Transmission Electron Microscopy Sample Research Articles

Chapter 3 - Imaging Yeast NPCs: From Classical Electron Microscopy to Immuno-SEM

Electron microscopy (EM) has been used extensively for the study of nuclear transport as well as the structure of the nuclear pore complex (NPC) and nuclear envelope. However, there are specific challenges faced when carrying out EM in one of the main model organisms used: the yeast, Saccharomyces cerevisiae. These are due to the presence of a cell wall, vacuoles, and a densely packed cytoplasm which, for transmission EM (TEM), make fixation, embedding, and imaging difficult. These also present problems for scanning EM (SEM) because cell wall removal and isolation of nuclei can easily damage the relatively fragile NPCs. We present some of the protocols we use to prepare samples for TEM and SEM to provide information about yeast NPC ultrastructure and the location of nucleoporins and transport factors by immunogold labeling within that ultrastructure.

Helium Implantation of Ultrafine Grained Tungsten within a TEM

ABSTRACTMany theoretical predictions have suggested that the confined length scales and increased interface density of various nanostructured materials may result in desired thermal, mechanical, and radiation properties. An important aspect of this for next generation nuclear reactors is understanding the change in swelling resulting from helium evolution in tungsten alloys, as a function of grain size and grain boundary type. This study investigated this using a new ion irradiation transmission electron microscope (TEM) facility that has been developed at Sandia National Laboratories and is capable of ion implanting helium at energies up to 20 keV. It was demonstrated in this feasibility study that helium could be implanted into an ultrafine grained tungsten TEM sample produced by severe plastic deformation. The size and density of the helium bubbles formed during the experiment appear nearly constant; while the larger voids formed appear to be dependent on the local microstructure. Future work is underway to both optimize the facility, as well as better understand the evolution of ultrafine grained tungsten resulting from both helium implantation and displacement damage.

Novel TEM sample preparation using XeF2 selective Etching

ABSTRACTWe present a new approach to prepare Transmission Electron Microscopy (TEM) nanowire (NW) samples that addresses the core drawbacks of conventional techniques, which are based on mechanical polishing. The proposed method is time efficient and uses XeF2 isotropic and selective dry etching of Si to remove the host substrate from the NWs, after their embedding into a poly(methyl-methacrylate) (PMMA) matrix. Scanning electron microscopy (SEM) data suggest that NWs were grown through the gaps between the parasitic layer islands and that the stems are in direct contact with the Si substrate. This technique does not adversely affect the NWs and offers a convenient means of transferring the GaAs NWs onto other surfaces for post-process TEM analysis. It also offers excellent potential to facilitate their integration into device fabrication via a bottom-up approach, using the PMMA layer as a transfer medium.

Effect of microstructure on adiabatic shear band bifurcation in Ti–6Al–4V alloys under ballistic impact

Adiabatic shear band (ASB) bifurcations in Ti–6Al–4V alloys with equiaxed, bimodal, and lamellar microstructures under ballistic impact were studied using transmission electron microscopy (TEM). Focused ion beam (FIB) technology was used to accurately prepare TEM samples in the ASB regions, which contained the regions in front of the ASB bifurcation, behind the ASB bifurcation, and the bifurcation regions. ASB consisted of dynamically recrystallized equiaxed grains and incompletely and dynamically recrystallized striped subgrains. ASB bifurcation occurred when the deformation incongruity between striped subgrains and surrounding equiaxed grains intensified sufficiently. Microstructure has an important effect on the number and morphology of ASB bifurcations. More ASB bifurcations formed in Ti–6Al–4V alloys with bimodal and lamellar microstructures than in the alloy with equiaxed microstructure because of the different amounts and distributions of striped subgrains. In the equiaxed microstructure, fewer subgrains were preserved in ASBs. Thus, forming deformation incongruities sufficiently intense to induce ASB bifurcation was difficult. In the bimodal microstructure, numerous striped subgrains and deformation incongruity locations were observed. More randomly distributed deformation incongruity locations would ultimately lead to more random ASB bifurcations. In the lamellar microstructure, the striped subgrains arranged along different directions in different colonies caused more intense deformation incongruity than when the subgrains were in the same colony. ASB bifurcation more commonly occurred at colony boundaries.

In situ TEM study of stability of TaRhx diffusion barriers using a novel sample preparation method

The atomic diffusion mechanisms associated with metallurgical failure of TaRhx diffusion barriers for Cu metallizations were studied by in situ transmission electron microscopy (TEM). The issues related to in situ heating of focused ion beam (FIB) prepared cross-sectional TEM samples that contain Cu thin films are discussed. The Cu layer in Si/(13nm)TaRhx/Cu stacks showed grain growth and formation of voids at temperatures exceeding 550°C. For Si/(43nm)TaRhx/Cu stacks, grain growth of Cu was delayed to higher temperatures, i.e., 700°C, and void formation was not observed. Extensive surface diffusion of Cu, however, preceded bulk diffusion. Therefore, a 10nm film of electron beam evaporated C was deposited on both sides of the TEM lamellae to limit surface diffusion. This processing technique allowed for direct observation of atomic diffusion and reaction mechanisms across the TaRhx interface. Failure occurred by nucleation of orthorhombic RhSi particles at the Si/TaRhx interface. Subsequently, the barrier at areas adjacent to RhSi particles was depleted in Rh. This created lower density areas in the barrier, which facilitated diffusion of Cu to the Si substrate to form Cu3Si. The morphology of an in situ annealed lamella was compared with an ex situ bulk annealed sample, which showed similar reaction morphology. The sample preparation method developed in this study successfully prevented surface diffusion/delamination of the Cu layer and can be employed to understand the metallurgical failure of other potential diffusion barriers.

FIB Plan and Side View Cross-Sectional TEM Sample Preparation of Nanostructures

Focused ion beam is a powerful method for cross-sectional transmission electron microscope sample preparation due to being site specific and not limited to certain materials. It has, however, been difficult to apply to many nanostructured materials as they are prone to damage due to extending from the surface. Here we show methods for focused ion beam sample preparation for transmission electron microscopy analysis of such materials, demonstrated on GaAs-GaInP core shell nanowires. We use polymer resin as support and protection and are able to produce cross-sections both perpendicular to and parallel with the substrate surface with minimal damage. Consequently, nanowires grown perpendicular to the substrates could be imaged both in plan and side view, including the nanowire-substrate interface in the latter case. Using the methods presented here we could analyze the faceting and homogeneity of hundreds of adjacent nanowires in a single lamella.

Reactive ion etching: Optimized diamond membrane fabrication for transmission electron microscopy

Commonly used preparation method for thin diamond membranes by focused ion beam (FIB) techniques results in surface damage. Here, the authors introduce an alternative method based on reactive ion etching (RIE). To compare these methods, cross-sectional samples are produced in single crystal diamond, a material that has generated growing interest for a variety of applications. The samples are examined by Raman spectroscopy and high-resolution transmission electron microscopy (TEM). Raman spectra indicate that the crystalline structure of the RIE-processed diamond is preserved, while the FIB-processed diamond membrane has a broad-background sp2 feature. Atomic-resolution TEM imaging demonstrates that the RIE-based process produces no detectable damage, while the FIB-processed sample has an amorphous carbon layer of about 11 nm thick. These findings show that the RIE-based process allows the production of diamond TEM samples with reduced near-surface damage and can thus enable direct examination of growth defects and crystallographic damage induced by processes such as ion implantation and bombardment.

EBSD and TEM characterization of high burn-up mixed oxide fuel

Understanding and studying the irradiation behavior of high burn-up oxide fuel is critical to licensing of future fast breeder reactors. Advancements in experimental techniques and equipment are allowing for new insights into previously irradiated samples. In this work dual column focused ion beam (FIB)/scanning electron microscope (SEM) was utilized to prepared transmission electron microscope samples from mixed oxide fuel with a burn-up of 6.7% FIMA. Utilizing the FIB/SEM for preparation resulted in samples with a dose rate of <0.5mRem/h compared to ∼1.1R/h for a traditionally prepared TEM sample. The TEM analysis showed that the sample taken from the cooler rim region of the fuel pellet had ∼2.5× higher dislocation density than that of the sample taken from the mid-radius due to the lower irradiation temperature of the rim. The dual column FIB/SEM was additionally used to prepared and serially slice ∼25μm cubes. High quality electron back scatter diffraction (EBSD) were collected from the face at each step, showing, for the first time, the ability to obtain EBSD data from high activity irradiated fuel.

Crystallography of surface precipitates associated with shape change in a Ti–5.26 wt.% Cr alloy

The crystallographic features of surface α precipitates accompanied by surface tilt(s) in a Ti–5.26wt.% Cr alloy have been comprehensively studied by transmission electron microscopy of samples prepared using a focused ion beam. For comparison, the bulk precipitates formed far below the free surface in the same alloy have also been examined. It is found that both the surface and the bulk α precipitates exhibit a lath-shaped morphology and their habit plane always contains a single set of misfit dislocations with the Burgers vector [11¯1]β/2|[21¯1¯3]α/6. However, the surface precipitates differ from the bulk ones in terms of their orientation relationship with the matrix, the habit plane and the long axis direction. As a result, the interphase interface between the surface precipitates and matrix contains glissile dislocations and the interface of bulk precipitates is associated with sessile dislocations. Such a glissile interface is one of the major common features of displacive-diffusional and martensitic transformations and can be used to further understand the mechanism of bainitic transformation in steels and other alloy systems.

Plant Tissues in 3D via X-Ray Tomography: Simple Contrasting Methods Allow High Resolution Imaging

Computed tomography remains strongly underused in plant sciences despite its high potential in delivering detailed 3D phenotypical information because of the low X-ray absorption of most plant tissues. Existing protocols to study soft tissues display poor performance, especially when compared to those used on animals. More efficient protocols to study plant material are therefore needed. Flowers of Arabidopsis thaliana and Marcgravia caudata were immersed in a selection of contrasting agents used to treat samples for transmission electron microscopy. Grayscale values for floral tissues and background were measured as a function of time. Contrast was quantified via a contrast index. The thick buds of Marcgravia were scanned to determine which contrasting agents best penetrate thick tissues. The highest contrast increase with cytoplasm-rich tissues was obtained with phosphotungstate, whereas osmium tetroxide and bismuth tatrate displayed the highest contrast increase with vacuolated tissues. Phosphotungstate also displayed the best sample penetration. Furthermore, infiltration with phosphotungstate allowed imaging of all plants parts at a high resolution of 3 µm, which approaches the maximum resolution of our equipment: 1.5 µm. The high affinity of phosphotungstate for vasculature, cytoplasm-rich tissue, and pollen causes these tissues to absorb more X-rays than the surrounding tissues, which, in turn, makes these tissues appear brighter on the scan data. Tissues with different brightness can then be virtually dissected from each other by selecting the bracket of grayscale to be visualized. Promising directions for the future include in silico phenotyping and developmental studies of plant inner parts (e.g., ovules, vasculature, pollen, and cell nuclei) via virtual dissection as well as correlations of quantitative phenotypes with omics datasets. Therefore, this work represents a crucial improvement of previous methods, allowing new directions of research to be undertaken in areas ranging from morphology to systems biology.

Detection of nanoparticles by means of reflection electron energy loss spectroscopy depth profiling

The various studies of nanoparticles are of great importance because of the wide application of nanotechnology. The shape and structure of the nanoparticles can be determined by transmission electron microscopy (TEM) and their chemistry by electron energy loss spectroscopy. TEM sample preparation is an expensive and difficult procedure, however. Surface sensitive, analytical techniques, such as Auger electron spectroscopy (AES) and x-ray photoelectron spectroscopy (XPS) are well applicable to detect the atoms that make up the nanoparticles, but cannot determine whether particle formation occurred. On the other hand, reflection electron energy loss spectroscopy (REELS) probes the electronic structures of atoms, which are strongly different for the atoms being in solution or in precipitated form. If the particle size is in the nm range, plasmon resonance can be excited in it, which appears as a loss feature in REELS spectrum. Thus, by measuring AES (XPS) spectra parallel with those of REELS, besides the atomic concentrations the presence of the nanoparticles can also be identified. As an example, the appearance of nanoparticles during ion beam induced mixing of C/Si layer will be shown.

Novel techniques of preparing TEM samples for characterization of irradiation damage

Focus ion beam preparation of transmission electron microscopy (TEM) samples has become increasingly popular due to the relative ease of extraction of TEM foils from specific locations within a larger sample. However the sputtering damage induced by Ga ion bombardment in focus ion beam means that traditional electropolishing may be a preferable method. First, we describe a special electropolishing method for the preparation of irregular TEM samples from ex-service nuclear reactor components, spring-shaped spacers. This method has also been used to prepare samples from a nonirradiated component for a TEM in situ heavy ion irradiation study. Because the specimen size is small (0.7 × 0.7 × 3 mm), a sandwich installation is adopted to obtain high quality polishing. Second, we describe some modifications to a conventional TEM cross-section sample preparation method that employs Ni electroplating. There are limitations to this method when preparing cross-section samples from either (1) metals which are difficult to activate for electroplating, or (2) a heavy ion irradiated foil with a very shallow damage layer close to the surface, which may be affected by the electroplating process. As a consequence, a novel technique for preparing cross-section samples was developed and is described.

Three-dimensional visualization of nanoscale structure and deformation

Abstract

Nanoporous Ni: electrodeposition synthesis, morphology, and magnetic property

Free-standing nanoporous Ni were successfully synthesized by electrodepositing Ni through the melamine–formaldehyde (MF) aerogels template which was subsequently removed during thermal decomposition process. The MF/Ni composites and the nanoporous Ni samples were investigated by X-ray diffraction, scanning electron microscopy, transmission electron microscopy, nitrogen sorption and vibration sample magnetometer. It was found that electrodepositing was successfully applied in growing Ni through the MF aerogels network structure. Burning out of the MF aerogels produced a free-standing nanoporous Ni with a relative high surface area (53.8 m2/g). In addition, the nanoporous Ni samples presented nearly superparamagnetic characteristics.

On oral calcifications: sialoliths, dental calculi and tonsilloliths

The oral cavity is susceptible to several calcifications such as salivary calculi (sialoliths), dental calculus (tartar) and tonsillar concretions (tonsilloliths). Although several individual studies had been already carried out, a comprehensive morphological and elemental comparison between them is still missing.Sialoliths are most commonly found in the submandibular glands and are composed of regions rich in Ca and P minerals, namely hydroxyapatite, whitlockite and brushite, and regions consisting of organic matter with high-sulphur content. These regions are organized in alternating concentric layers. Several bacterial species have also been identified in sialoliths microstructure showing that infection occurs recurrently throughout the stone formation.Generally, tartar presents an inorganic structure rich in Ca and P minerals, such as brushite, octacalcium phosphate, hydroxyapatite and whitlockite, and an organic matrix, mainly constituted by aerobic bacteria and yeast or just anaerobic bacteria.Tonsilloliths occur most commonly on the crypts of the palatal tonsils and are composed of a mixture of organic matter, namely bacterial cells and epithelial debris, as well as inorganic material rich in Ca and P minerals such as hydroxyapatite. Volatile sulphur compounds produced by anaerobic bacteria are usually associated to these, in general, innocuous structures.The current study involved the ultrastructure and chemical characterization of the calcified structures by scanning electron microscopy (SEM) combined with energy dispersive spectroscopy carried out with a JEOL JSM 7001F instrument with an INCA pentaFetx3 Oxford spectrometer operated at 15 kV. Higher resolution characterization has been performed by transmission electron microscopy (TEM) using a H8100 Hitachi instrument operated at 200 kV. SEM samples were prepared following metallographic procedures, whereas TEM samples were obtained following standard biological sample preparation procedures.The results show that sialoliths present the most complex structure, with a central core surrounded by concentric layers, while tartar and tonsilloliths do not have a distinctive architecture (Figures 1 (a), 2 (a) and 3 (a). At higher magnifications, layered structures, as well as crystals could be found in sialoliths and tartar (Figures 1 (b) and 2 (b). Bacteria were common in all the calcified structures, although in tonsilloliths their abundance is higher (Figure 3 (b)). All calcifications have similar elemental constitution, with Ca and P, indicating the presence of calcium phosphates (Figures 1 (c), 2 (c) and 3 (c). Sulphur was also found associated with the organic matter in sialoliths and tonsilloliths, though the amounts found in the latter were much smaller than initially expected.Based on the similarities found, new correlations between these calcification will be available. For instance, the mineralization process described in tartar can help understand the similar processes occurring in sialoliths and tonsilloliths, while the association between bacteria and sulphur in tonsilloliths can be a clue for their presence in sialoliths.The work was carried out with financial support of the Portuguese Foundation for Science and Technology through PTDC/SAU-ENB/111941/2009 and PEst-OE/CTM-UI0084/2011 grants.

Optimization of electron beam induced deposition process for the fabrication of diode-like Pt/SiO2/W devices

Pt/SiO2/W devices of controlled size, thickness, and interfaces were fabricated by electron beam induced deposition (EBID) in a focused ion beam (FIB) system, for possible applications as metal-insulator-metal diodes. A systematic study of the deposition parameters used for the fabrication of the devices was carried out by scanning electron microscopy, energy dispersive x-ray spectroscopy, and transmission electron microscopy (TEM). In particular, the morphological and structural quality of the deposited layers, their mutual interfaces, and the thickness of the insulating layer were accurately investigated, being key issues for the application of these multilayer structures as tunneling diodes; to this aim, a novel TEM sample preparation approach was developed, which allows the in situ FIB preparation of ready-to-be-observed multilayer structures; by using this approach, TEM samples can be directly made, by depositing the structures in the same conditions used for the device fabrication, in the correct geometry for the cross sectional investigations and, without any further preparation steps. The capability of the EBID technique to fabricate submicrometer electronic devices with promising electrical properties, such as asymmetry and nonlinearity, was demonstrated.

New sampling technique for collection of nanoparticles from liquids

Transmission electron microscopy (TEM) is the only technique which can give accurate information on the individual size, shape and composition of NPs. However, in dilute forms it is difficult to collect a sufficient number of NPs on an electron microscope grid for TEM analysis without some means of concentrating the dispersion of NPs. This study introduces a novel TEM sampling technique which overcomes this shortcoming and allows for both the natural and engineered NPs in dilute dispersions as found in waste and contaminated waters to be selectively sampled, based on their electrical charge. The technique is based on the use of carefully functionalized collector surfaces built by using the layer-by-layer assembly of polyelectrolytes on TEM grids. The layers are electrically charged and the top layer will attract and retain oppositely charged sampled NPs from aqueous solutions. The ability of the collector to attract both single particles and particle aggregates on the top polyelectrolyte layer collected from dilute aqueous dispersions after a short sampling time is demonstrated from the TEM images. The nature of the particles attracted to the collector can be identified from the TEM images, their diffraction pattern and the EDX spectrum. A large variety of equipment is nowadays being used worldwide for the detection and characterization of nanoparticles (NPs) dispersed in aqueous environmental liquids. To take advantage of these advanced analytical tools there is an increased need for the development of sampling technologies capable of collecting nanoparticle samples directly on suitable substrates (TEM grids or filters).

Mesoporous β-MnO2/sulfur composite as cathode material for Li–S batteries

Highly ordered mesoporous β-MnO2 (HMM) was prepared using KIT-6 templates, and mesoporous β-MnO2/S (HMM/S) composites with four sulfur content were synthesized by thermally treating a mixture of sublimed sulfur and HMM for use as cathode in Li–S batteries. The results of thermal gravimetric analysis, Brunauer–Emmett–Teller measurements, X-ray diffraction, environmental scanning electron microscopy and transmission electron microscopy for samples of HMM and HMM/S material indicate that HMM has a highly ordered pore structure and highly crystalline walls, facilitating electrolyte flooding of these pores within the cathode. The mesopores of HMM contain the majority of the sulfur mass, and effectively suppress the diffusion of polysulfide species into the electrolyte. The thin (nm scale) walls of HMM ensure short diffusion distances for Li+ on intercalation in charge/discharge cycles. Electrochemical experiments show that HMM/S composites with low sulfur content exhibit high discharge capacities and good cycling stability.

XEDS STEM tomography for 3D chemical characterization of nanoscale particles

We present a tomography technique which couples scanning transmission electron microscopy (STEM) and X-ray energy dispersive spectrometry (XEDS) to resolve 3D distribution of elements in nanoscale materials. STEM imaging when combined with XEDS mapping using a symmetrically arranged XEDS detector design around the specimen overcomes many of the obstacles in 3D chemical imaging of nanoscale materials and successfully elucidates the 3D chemical information in a large field of view of the transmission electron microscopy (TEM) sample. We employed this technique to investigate 3D distribution of Nickel (Ni), Manganese (Mn) and Oxygen (O) in a Li1.2Ni0.2Mn0.6O2 (LNMO) nanoparticle used as a cathode material in Lithium (Li) ion batteries. For this purpose, 2D elemental maps were acquired for a range of tilt angles and reconstructed to obtain 3D elemental distribution in an isolated LNMO nanoparticle. The results highlight the strength of this technique in 3D chemical analysis of nanoscale materials by successfully resolving Ni, Mn and O elemental distributions in 3D and discovering the new phenomenon of Ni surface segregation in this material. Furthermore, the comparison of simultaneously acquired high angle annular dark field (HAADF) STEM and XEDS STEM tomography results shows that XEDS STEM tomography provides additional 3D chemical information of the material especially when there is low atomic number (Z) contrast in the material of interest.

Introduction to the series of reactive and abnormal lymphoid proliferations mimicking malignant lymphoma

The intersection of adiabatic shear band (ASB) and its effect on the ballistic performance of the Ti–6Al–4V plates with equiaxed, bimodal, and lamellar microstructures were studied using transmission electron microscopy (TEM). Focused ion beam (FIB) technology was used to accurately prepare TEM samples at specific locations around the ASB intersections. The effect of different microstructures on the number and morphology of subgrains in ASB intersections was investigated.Multiple ASBs and their interactions were observed in the Ti–6Al–4V plates with equiaxed, bimodal, and lamellar microstructures impacted by armor-piercing projectiles. The ASBs mainly expanded along two directions, which were top-to-down and left-to-right. All the ASBs comprised dynamically recrystallized equiaxed grains and some partial dynamically recrystallized strip subgrains. The arrangement directions of these subgrains were different between the top-to-bottom and left-to-right ASBs. The expansion range of the left-to-right ASBs was the largest in the bimodal microstructure and the smallest in the equiaxed microstructure. The existing subgrains in the previously formed ASBs were elongated and fragmented during the intersection process, and decreased with the expansion of later-formed ASBs. Some of the subgrains were sheared again, which probably inhibited the expansion of the later-formed ASBs. The least amount of fragments and the smallest craters were observed in the Ti–6Al–4V plates with the equiaxed microstructure because of the lowest amount of ASB intersections and cracks, as well as the smallest expansion range of sideward ASBs. More and larger fragments and the largest craters were observed in the bimodal and lamellar microstructures because of more intersections, cracks and the largest expansion range of sideward ASBs.