Carbon Support Film Research Articles

Downstream Plasma Technology for Cleaning TEM Samples on Carbon Films

Introduction With the advent of modern scanning/transmission electron microscopy (S/TEM) capable of higher resolution, better contrast, and faster throughput, it is imperative to ensure the cleanliness of the TEM sample under the ultrahigh vacuum conditions of the microscopes [1–5]. It is well known that sample contamination can severely deteriorate the quality of electron microscopy analysis of materials, especially as the sample regions of interest decrease in size. The adverse effects of sample contamination include obscuring the area of the sample being analyzed by buildup of a carbonaceous layer, interfering with focusing and astigmatism correction, and generating unexpected microanalysis signals [2, 3]. A variety of cleaning methods, including electron beam flooding, heating and/or cooling, ultraviolet light exposure, and plasma cleaning, have been developed to minimize sample contamination [4, 5]. Among them, plasma cleaning is considered the most effective way to prepare samples for electron microscopy. As shown in Figure 1a, a plasma can be described as an ionized gaseous state created by direct current (DC), radio frequency (RF), or microwave glow discharge, in which electrons, ions, and radicals coexist. The interaction of these plasma species with a solid surface causes three basic phenomena that lead to surface cleaning: heating from the electron-specimen interaction, sputtering from the ion-specimen interaction, and etching from the radical-specimen interaction [2]. Although the combination of all three plasma species is efficient in terms of cleaning rates, it can cause irreversible surface modification and undesirable heating. More problematically, as the plasma cleaning process removes the hydrocarbon contamination layer, it removes other carbon structures at the same time. This can be an issue for TEM carbon film users because the carbon film is extensively used as a support on TEM grids for both materials science and biological applications. For example, our laboratory routinely loads from 10 to 50 ex-situ lifted-out TEM samples on a carbon film Cu-grid in order to support the high volume wafer-based manufacturing process [6]. If the carbon support film is damaged before the hydrocarbon contamination layer on the TEM samples is removed, valuable information in TEM samples will be lost. Therefore, it is essential to develop a method that removes only the hydrocarbon contamination layer while preserving the carbon support film. Downstream oxygen plasma is one technology that has been extensively used in modern semiconductor and

Carbon sandwich preparation preserves quality of two-dimensional crystals for cryo-electron microscopy

Received 7 June 2013; accepted 21 June 2013Electron crystallography is an important method for determining the structure of membrane proteins. In this paper, we show the impact of a carbon sandwich preparation on the preservation of crystalline sample quality, using characteristic examples of two-dimensional (2D) crystals from gastric H+,K+-ATPase and their analyzed images. Compared with the ordinary single carbon support film preparation, the carbon sandwich preparation dramatically enhanced the resolution of images from flat sheet 2D crystals. As water evaporation is restricted in the carbon-sandwiched specimen, the improvement could be due to the strong protective effect of the retained water against drastic changes in the environment surrounding the specimen, such as dehydration and increased salt concentrations. This protective effect by the carbon sandwich technique helped to maintain the inherent and therefore best crystal conditions for analysis. Together with its strong compensation effect for the image shift due to beam-induced specimen charging, the carbon sandwich technique is a powerful method for preserving crystals of membrane proteins with larger hydrophilic regions, such as H+,K+-ATPase, and thus constitutes an efficient and high-quality method for collecting data for the structural analysis of these types of membrane proteins by electron crystallography.

Deformed grids for single-particle cryo-electron microscopy of specimens exhibiting a preferred orientation

For biological samples showing a preferred orientation on the carbon support film of an electron microscope (EM) grid, accurate three-dimensional (3D) reconstructions by single-particle cryo-EM require data collection in which the specimen grids are tilted in the microscope, to obtain adequate numbers of particles that cover the high-degree angular distribution. However, image drift caused by the electron beam interacting with the cryo specimen becomes severe when grids are tilted to high angles (>30°). We produced deformed grids by applying a deliberate mechanical deformation to EM grids containing a thin carbon film supported by a thick holey carbon film. We applied cryo-EM using deformed grids to the isolated cardiac ryanodine receptor, an ion channel complex known to assume a preferred orientation on the carbon support film. These grids contained more particles having high Euler angle orientations without the need to tilt the specimen grids. Meanwhile, the drifting that was apparent in the images was reduced from that typical of images from tilted regular EM grids. This was achieved by imaging particles in holes close to the deformed areas, where carbon films were locally bent, offering planes of inclination with various angles. The deformed grids improve the efficiency and quality of data collection for single-particle cryo-EM of samples showing a limited range of orientations.

Molecular Surveillance of Viral Processes Using Silicon Nitride Membranes

Here we present new applications for silicon nitride (SiN) membranes to evaluate biological processes. We determined that 50-nanometer thin films of SiN produced from silicon wafers were sufficiently durable to bind active rotavirus assemblies. A direct comparison of SiN microchips with conventional carbon support films indicated that SiN performs equivalent to the traditional substrate to prepare samples for Electron Microscopy (EM) imaging. Likewise, SiN films coated with Ni-NTA affinity layers concentrated rotavirus particles similarly to affinity-coated carbon films. However, affinity-coated SiN membranes outperformed glow-discharged conventional carbon films 5-fold as indicated by the number of viral particles quantified in EM images. In addition, we were able to recapitulate viral uncoating and transcription mechanisms directed onto the microchip surfaces. EM images of these processes revealed the production of RNA transcripts emerging from active rotavirus complexes. These results were confirmed by the functional incorporation of radiolabeled nucleotides into the nascent RNA transcripts. Collectively, we demonstrate new uses for SiN membranes to perform molecular surveillance on life processes in real-time.

Characterization of an amorphous carbon film covering a Mo grid during in situ heating TEM study

In the present study, we investigated the behavior of an amorphous carbon film covering a Mo grid at high temperature. It was found that metastable nanosized particles were formed on the carbon film at 400 °C. They finally became nanosized α-Mo 2 C crystals with an average size of 3.5 nm at 700 °C. Moreover, modification of the amorphous carbon support film was observed. High-resolution TEM images showed that the carbon film was transformed to irregular and disordered lamellar structures at 300 °C and to short-range ordered and crystallized structures at 700 °C. Based on these experimental results, we suggest selecting suitable metal grids and support films for the TEM specimen preparation and performing a chemical analysis of nanostructure materials in in situ heating experiment. Finally, it is expected that these results will be helpful for the appropriate experimental designs for the in situ TEM studies. ► Amorphous carbon film covering a Mo grid was investigated by the in situ TEM technique. ► Unexpected nanosized α-Mo 2 C crystals were formed at 700 °C. ► The amorphous carbon film was transformed into the crystalline phase during the in situ heating process.

A method to achieve homogeneous dispersion of large transmembrane complexes within the holes of carbon films for electron cryomicroscopy

Difficulties associated with using X-ray crystallography for structural studies of large macromolecular complexes have made single particle cryo-electron microscopy (cryoEM) a key technique in structural biology. The efficient application of the single particle cryoEM approach requires the sample to be vitrified within the holes of carbon films, with particles well dispersed throughout the ice and adopting multiple orientations. To achieve this, the carbon support film is first hydrophilised by glow discharge, which allows the sample to spread over the film. Unfortunately, for transmembrane complexes especially, this procedure can result in severe sample adsorption to the carbon support film, reducing the number of particles dispersed in the ice. This problem is rate-limiting in the single particle cryoEM approach and has hindered its widespread application to hydrophobic complexes. We describe a novel grid preparation technique that allows for good particle dispersion in the ice and minimal hydrophobic particle adhesion to the support film. This is achieved by hydrophilisation of the carbon support film by the use of selected detergents that interact with the support so as to achieve a hydrophilic and neutral or selectively charged surface.

Binderless thin films of zeolite-templated carbon electrodes useful for electrochemical microcapacitors with ultrahigh rate performance

Controlled nanozeolite deposits are prepared by electrochemical techniques on a macroporous carbon support and binderless thin film electrodes of zeolite-templated carbon are synthesized using the deposits as templates. The obtained film electrodes exhibit extremely high area capacitance (10-12 mF cm(-2)) and ultrahigh rate capability in a thin film capacitor.

Development for Dynamic Live Cell Imaging by Cryo-Electron Tomography and Stem

Cryo-EM tomography of intact cells is an emerging technology that compliments crystallography, NMR and single molecule imaging techniques. Its strength is in that it reveals the spatial arrangements of key proteins and complexes during intracellular signaling and mechanical events like motility and division. In this meeting, we describe dynamical Live Cell imaging by Cryo-EM tomography using the Titan Krios microscope with 300 kV electron column and HAADEF detector. Observed living cells were grown directly on Holey Carbon Support Film. In order to make sure that the cells are suitable for observation we analyzed the some parameters of cell movement and cell division on Quantifoil using Fiji and compared these properties with those of cells grown on a normal cell culture plastic plate. After phosphate-buffered saline washing, the cells were done rapid freeze fixation (vitrification) by dropping in liquid ethane using VitrobotTM Mark IV instead of the usual chemical fixation and were transferred onto the microscope immediately while keeping the environment under liquid N2. Cells were imaged over an angular range from −70 degrees to 70 degrees at 2 degrees x cos θ tilt increments automatically and analyzed with Inspect 3D and Amira software to provide 3D images and Volume rendering respectively.We observed some unique architectures of a part of Lamellipodium in GFP-Myosin X expressed COS 7 cell and them from near the nuclear membrane to plasma membrane in COS 7 and several eukaryotic cells (HeLa, NIH3T3⋯ etc.) by Cryo-EM tomography using STEM using intact cells and vitreous cell sections.At future, our system can provide any new information about many kinds of cells and organelles during important events.

Atomic motion on various surfaces of ceria nanoparticles in comparison

Aberration corrected HRTEM has been successfully applied to image the atomic motion at the edges and surfaces of nanoparticles of ceria of various types under electron irradiation. Here we identify movements not only on {100} facets, but also on {110} and even {111} facets, previously considered stable. However, the degree of movement varies strongly and HRTEM is evidently the preferred technique to measure relative stability at high spatial resolution as it does not require extended surfaces as in scanning probe microscopy (SPM) or chemical methods. The advantage of aberration correction shows in suppression of contrast from the carbon support films and the absence of delocalisation fringes at particle edges, apart from improving point resolution.

Paraxial charge compensator for electron cryomicroscopy

We describe a multi-hole condenser aperture for the production of several electron beams in the transmission electron microscope (TEM) making it possible to simultaneously image and irradiate spatially separated regions of a specimen. When the specimen is a thin film of vitreous ice suspended over a holey carbon film, simultaneous irradiation of the adjacent carbon support with the off-axis beam compensates for some of the effects of charging in the image formed by a beam irradiating only the ice. Because the intervening region is not irradiated, charge-neutralization of frozen-hydrated specimens can occur by a through-space mechanism such as the emission of secondary electrons from a grounded carbon support film. We use paraxial charge compensation (PCC) to control the amount of charge build-up on the specimen and observe the effects of charge on images. The multi-hole aperture thus provides a tool for investigating the mechanism of charging and charge mitigation during the imaging of radiation sensitive biological specimens by cryomicroscopy.

Immersion Freezing of Cell Monolayers for Cryo-Electron Tomography

INTRODUCTIONCryo-transmission electron microscopy (cryo-TEM) is not limited to the visualization of suspended macromolecules in their native hydrated state. It can also be used to elucidate the molecular architecture of peripheral parts of adherent cells: After cultivation of the cells directly on EM grids, they are physically fixed by immersion freezing to yield cells embedded in thin, crystal-free layers of frozen water. Subsequently, specimens can be visualized using cryo-electron tomography (cryo-ET). This protocol outlines the production of protein-coated gold particles as suitable fiducial markers for electron tomography, and how to cultivate cells on grids with a carbon support film. It also describes how to freeze cells to obtain optimal and reproducible results using the Leica EM GP immersion freezer, and how to assess the results.

Images of paraffin monolayer crystals with perfect contrast: Minimization of beam-induced specimen motion

Quantitative analysis of electron microscope images of organic and biological two-dimensional crystals has previously shown that the absolute contrast reached only a fraction of that expected theoretically from the electron diffraction amplitudes. The accepted explanation for this is that irradiation of the specimen causes beam-induced charging or movement, which in turn causes blurring of the image due to image or specimen movement. In this paper, we used three different approaches to try to overcome this image-blurring problem in monolayer crystals of paraffin. Our first approach was to use an extreme form of spotscan imaging, in which a single image was assembled on film by the successive illumination of up to 50,000 spots, each of a diameter of around 7 nm. The second approach was to use the Medipix II detector with its zero-noise readout to assemble a time-sliced series of images of the same area in which each frame from a movie with up to 400 frames had an exposure of only 500 electrons. In the third approach, we simply used a much thicker carbon support film to increase the physical strength and conductivity of the support. Surprisingly, the first two methods involving dose fractionation in space or time produced only partial improvements in contrast whereas the third approach produced many virtually perfect images, where the absolute contrast predicted from the electron diffraction amplitudes was observed in the images. We conclude that it is possible to obtain consistently almost perfect images of beam-sensitive specimens if they are attached to an appropriately strong and conductive support; however great care is needed in practice and the problem remains of how to best image ice-embedded biological structures in the absence of a strong, conductive support film.

Cholesterol microcrystals and cochleate cylinders: Attachment of pyolysin oligomers and domain 4

Using an established organic solvent injection procedure for the preparation of aqueous cholesterol microcrystal suspensions, it has now been shown that a new, hollow, cylindrical, tightly-coiled, multi-bilayer form of cholesterol can be generated, termed the cochleate cylinder. Cholesterol cochleate cylinders are formed in larger numbers at intermediate temperatures (40-75°C) but are not formed at 100°C. The structure of the cholesterol microcrystals and cochleate cylinders is shown in negatively stained electron micrographs. Oligomerization and attachment of pyolysin to cholesterol microcrystals and cochleate cylinders is shown, as is the attachment of the pyolysin "cholesterol-binding" domain 4 (D4) fragment. The bound D4 domain forms a linear array on the two planar surfaces and edges of the cholesterol microcrystals and a quasi helical array on the surface of the cochleate cylinders. Little evidence has been obtained to support the possibility that interaction or hetero-oligomerization can occur between intact pyolysin and the pyolysin D4 fragment on the surface of cholesterol microcrystals. Using immobilized cholesterol crystals attached to a carbon support film, single-sided linear labelling of the cholesterol surface with pyolysin D4 has been achieved, which correlates well with the images from the microcrystal suspensions and our earlier data using non-cytolytic streptolysin O mutants.

Ultrathin conductive carbon nanomembranes as support films for structural analysis of biological specimens

Ultrathin carbon nanomembranes (CNM) have been tested as supports for both cryogenic high-resolution transmission electron microscopy (cryo-EM) as well as atomic force microscopy (AFM) of biological specimens. Purple membrane (PM) from Halobacterium salinarum, a 2-D crystalline monolayer of bacteriorhodopsin (BR) and lipids, was used for this study. Due to their low thickness of just 1.6 nm CNM add virtually no phase contrast to the transmission pattern. This is an important advantage over commonly used amorphous carbon support films which become instable below a thickness of approximately 20 nm. Moreover, the electrical conductivity of CNM can be tuned leading to conductive carbon nanomembranes (cCNM). cCNM support films were analyzed for the first time and were found to ideally meet all requirements of cryo-EM of insulating biological samples. A projection map of PM on cCNM at 4 A resolution has been calculated which proves that the structural integrity of biological samples is preserved up to the high-resolution range. CNM have also proven to be suitable supports for AFM analysis of biological samples. PM on CNM was imaged at molecular resolution and single molecule force spectra were recorded which show no differences compared to force spectra of PM obtained with other substrates. This is the first demonstration of a support film material which meets the requirements of both, cryo-EM and AFM, thus enabling comparative structural studies of biomolecular samples with unchanged sample-substrate interactions. Beyond high-resolution cryo-EM of biological samples, cCNM are attractive new substrates for other biophysical techniques which require conductive supports, i.e. scanning tunneling microscopy (STM) and electrostatic force microscopy (EFM).

Few-Layer Graphene as a Support Film for Transmission Electron Microscopy Imaging of Nanoparticles

One consistent limitation for high-resolution imaging of small nanoparticles is the high background signal from the amorphous carbon support film. With interest growing for smaller and smaller nanostructures, state of the art electron microscopes are becoming necessary for rudimentary tasks, such as nanoparticle sizing. As a monolayer of carbon, free-standing graphene represents the ultimate support film for nanoparticle imaging. In this work, conventional high-resolution transmission electron microscopy (HRTEM) and aberration-corrected scanning transmission electron microscopy (STEM) were used to assess the benefits and feasibility of few-layer graphene support films. Suspensions of few-layer graphene to produce the support films were prepared by simple sonication of exfoliated graphite. The greatest benefit was observed for conventional HRTEM, where lattice resolved imaging of sub 2 nm CdSe nanocrystals was achieved. The few-layer graphene films were also used as a support film for C(s)-corrected STEM and electron energy loss spectroscopy of CuInSe(2) nanocrystals.

Nucleation and Growth of Crystalline Grains in RF-Sputtered TiO2Films

AmorphousTiO2thin films were radio frequency sputtered onto siliconmonoxide and carbon support films on molybdenum transmission electron microscope (TEM) grids and observed during in situ annealing in a TEM heating stage at250∘C. The evolution of crystallization is consistent with a classical model of homogeneous nucleation and isotropic grain growth. The two-dimensional grain morphology of the TEM foil allowed straightforward recognition of amorphous and crystallized regions of the films, for measurement of crystalline volume fraction and grain number density. By assuming that the kinetic parameters remain constant beyond the onset of crystallization, the final average grain size was computed, using an analytical extrapolation to the fully crystallized state. Electron diffraction reveals a predominance of the anatase crystallographic phase.

Structural stability of icosahedral FePt nanoparticles

The structural stability of FePt nanoparticles of about 5-6 nm diameter was investigated by dynamic high resolution transmission electron microscopy. The FePt icosahedra were very stable under an electron beam flux of approximately 20 A/cm(2) at 300 kV. Surface sputtering was suppressed due to the large sputtering threshold energy of a Pt-rich shell. Under a flux of approximately 50 A/cm(2), the trapping potential well of the FePt particle on the supporting carbon film was lowered by the magnetic interaction between the electron beam and the particle, which leads to rotational and translational motions of the particle. A large dose of electrons (approximately 200 A/cm(2)) initiated melting and recrystallization of the FePt particle. The structure of the FePt nanoparticle, a Pt enriched shell around an Fe/Pt magnetic core, is believed to be responsible for its dynamic behaviour under different beam conditions.

Streptavidin crystals as nanostructured supports and image-calibration references for cryo-EM data collection

For cryo-EM structural studies, we seek to image membrane proteins as single particles embedded in proteoliposomes. One technical difficulty has been the low density of liposomes that can be trapped in the approximately 100nm ice layer that spans holes in the perforated carbon support film of EM grids. Inspired by the use of two-dimensional (2D) streptavidin crystals as an affinity surface for biotinylated DNA (Crucifix et al., 2004), we propose to use the crystals to tether liposomes doped with biotinylated lipids. The 2D crystal image also serves as a calibration of the image formation process, providing an absolute conversion from electrostatic potentials in the specimen to the EM image intensity, and serving as a quality control of acquired cryo-EM images. We were able to grow streptavidin crystals covering more than 90% of the holes in an EM grid, and which remained stable even under negative stain. The liposome density in the resulting cryo-EM sample was uniform and high due to the high-affinity binding of biotin to streptavidin. Using computational methods, the 2D crystal background can be removed from images without noticeable effect on image properties.

Ribosome Binding of a Single Copy of the SecY Complex: Implications for Protein Translocation

The SecY complex associates with the ribosome to form a protein translocation channel in the bacterial plasma membrane. We have used cryo-electron microscopy and quantitative mass spectrometry to show that a nontranslating E. coli ribosome binds to a single SecY complex. The crystal structure of an archaeal SecY complex was then docked into the electron density maps. In the resulting model, two cytoplasmic loops of SecY extend into the exit tunnel near proteins L23, L29, and L24. The loop between transmembrane helices 8 and 9 interacts with helices H59 and H50 in the large subunit RNA, while the 6/7 loop interacts with H7. We also show that point mutations of basic residues within either loop abolish ribosome binding. We suggest that SecY binds to this primary site on the ribosome and subsequently captures and translocates the nascent chain.

Negative staining across holes: Application to fibril and tubular structures

The negative staining technique, when used with holey carbon support films, presents superior imaging conditions than is the case when samples are adsorbed to continuous carbon films. A demonstration of this negative staining approach is presented, using ammonium molybdate in combination with trehalose, applied to several fibrillar and tubular samples. Fibrils formed from the amyloid-β peptide and the protease inhibitor pepstain A spread very well unsupported across holes and the different polymorphic fibril forms can be readily assessed. However, tubular forms of amyloid-β have a tendency to be flattened, due to surface tension forces prior to and during specimen drying. Sub-fibril assembly forms and D-banded rat tail type 1 collagen fibres are presented. The air-dried collagen images produced are shown to contain almost as much detail as those obtainable by cryo-negative staining. Fragile DNA and DNA-protein nanotubes are also shown to yield superior quality images to those produced on continuous carbon films. The iron-storage protein, frataxin, creates elongated oligomeric assemblies, containing bound ferrihydrite microcrystals. The iron particles within these flexuous oligomers can be defined in the presence of ammonium molybdate, but they are more readily demonstrated if the frataxin is spread across holes in the presence of trehalose alone. The samples used here serve to show the likely benefit obtainable from negative staining across holes for a range of other fibrillar and tubular samples in biology, medicine and nanobiotechnology.