Transmission Electron Tomography Research Articles

CuxO and carbon–modified TiO2–based hybrid materials for photocatalytically assisted H2 generation

Hydrogen, as an energy carrier, is a zero-emission fuel. Being green and clean, it is considered to play an important role in energy and environmental issues. Photocatalytic water splitting is a process used to generate hydrogen from the dissociation of water. Titanium dioxide is the archetype material for photocatalytic water splitting. However, because of the fast recombination of the photo-generated exciton, the yield of the reaction is typically low. To overcome this limit, in this work, the surface of the TiO2 nanoparticles was modified with copper and graphene to give hybrid nanostructures. Synthesized materials were tested in the photocatalytic hydrogen generation using methanol as the sacrificial agent. X-ray diffraction and spectroscopic results showed that copper did not enter the TiO2 structure, and that neither copper nor graphene substantially altered the optical band-gap of prepared photocatalysts. Detailed aberration-corrected high-resolution electron transmission electron imaging and spatially resolved energy-loss spectroscopy experiments showed the oxidation and amorphization of graphene nanoplatelets, probably due to the combined action of the acidic media of the solution with the thermal treatment necessary to produce the hybrid materials. Hydrogen generation from methanol/water mix proved that exists an optimum concentration of both copper and graphene (i.e. 0.5 mol% copper plus 0.5 wt% graphene) to grant a two-fold increase in hydrogen production compared with that of unmodified titania when using UVA irradiation. A higher amount of initial graphene (i.e. 1.0 wt% graphene and 0.5 mol% copper) was instead necessary for granting higher H2 generation on visible-light exposure. Hybrid materials based on titania modified with both copper oxide and carbon-based materials could therefore be exploited as ideal candidates for light-to-energy applications.

Transmission Electron Microscopy and Tomography on Plasma Membrane Sheets to Study Secretory Docking.

To study the formation and the architecture of exocytotic site, we generated plasma membrane (PM) sheets on electron microscopy grids to visualize the membrane organization and quantitatively analyze distributions of specific proteins and lipids. This technique allows observing the cytoplasmic face of the plasma membrane by transmission electron microscope. The principle of this approach relies on application of mechanical forces to break open cells. The exposed inner membrane surface can then be visualized with different electron-dense colorations, and specific proteins or lipids can be detected with gold-conjugated probes. Moreover, the membrane sheets are sufficiently resistant to support automated acquisition of multiple-tilt projections, and thus electron tomography allows to obtain three-dimensional (3D) ultrastructural images of secretory granule docked to the plasma membrane.

Scalar nanostructure of the Candida albicans cell wall; a molecular, cellular and ultrastructural analysis and interpretation

Despite the importance of fungal cell walls as the principle determinant of fungal morphology and the defining element determining fungal interactions with other cells, few scalar models have been developed that reconcile chemical and microscopic attributes of its structure. The cell wall of the fungal pathogen Candida albicans is comprised of an amorphous inner skeletal layer of β(1,3)- and β(1,6)-glucan and chitin and an outer fibrillar layer thought to be dominated by highly mannosylated cell wall proteins. The architecture of these two layers can be resolved at the electron microscopy level, but the visualised structure of the wall has not yet been defined precisely in chemical terms. We have therefore examined the precise structure, location and molecular sizes of the cell wall components using transmission electron microscopy and tomography and tested predictions of the cell wall models using mutants and agents that perturb the normal cell wall structure. We demonstrate that the fibrils are comprised of a frond of N-linked outer chain mannans linked to a basal layer of GPI-proteins concentrated in the mid-wall region and that the non-elastic chitin microfibrils are cantilevered with sufficient lengths of non-fibrillar chitin and/or β-glucan to enable the chitin-glucan cage to flex, e.g. during morphogenesis and osmotic swelling. We present the first three-dimensional nano-scalar model of the C. albicans cell wall which can be used to test hypotheses relating to the structure–function relationships that underpin the pathobiology of this fungal pathogen.

Three-dimensional deconvolution processing for STEM cryotomography

The complex environment of biological cells and tissues has motivated development of three-dimensional (3D) imaging in both light and electron microscopies. To this end, one of the primary tools in fluorescence microscopy is that of computational deconvolution. Wide-field fluorescence images are often corrupted by haze due to out-of-focus light, i.e., to cross-talk between different object planes as represented in the 3D image. Using prior understanding of the image formation mechanism, it is possible to suppress the cross-talk and reassign the unfocused light to its proper source post facto. Electron tomography based on tilted projections also exhibits a cross-talk between distant planes due to the discrete angular sampling and limited tilt range. By use of a suitably synthesized 3D point spread function, we show here that deconvolution leads to similar improvements in volume data reconstructed from cryoscanning transmission electron tomography (CSTET), namely a dramatic in-plane noise reduction and improved representation of features in the axial dimension. Contrast enhancement is demonstrated first with colloidal gold particles and then in representative cryotomograms of intact cells. Deconvolution of CSTET data collected from the periphery of an intact nucleus revealed partially condensed, extended structures in interphase chromatin.

Preparation, Characterization and In Vivo Biocompatibility Studies of Cotton Cellulose Nanofibers.

Cellulose nanofibers have mechanical properties that make them very attractive in a myriad of fields such as biomedicine, tissue engineering, biosensors, cosmetics and food packet products. To evaluate the potential health risks of airborne cellulose nanofibers, the cellulose nanofiber was prepared and characterized and then its pulmonary potential toxicity to a mouse model was studied. Cellulose nanofiber has been prepared by acid hydrolysis of cotton cellulose and characterized by transmission electron microscopy, zeta potential and X-ray diffraction analysis. Then, using a short-term inhalation test, the pulmonary biocompatibility of cotton cellulose nanofibers at different concentrations (0.5 mg/mL, 1 mg/mL and 2 mg/mL) were evaluated. Transmission electron images showed needle-shaped particle with a diameter of about 6-18 nm and a length of 85-225 μm. Zeta potential was -25.3±7.80 mV and the X-ray diffraction patterns indicate that cotton cellulose nanofiber has pure structural characteristics. The In Vivo results revealed that the exposure to cotton cellulose nanofiber did not alter the number of inflammatory cells or cytokine secretion by lung cells (p > 0.05). The results demonstrate that the cotton cellulose nanofiber is biocompatible and it is an environment-friendly nanomaterial with promise in various industrial sectors.

Nanoscale structural and morphological features of kaolinite nanoscrolls

One-dimensional kaolinite nanoscrolls have been arousing great interest due to their applicability in advanced materials for adsorption and slow release of reagents, as well as in nanoscale reactors and carriers. Production of high-quality halloysite-like nanoscrolls with controlled morphology, however, remains challenging as there is a lack of understanding of the curling process of kaolinite layers on the atomic scale. In the present work, the nanoscrolls were efficiently produced from the readily available natural kaolinite using a two-pot solvothermal exfoliation of the kaolinite-dimethyl sulfoxide, kaolinite-urea, and kaolinite-N-methylformamide precursors. The structures, sizes, shapes and crystallographic properties of the produced halloysite-like nanoscrolls were characterized using X-ray diffraction (XRD), high-resolution transmission electron microscopy (HRTEM) and electron tomography in scanning transmission mode (STEM). In order to avoid structural disintegration of the highly electron beam-sensitive nanoscrolls, we used a low imaging current (70–240 e/A2); as a result, the acquisition of structure images was possible. In contrast to the triclinic symmetry of kaolinite, electron diffraction patterns suggest a hexagonal symmetry of exfoliated layers. Both HRTEM and STEM tomography show partially and completely rolled up layers, with the axis of curvature being parallel to either the a or b axes. The methanol-grafted nanoscrolls have a variable but larger basal spacing (from 0.76 to 0.90 nm) than the value in ordered kaolinite (0.72 nm). Their external diameters range from 22 to 75 nm, lengths from 218 to 2287 nm, aspect ratios from 5 to 74, and the scrolls have recognizable chirality. Crystallographic image processing of HRTEM structure images suggest that the tetrahedral sheet can be on either the outer or the inner sides of the nanoscrolls. Molecular simulation results for the curled kaolinite layers are consistent in their details with the experimental observations, suggesting that the layers may roll up either way.

In Situ Formation of Free-Standing Single-Atom-Thick Antiferromagnetic Chromium Membranes

Compared to van der Waals two-dimensional (2D) layers with lateral covalent bonds, metallic bonding systems favor close-packed structures, and thus, free-standing 2D metals have remained, for the most part, elusive. However, a number of theoretical studies suggest a number of metals can exist as 2D materials and a few early experiments support this notion. Here we demonstrate free-standing single-atom-thick crystalline chromium (Cr) suspended membranes using aberration-corrected transmission electron microscopy and image simulations. Density functional theory studies confirm the 2D Cr membranes have an antiferromagnetic ground state making them highly attractive for spintronic applications. Moreover, the work also helps consolidate the existence of a new family of 2D metal layers.

Megapinosome: Morphological description of a novel organelle

The megapinosome is an endocytic cell organel that we observed in human macrophages with electron microscopy. In a previous work we showed that it is formed by an endocytic event that we called megapinocytosis. The megapinosome is filled with a membrane surrounded trabecular meshwork that is topologically part of the cytosol. In this work we used scanning transmission electron tomography on high pressure frozen and freeze substituted human macrophages in order to unravel the three-dimensional structure of both the megapinosome and the adjacent structures. The megapinosome consists of the trabecular meshwork and the lacunae which are connected with and topologically equivalent to the cytosol. The surrounding lumen is topologically equivalent to the structures of the vesicular pathway. In addition, we show the connections of the trabecular meshwork with the cytosol and the connection of the megapinosomes to a complex tubular and cisternal system covering a large part of the macrophages that we named megapinosome complex. We assume that our methodological approach, based on high pressure freezing from a defined physiological state and three-dimensional imaging, renders the tubular components of the macrophages better visible than the classical two-dimensional imaging of chemically fixed cells used as a “blueprint” for textbook illustrations. The cell biological functions of the megapinosome are largely enigmatic. Probably, megapinosomes assures storage of surface membranes that can be promptly made available when a macrophage needs to change shape to move through a tissue, to uptake extracellular material or dead cells as well as to fight against microbes.

Correlative Light and Electron Microscopy (CLEM) Analysis of Nuclear Reorganization Induced by Clustered DNA Damage Upon Charged Particle Irradiation.

Chromatin architecture plays major roles in gene regulation as well as in the repair of DNA damaged by endogenous or exogenous factors, such as after radiation. Opening up the chromatin might provide the necessary accessibility for the recruitment and binding of repair factors, thus facilitating timely and correct repair. The observed formation of ionizing radiation-induced foci (IRIF) of factors, such as 53BP1, upon induction of DNA double-strand breaks have been recently linked to local chromatin decompaction. Using correlative light and electron microscopy (CLEM) in combination with DNA-specific contrasting for transmission electron microscopy or tomography, we are able to show that at the ultrastructural level, these DNA damage domains reveal a chromatin compaction and organization not distinguishable from regular euchromatin upon irradiation with carbon or iron ions. Low Density Areas (LDAs) at sites of particle-induced DNA damage, as observed after unspecific uranyl acetate (UA)-staining, are thus unlikely to represent pure chromatin decompaction. RNA-specific terbium-citrate (Tb) staining suggests rather a reduced RNA density contributing to the LDA phenotype. Our observations are discussed in the view of liquid-like phase separation as one of the mechanisms of regulating DNA repair.

Nanostructure of mouse otoconia

Mammalian otoconia of the inner ear vestibular apparatus are calcium carbonate-containing mineralized structures critical for maintaining balance and detecting linear acceleration. The mineral phase of otoconia is calcite, which coherently diffracts X-rays much like a single-crystal. Otoconia contain osteopontin (OPN), a mineral-binding protein influencing mineralization processes in bones, teeth and avian eggshells, for example, and in pathologic mineral deposits. Here we describe mineral nanostructure and the distribution of OPN in mouse otoconia. Scanning electron microscopy and atomic force microscopy of intact and cleaved mouse otoconia revealed an internal nanostructure (~50 nm). Transmission electron microscopy and electron tomography of focused ion beam-prepared sections of otoconia confirmed this mineral nanostructure, and identified even smaller (~10 nm) nanograin dimensions. X-ray diffraction of mature otoconia (8-day-old mice) showed crystallite size in a similar range (73 nm and smaller). Raman and X-ray absorption spectroscopy – both methods being sensitive to the detection of crystalline and amorphous forms in the sample – showed no evidence of amorphous calcium carbonate in these mature otoconia. Scanning and transmission electron microscopy combined with colloidal-gold immunolabeling for OPN revealed that this protein was located at the surface of the otoconia, correlating with a site where surface nanostructure was observed. OPN addition to calcite growing in vitro produced similar surface nanostructure. These findings provide details on the composition and nanostructure of mammalian otoconia, and suggest that while OPN may influence surface rounding and surface nanostructure in otoconia, other incorporated proteins (also possibly including OPN) likely participate in creating internal nanostructure.

New insights on the structure of alpha-synuclein fibrils using cryo-electron microscopy.

Fibrils of alpha-synuclein are significant components of cellular inclusions associated with several neuropathological disorders including Parkinson's disease, multiple system atrophy and dementia with Lewy bodies. In recent years, technological advances in the field of transmission electron microscopy and image processing have made it possible to solve the structure of alpha-synuclein fibrils at high resolution. This review discusses the results of structural studies using cryo-electron microscopy, which revealed that in-vitro produced fibrils vary in diameter from 5nm for single-protofilament fibrils, to 10nm for two-protofilament fibrils. In addition, the atomic models hint at contributions of the familial Parkinson's disease mutation sites to inter-protofilament interaction and the locations where post-translational modifications take place. Here, we propose a nomenclature system that allows identifying the existing alpha-synuclein polymorphs and that will allow to incorporate additional high-resolution structures determined in the future.

Microstructural features affecting mechanical properties: Effect of processing on dispersion of carbon black (N220) nanoparticles reinforcement in poly (lactic acid)

The interrelation between microstructure and macroscopic behavior is important information, which is used to characterize generally inhomogeneity, orientations, distributions; ultimately its structure relationship with specific properties. Carbon black (N220) filled poly (lactic acid) composites were processed by using melt mixing (i.e. MMCM – Melt Mixing followed by Compression Molding) and solution mixing technique (i.e. DDCS – Dissolution Dispersion followed by Casting Sheet. Carbon black filled poly (lactic acid) composites were characterized for microscopic – structural analysis and compared with mechanical properties. Microstructural analysis on tensile fractured specimens by optical microscope and scanning electron microscope was done in order to determine the microstructural features affecting mechanical properties; transmission electron microscopy and atomic force microscopy were performed to evaluate the effect of processing techniques on the dispersion of carbon black nanoparticles reinforcement in poly (lactic acid). The analysis of the mechanical properties showed an increase up to the loading of 2–3 wt% of carbon black followed by a decrease in mechanical properties at 5 wt% and above due to microstructural changes induced by low structure carbon black in the poly (lactic acid) matrix. The dominance of filler-filler interaction beyond 2.5 wt% was clearly visible in optical, electron and atomic force microscopic studies. The appearance of smooth and rough surfaces in optical and scanning electron microscopic images, presence of agglomerated carbon black in transmission electron microscopic images and the decrease in average roughness analyzed via atomic force microscopic images indicates microstructural changes in poly (lactic acid) after addition of carbon black. The filler-filler interaction as imaged and recorded from the transmission electron images and atomic force microscopic roughness values was the cause for a decrease in mechanical properties of carbon black filled poly (lactic acid) composites at 5.0wt% and beyond.

Fast Synthesis of Co3O4 by Microwave-Assisted Hydrothermal Treatment

This work describes a fast and simple procedure that combines the virtues of the microwave-assisted hydrothermal method with an oxidizing agent to produce Co3O4 nanocubes and nanoplates. We observed that particle morphology and size depend on the synthesis time and oxidizing agent (urea and hydrogen peroxide). The X-ray diffraction results showed that the samples are single phase, with crystallite sizes of approximately 30 nm. A similar crystalline domain is observed in the transmission electronic images. Magnetic measurements revealed the influence of the size and morphology of the particles on the magnetic curves. These measurements on the nanoplate samples revealed a paramagnetic behaviour at higher temperatures, and the presence of a cusp at that temperature was defined as TP. The TP decreases from 36 K to 21 K when the size of the plate particles decreases from ~70 nm to 10 nm. These samples also present weak ferromagnetism below TP, which is attributed to a superparamagnetic blockade state. The nanocube samples have a lower magnetic susceptibility magnitude and weak ferromagnetism behaviour at room temperature. Our results show that this synthesis produces Co3O4 nanoplates and nanocubes with interesting magnetic properties related to their shape and size.

The optical nanosizer - quantitative size and shape analysis of individual nanoparticles by high-throughput widefield extinction microscopy.

Nanoparticles are widely utilised for a range of applications, from catalysis to medicine, requiring accurate knowledge of their size and shape. Current techniques for particle characterisation are either not very accurate or time consuming and expensive. Here we demonstrate a rapid and quantitative method for particle analysis based on measuring the polarisation-resolved optical extinction cross-section of hundreds of individual nanoparticles using wide-field microscopy, and determining the particle size and shape from the optical properties. We show measurements on three samples consisting of nominally spherical gold nanoparticles of 20 nm and 30 nm diameter, and gold nanorods of 30 nm length and 10 nm diameter. Nanoparticle sizes and shapes in three dimensions are deduced from the measured optical cross-sections at different wavelengths and light polarisation, by solving the inverse problem, using an ellipsoid model of the particle polarisability in the dipole limit. The sensitivity of the method depends on the experimental noise and the choice of wavelengths. We show an uncertainty down to about 1 nm in mean diameter, and 10% in aspect ratio when using two or three color channels, for a noise of about 50 nm2 in the measured cross-section. The results are in good agreement with transmission electron microscopy, both 2D projection and tomography, of the same sample batches. Owing to its combination of experimental simplicity, ease of access to statistics over many particles, accuracy, and geometrical particle characterisation in 3D, this "optical nanosizer" method has the potential to become the technique of choice for quality control in next-generation particle manufacturing.

In situ structural analysis of the flagellum attachment zone in Trypanosoma brucei using cryo-scanning transmission electron tomography

The flagellum of Trypanosoma brucei is a 20 µm-long organelle responsible for locomotion and cell morphogenesis. The flagellum attachment zone (FAZ) is a multi-protein complex whose function is to attach the flagellum to the cell body but also to guide cytokinesis. Cryo-transmission electron microscopy is a tool of choice to access the structure of the FAZ in a close-to-native state. However, because of the large dimension of the cell body, the whole FAZ cannot be structurally studied in situ at the nanometre scale in 3D using classical transmission electron microscopy approaches. In the present work, cryo-scanning transmission electron tomography, a new method capable of investigating cryo-fixed thick biological samples, has been used to study whole T. brucei cells at the bloodstream stage. The method has been used to visualise and characterise the structure and organisation of the FAZ filament. It is composed of an array of cytoplasmic stick-like structures. These sticks are heterogeneously distributed between the posterior part and the anterior tip of the cell. This cryo-STET investigation provides new insights into the structure of the FAZ filament. In combination with protein structure predictions, this work proposes a new model for the elongation of the FAZ.

Improvements on marker-free images alignment for electron tomography

Electron tomography is a technique to obtain three-dimensional structural information of samples. However, the technique is limited by shifts occurring during acquisition that need to be corrected before the reconstruction process. In 2009, we proposed an approach for post-acquisition alignment of tilt series images. This approach was marker-free, based on patch tracking and integrated in free software. Here, we present improvements to the method to make it more reliable, stable and accurate. In addition, we modified the image formation model underlying the alignment procedure to include different deformations occurring during acquisition. We propose a new way to correct these computed deformations to obtain reconstructions with reduced artifacts. The new approach has demonstrated to improve the quality of the final 3D reconstruction, giving access to better defined structures for different transmission electron tomography methods: resin embedded STEM-tomography and cryo-TEM tomography. The method is freely available in TomoJ software.

Collagen fibrils and proteoglycans of peripheral and central stroma of the keratoconus cornea - Ultrastructure and 3D transmission electron tomography

Keratoconus (KC) is a progressive corneal disorder in which vision gradually deteriorates as a result of continuous conical protrusion and the consequent altered corneal curvature. While the majority of the literature focus on assessing the center of this diseased cornea, there is growing evidence of peripheral involvement in the disease process. Thus, we investigated the organization of collagen fibrils (CFs) and proteoglycans (PGs) in the periphery and center of KC corneal stroma. Three-dimensional transmission electron tomography on four KC corneas showed the degeneration of microfibrils within the CFs and disturbance in the attachment of the PGs. Within the KC corneas, the mean CF diameter of the central-anterior stroma was significantly (p ˂ 0.001) larger than the peripheral-anterior stroma. The interfibrillar distance of CF was significantly (p ˂ 0.001) smaller in the central stroma than in the peripheral stroma. PGs area and the density in the central KC stroma were larger than those in the peripheral stroma. Results of the current study revealed that in the pre- Descemet’s membrane stroma of the periphery, the degenerated CFs and PGs constitute biomechanically weak lamellae which are prone to disorganization and this suggests that the peripheral stroma plays an important role in the pathogenicity of the KC cornea.

Three-dimensional morphology of anatase nanocrystals obtained from supercritical flow synthesis with industrial grade TiOSO4 precursor

Anatase TiO2 (a-TiO2) nanocrystals are vital in catalytic applications both as catalysts (e.g. photodegradation) and as a carrier material (e.g. NOx removal from exhaust). The synthesis of a-TiO2 nanocrystals and their properties have been heavily scrutinized, but there exists a clear gap between the scientific literature, and the scale and price expectation of industrial application. Here it is demonstrated that the industrially most attractive Ti precursor, titanyl sulfate (TiOSO4), can be combined with the green, scalable and fast supercritical flow method to produce phase pure and highly crystalline a-TiO2 nanoparticles with high specific surface area. Control of the nanocrystal morphology is important since it is known that certain facets substantially promote catalytic activity. It is, however, in itself challenging to determine nanocrystal morphology to provide a rational basis for the synthesis control. Here we advocate the use of advanced Rietveld refinement of powder X-ray diffraction data including anisotropic size broadening models in aiding to establish the sample three-dimensional morphology. This relatively quick and robust method assists in overcoming the often encountered ambiguity inherent in two-dimensional to three-dimensional reconstruction of selected particle morphologies with transmission electron microscopy and tomography techniques.

Natural versus synthetic quasicrystals: analogies and differences in the optical behavior of icosahedral and decagonal quasicrystals

The optical behavior of both synthetic and natural icosahedral and decagonal quasicrystals in the range 400–700 nm has been explored by repeated measurements of the reflectance values on crystallographically oriented fragments. We found differences in the optical behavior between natural and synthetic quasicrystals, and discuss the possible reasons to justify this feature. Natural quasicrystals, both icosahedral and decagonal, exhibit an almost identical chemical composition to their synthetic analogs, but seem to show a higher degree of structural perfection, as indicated from their electron and X-ray diffraction patterns. Such perfection is mainly due to the very low phason strain shown by the minerals with respect to the synthetic compounds and substantiates the differences in the measured reflectance values. The new finding constitutes significant new support for the original proposal that icosahedral and decagonal quasicrystals can be energetically stable states of matter, on the same footing as crystals. Electron diffraction pattern of icosahedrite (left) and decagonite (right). A high-resolution transmission electron image of icosahedrite is on the background.

Decoding crystallography from high-resolution electron imaging and diffraction datasets with deep learning.

While machine learning has been making enormous strides in many technical areas, it is still massively underused in transmission electron microscopy. To address this, a convolutional neural network model was developed for reliable classification of crystal structures from small numbers of electron images and diffraction patterns with no preferred orientation. Diffraction data containing 571,340 individual crystals divided among seven families, 32 genera, and 230 space groups were used to train the network. Despite the highly imbalanced dataset, the network narrows down the space groups to the top two with over 70% confidence in the worst case and up to 95% in the common cases. As examples, we benchmarked against alloys to two-dimensional materials to cross-validate our deep-learning model against high-resolution transmission electron images and diffraction patterns. We present this result both as a research tool and deep-learning application for diffraction analysis.