, this article mainly studied the application of adhesives loaded with amorphous nanocalcium phosphate in the prevention of enamel demineralization in orthodontic patients. In the experiment, we used spray‐drying technology to synthesize NACP. The collected dry particles were dispersed with absolute ethanol, sonicated for 10 minutes, dropped on a 200 ordinary carbon support film, and then dried and observed with a transmission electron microscope (TEM). Prevention of enamel demineralization in orthodontic patients was by adhesive‐loaded amorphous nanocalcium phosphate. In this experiment, the membrane dialysis method was used to release the drug‐loaded nanoparticles. The MH‐5 microhardness tester was used to randomly select 3 positions on the buccal area of the enamel surface to measure the microhardness. After measuring the microhardness of each point, take the average of the readings of the 3 positions. Before and after the experiment, the difference of the enamel surface microhardness before and after the experiment was statistically analyzed. Before the scanning electron microscope observation, to avoid contamination of the window area of the enamel surface, which will cause interference with the scanning electron microscope observation, we use acetone to remove the acid‐resistant nail polish coated on the enamel surface. Clean the attachments on the surface of the teeth first, then fix, dehydrate, and dry. When the release time reaches 52 h, the cumulative release rates of Cur in pH 5.4 and pH 7.4 buffers are 85.84 and 64.68%, respectively. The results show that by adjusting the concentration of PAA, it is possible to configure a mineralized liquid that can not only use the fluidity of NACP to penetrate into the collagen fibers but also transform into HAP within a suitable time, to achieve the purpose of repairing demineralized dentin.

A Sample Preparation Pipeline for Microcrystals at the VMXm Beamline.

The mounting of microcrystals (<10 µm) for single crystal cryo-crystallography presents a non-trivial challenge. Improvements in data quality have been seen for microcrystals with the development of beamline optics, beam stability and variable beam size focusing from submicron to microns, such as at the VMXm beamline at Diamond Light Source1. Further improvements in data quality will be gained through improvements in sample environment and sample preparation. Microcrystals inherently generate weaker diffraction, therefore improving the signal-to-noise is key to collecting quality X-ray diffraction data and will predominantly come from reductions in background noise. Major sources of X-ray background noise in a diffraction experiment are from their interaction with the air path before and after the sample, excess crystallization solution surrounding the sample, the presence of crystalline ice and scatter from any other beamline instrumentation or X-ray windows. The VMXm beamline comprises instrumentation and a sample preparation protocol to reduce all these sources of noise. Firstly, an in-vacuum sample environment at VMXm removes the air path between X-ray source and sample. Next, sample preparation protocols for macromolecular crystallography at VMXm utilize a number of processes and tools adapted from cryoTEM. These include copper grids with holey carbon support films, automated blotting and plunge cooling robotics making use of liquid ethane. These tools enable the preparation of hundreds of microcrystals on a single cryoTEM grid with minimal surrounding liquid on a low-noise support. They also minimize the formation of crystalline ice from any remaining liquid surrounding the crystals. We present the process for preparing and assessing the quality of soluble protein microcrystals using visible light and scanning electron microscopy before mounting the samples on the VMXm beamline for X-ray diffraction experiments. We will also provide examples of good quality samples as well as those which require further optimization and strategies to do so.

A Sample Preparation Pipeline for Microcrystals at the VMXm Beamline

The mounting of microcrystals (<10 µm) for single crystal cryo-crystallography presents a non-trivial challenge. Improvements in data quality have been seen for microcrystals with the development of beamline optics, beam stability and variable beam size focusing from submicron to microns, such as at the VMXm beamline at Diamond Light Source1. Further improvements in data quality will be gained through improvements in sample environment and sample preparation. Microcrystals inherently generate weaker diffraction, therefore improving the signal-to-noise is key to collecting quality X-ray diffraction data and will predominantly come from reductions in background noise. Major sources of X-ray background noise in a diffraction experiment are from their interaction with the air path before and after the sample, excess crystallization solution surrounding the sample, the presence of crystalline ice and scatter from any other beamline instrumentation or X-ray windows. The VMXm beamline comprises instrumentation and a sample preparation protocol to reduce all these sources of noise. Firstly, an in-vacuum sample environment at VMXm removes the air path between X-ray source and sample. Next, sample preparation protocols for macromolecular crystallography at VMXm utilize a number of processes and tools adapted from cryoTEM. These include copper grids with holey carbon support films, automated blotting and plunge cooling robotics making use of liquid ethane. These tools enable the preparation of hundreds of microcrystals on a single cryoTEM grid with minimal surrounding liquid on a low-noise support. They also minimize the formation of crystalline ice from any remaining liquid surrounding the crystals. We present the process for preparing and assessing the quality of soluble protein microcrystals using visible light and scanning electron microscopy before mounting the samples on the VMXm beamline for X-ray diffraction experiments. We will also provide examples of good quality samples as well as those which require further optimization and strategies to do so.

Investigation of the Effects of Low-Energy Plasma Treatment on Amorphous Carbon and Graphene Support Films for Cryo-Electron Microscopy

Journal Article Investigation of the Effects of Low-Energy Plasma Treatment on Amorphous Carbon and Graphene Support Films for Cryo-Electron Microscopy Get access MJ Campin, MJ Campin E.A. Fischione Instruments, Inc., Export, PA, USA Corresponding author: mj_campin@fischione.com Search for other works by this author on: Oxford Academic Google Scholar M Stumpf, M Stumpf E.A. Fischione Instruments, Inc., Export, PA, USA Search for other works by this author on: Oxford Academic Google Scholar PE Fischione PE Fischione E.A. Fischione Instruments, Inc., Export, PA, USA Search for other works by this author on: Oxford Academic Google Scholar Microscopy and Microanalysis, Volume 25, Issue S2, 1 August 2019, Pages 996–997, https://doi.org/10.1017/S1431927619005713 Published: 01 August 2019

Phase Separation in Liquid Metal Nanoparticles

Nanoparticles produced from gallium-based liquid metal alloys have been explored for developing applications in the fields of electronics, catalysis, and biomedicine. Nonetheless, physical properties, such as phase behavior at micro-/nanosize scale, are still significantly underexplored for such nanoparticles. Here, we conduct an in situ investigation of phase behavior for gallium-based liquid metal nanoparticles and discover the unprecedented coexistence of solid particles in spherical liquid metal shells without the support of a crystalline substrate. The particles can also transform into solid Janus nanoparticles after temperature cycling. In addition, we investigate the optical properties of the nanoparticles before and after phase separation using in situ electron energy-loss spectroscopy. Most importantly, we discover that increasing the content of indium within the nanoparticle can stabilize the solid-core/liquid-shell structure at room temperature. This study provides a foundation to engineer liquid metal nanoparticles for developing new applications in nanoscale optical platforms and shape-configurable transformers.Although various electronic, chemical, and biomedical applications have been demonstrated for nanoparticles made from gallium-based liquid metal alloys, fundamental physical properties such as phase behavior of such nanoparticles are still significantly underexplored. Here, Tang and coworkers present the in situ investigation of phase separation in binary and ternary spherical liquid metal nanoparticles upon cooling, and demonstrate the coexistence of solid core/liquid shell without the support of a crystalline substrate. This study provides insight into engineering such nanoparticles for the development of new applications.Despite gallium-based liquid metal alloys attracting extensive attention for various applications, their phase behavior at the nanoscale is still underexplored. Understanding the impact of phase separation in nanoparticles can be extremely useful for developing new structures and exploring suitable applications. Here, we report on the investigation of phase behavior for spherical nanoparticles made from gallium-based liquid metal alloys upon cooling. We discover the thermally stable coexistence of solid cores in spherical liquid metal shells without the support of a crystalline substrate at room temperature. Given the unique properties of liquid metal, together with the facile process for producing core-shell and Janus nanoparticles, this work will encourage further investigation of the properties of such nanoparticles for developing applications in the fields of electronics, catalysis, nanomedicine, and beyond.

Lotus-like nano-architectures constructed from self-assembled micelles via hierarchical assembly

Dopamine modified γ-polyglutamic acid (γ-PGA-DA) copolymer and melamine (Mel) can self-assemble into Mel/γ-PGA-DA micelles via weak intermolecular interactions in aqueous solution containing small amount of methanol. When Mel/γ-PGA-DA micellar solution was cast on the surface of formvar stabilized with carbon support films, the lotus-like nano-architectures were formed on the surface of substrate via hierarchical assembly of micelles. The size of lotus-like nanoarchitectures was approximately 200 nm×500 nm. The possible driving forces for hierarchical assembly of micelles were the solution fluid and interaction between micelles. The hierarchical assembly of micelles was similar to the fractal aggregation of inorganic particles and a possible reason was discussed.

Observation of metal nanoparticles at atomic resolution in Pt-based cancer chemotherapeutics.

The chemotherapeutics cisplatin and oxaliplatin are important tools in the fight against cancer. Both compounds are platinum complexes. Aberration-corrected scanning transmission electron microscopy using the annular dark-field imaging mode now routinely provides single-atom sensitivity with atomic number contrast. Here, this imaging mode is used to directly image the platinum within the two drugs in their dried form on an amorphous carbon support film. The oxaliplatin is found to have wetted the supporting amorphous carbon, forming disordered clusters suggesting that the platinum has remained within the complex. Conversely, the cisplatin sample reveals 1.8-nm-diameter metallic platinum clusters. The size and shape of the clusters do not appear to be dependent on drying rate nor formed by beam damage, which may suggest that they were present in the original drug solution.

Vitrification after multiple rounds of sample application and blotting improves particle density on cryo-electron microscopy grids.

Single particle cryo-electron microscopy (cryoEM) is becoming widely adopted as a tool for structural characterization of biomolecules at near-atomic resolution. Vitrification of the sample to obtain a dense distribution of particles within a single field of view remains a major bottleneck for the success of such experiments. Here, we describe a simple and cost-effective method to increase the density of frozen-hydrated particles on grids with holey carbon support films. It relies on performing multiple rounds of sample application and blotting prior to plunge freezing in liquid ethane. We show that this approach is generally applicable and significantly increases particle density for a range of samples, such as small protein complexes, viruses and filamentous assemblies. The method is versatile, easy to implement, minimizes sample requirements and can enable characterization of samples that would otherwise resist structural studies using single particle cryoEM.

Influence of supporting amorphous carbon film thickness on measured strain variation within a nanoparticle.

Strain variation within a nanoparticle plays a crucial role in tuning its properties. High Resolution Transmission Electron Microscopy (HRTEM) images of a nanoparticle supported on amorphous carbon film are used to determine the strain variation. Experimental measurements in this present study on a single crystalline silver nanoparticle exhibited an unexpected high strain variation. Generally, the influence of carbon film is not accounted for during interpretation of measured strain variation. However, experimental observations raise the question whether the supporting carbon film alters the measured strain variation. In order to address this, strain variation within a simulated Ag nanoparticle supported on an amorphous carbon is measured with varying film thicknesses. The results show that supporting carbon film thickness introduces an artefact leading to more strain variation than what is present within an unsupported nanoparticle. Moreover, the variation increases with increasing supporting carbon film thickness. This effect is more pronounced in a thinner nanoparticle. Without considering this influence, the interpretation of strain within a nanoparticle may introduce severe errors which in turn will affect the tunability of desirable properties for different applications. Since strain measurement depends on the accuracy of the atomic position, the interpretation of any result using the atomic position from HRTEM images of a nanoparticle should consider the influence of supporting film.

Negative Staining and Transmission Electron Microscopy of Bacterial Surface Structures.

Negative staining is an essential and versatile staining technique in transmission electron microscopy that can be employed for visualizing bacterial cell morphology, size, and surface architecture at high resolution. Bacteria are usually transferred by passive electrostatic adsorption from suspensions in physiological saline onto suitable hydrophilic support films on electron microscopic grids. There they are contrasted, or "stained," by heavy metal ions in solution such as tungsten, uranyl, molybdate, or vanadate compounds. Here, I describe how to visualize the interaction between the bacterial M1 protein and complement factors C1q and C3 on the surface of group A streptococcus by negative staining with uranyl formate on carbon support films. The methodology should be generally applicable to the study of a large number of other bacteria-protein interactions.

The benefit of thresholding carbon layers in electron tomographic tilt series by intensity downshifting.

When performing electron tomography, tilt series of images are often acquired from samples that contain unwanted carbonaceous material, such as an embedding resin, a thin carbon support film or hydrocarbon contamination. The presence of such layers can introduce artefacts in reconstructions, obscuring features of interest. Here, we illustrate the benefit of preprocessing a high-angle annular dark-field tomographic tilt series by thresholding unwanted low-density materials using a simple intensity downshifting procedure. The resulting tomograms have fewer artefacts and segmentation can be performed more accurately. We present two representative examples taken from studies of catalyst nanoparticles and amyloid plaque core material from the human brain.

Quantitative measurement of hydrophilicity/hydrophobicity of the plasma-polymerized naphthalene film (Super Support Film) and other support films and grids in electron microscopy.

Hydrophilicity/hydrophobicity of the surfaces of plasma-polymerized naphthalene film (Super Support Film, Nisshin EM Co. Ltd., Tokyo), carbon and formvar support films, and copper and nickel grids were quantitatively estimated by contact angles measured from diameters and heights of water droplets placed on various specimens. With treatment of glow discharge, the surfaces of plasma-polymerized naphthalene and carbon support films became fully hydrophilic in 20 s. They remained hydrophilic for 6 h. The surfaces of copper and nickel grids became fully hydrophilic with 60 s of glow discharge treatment. They remained hydrophilic for only 1 h. This information is useful for negative staining, ultrathin sectioning and rapid freezing of biological specimens using the sandwich method.

Contamination mitigation strategies for scanning transmission electron microscopy

Modern scanning transmission electron microscopy (STEM) enables imaging and microanalysis at very high magnification. In the case of aberration-corrected STEM, atomic resolution is readily achieved. However, the electron fluxes used may be up to three orders of magnitude greater than those typically employed in conventional STEM. Since specimen contamination often increases with electron flux, specimen cleanliness is a critical factor in obtaining meaningful data when carrying out high magnification STEM. A range of different specimen cleaning methods have been applied to a variety of specimen types. The contamination rate has been measured quantitatively to assess the effectiveness of cleaning. The methods studied include: baking, cooling, plasma cleaning, beam showering and UV/ozone exposure. Of the methods tested, beam showering is rapid, experimentally convenient and very effective on a wide range of specimens. Oxidative plasma cleaning is also very effective and can be applied to specimens on carbon support films, albeit with some care. For electron beam-sensitive materials, cooling may be the method of choice. In most cases, preliminary removal of the bulk of the contamination by methods such as baking or plasma cleaning, followed by beam showering, where necessary, can result in a contamination-free specimen suitable for extended atomic scale imaging and analysis.

Multicomponent signal unmixing from nanoheterostructures: overcoming the traditional challenges of nanoscale X-ray analysis via machine learning.

The chemical composition of core–shell nanoparticle clusters have been determined through principal component analysis (PCA) and independent component analysis (ICA) of an energy-dispersive X-ray (EDX) spectrum image (SI) acquired in a scanning transmission electron microscope (STEM). The method blindly decomposes the SI into three components, which are found to accurately represent the isolated and unmixed X-ray signals originating from the supporting carbon film, the shell, and the bimetallic core. The composition of the latter is verified by and is in excellent agreement with the separate quantification of bare bimetallic seed nanoparticles.

Characterization of the Dehydrogenation Process of LiBH4 Confined in Nanoporous Carbon

Annealing in the transmission electron microscope allows direct comparison of the same nanoporous carbon scaffold particles following dehydrogenation of the confined LiBH4. At a nominal temperature of 200 °C, a granular crust of cubic and cuboidal LiH nanocrystals ∼8–15 nm wide grew on the outer surface of the scaffold. Furthermore, these crystals migrated on the carbon support film away from the scaffold. Ejection of material from the scaffold also occurred upon dehydrogenation in a differential scanning calorimeter. The form of the ejected material was different, being bristles instead of cubes or cuboids. This ejection of LiH and thus preferential segregation of lithium from boron is proposed to explain the observed continual decrease in hydrogen storage capacity with the number of cycles.