Prospects of anionic nanolipoplexes in nanotherapy: transmission electron microscopy and light scattering studies

Currently nanosystems composed of polynucleotides and lipid vesicles (nanolipoplexes) are considered to be promising tools for gene therapeutics. Successful in vivo application of these vectors depends on their physicochemical, technological and biological characteristics including morphology, size distribution, molecular interactions and stability. Anionic nanoliposomes (DPPC:DCP:CHOL) were prepared by two different techniques, namely the conventional thin-film hydration method followed by extrusion, and the heating method (HM), in which no volatile solvent or detergent is used. A non-viral and non-cationic gene transfer vector was constructed by incorporating plasmid DNA (pcDNA3.1/His B/lacZ) to the HM-nanoliposomes by the electrostatic mediation of Ca²⁺ ions. Transfection efficiency of the nanolipoplexes was evaluated using a human bronchial epithelial cell line (16HBE14o-) in the presence of serum. Particle characterisation, stability of the formulations and lipid–DNA interaction studies were performed using transmission electron microscopy (TEM) and light scattering. TEM pictures of nanolipoplexes showed presence of two to four closely packed vesicles with signs of fusion. Efficient delivery of plasmid DNA and subsequent β-galactosidase expression was achieved using the anionic nanolipoplexes. Transfection efficiency increased with lipid:DNA ratio up to 7:1 (w/w), where transfection efficiency was 12-fold higher than in untreated cells. Further increase in lipid ratio decreased transfection. These nanolipoplexes appear to be safe, stable and efficient in the protection and delivery of DNA to different cells and tissues.     

Intracellular papillae of Actinocephalus (Eriocaulaceae-Poales) roots and their interaction with fungi: a light and transmission electron microscopy study

The genus Actinocephalus comprises 25 species and is restricted to Brazil, occurring mainly in the Espinhaço Mountains of Minas Gerais and Bahia States. Previous anatomical studies have reported the occurrence of intracellular papillae in the Actinocephalus roots, without dealing with their ultrastructure and function. The purpose of this paper is to investigate the structure, the composition and the probable function of the intracellular papillae of Actinocephalus roots, based on light microscopy, transmission electron microscopy and histochemical tests. The intracellular papillae occurred in all root tissues, from the rhizodermis to the vascular cylinder; they presented different forms and sizes and, ultrastructurally, they corresponded to material deposited between the cell wall and the plasma membrane. The histochemical tests carried out were positive for cellulose, pectin and callose. The intracellular papillae are responses of the plant cells to the interaction with fungi. They work as a physical barrier restricting fungal penetration, and they may also favor the supply of water and nutrients to the plant, since they increase root absorption surface. This might explain why the species of Actinocephalus are among the tallest Eriocaulaceae despite their reduced radicular system and the nutritional deficiency of the soil in which they grow.     

Quantitative atomic-scale analysis of interface structures: transmission electron microscopy and local density functional theory

Transmission electron microscopy (TEM) and local density functional theory (LDFT) are combined to analyze the microscopic structure of the rhombohedral twin interface in alpha-Al2O3. LDFT provides interfacial energetics and atomic and electronic structures for three competing models. With high-resolution TEM the atomic structure at the interface is imaged quantitatively along two orthogonal zone axes. Electron energy loss spectroscopy in TEM with nanoscale spatial resolution yields the interfacial electronic structure. Both experiments confirm the theoretically preferred model quantitatively.     

Application of transmission electron microscopy to the clinical study of viral and bacterial infections: present and future

Transmission electron microscopy has had a profound impact on our knowledge and understanding of viruses and bacteria. The 1000-fold improvement in resolution provided by electron microscopy (EM) has allowed visualization of viruses, the existence of which had previously only been suspected as the causative agents of transmissible infectious disease. Viruses are grouped into families based on their morphology. Viruses from different families look different and these morphological variances are the basis for identification of viruses by EM. Electron microscopy initially came to prominence in diagnostic microbiology in the late 1960s when it was used in the rapid diagnosis of smallpox, by differentiating, on a morphological basis, poxviruses from the less problematic herpesviruses in skin lesions. Subsequently, the technique was employed in the diagnosis of other viral infections, such as hepatitis B and parvovirus B19. Electron microscopy has led to the discovery of many new viruses, most notably the various viruses associated with gastroenteritis, for which it remained the principal diagnostic method until fairly recent times. Development of molecular techniques, which offer greater sensitivity and often the capacity to easily process large numbers of samples, has replaced EM in many areas of diagnostic virology. Hence the role of EM in clinical virology is evolving with less emphasis on diagnosis and more on research, although this is likely only to be undertaken in specialist centres. However, EM still offers tremendous advantages to the microbiologist, both in the speed of diagnosis and the potential for detecting, by a single test, any viral pathogen or even multiple pathogens present within a sample. There is continuing use of EM for the investigation of new and emerging agents, such as SARS and human monkeypox virus. Furthermore, EM forms a vital part of the national emergency response programme of many countries and will provide a frontline diagnostic service in the event of a bioterrorism incident, particularly in the scenario of a deliberate release of smallpox virus. In the field of bacteriology, EM is of little use diagnostically, although some bacterial pathogens can be identified in biopsy material processed for EM examination. Electron microscopy has been used, however, to elucidate the structure and function of many bacterial features, such as flagellae, fimbriae and spores and in the study of bacteriophages. The combined use of EM and gold-labelled antibodies provides a powerful tool for the ultrastructural localisation of bacterial and viral antigens.     

Nanoscale experimental study of the morphology of a microcrack in silicon by transmission electron microscopy

A microcrack in a silicon single crystal was experimentally investigated using high-resolution transmission electron microscopy (HRTEM). In particular, the numerical Moiré (NM) method was used to visualize the deformations and defects. The lattice structure of the microcrack was carefully observed at the nanoscale. HRTEM images of the microcrack demonstrated that the lattice structure of most of the microcrack regions is regular with good periodicity. In addition, the microcrack cleavage expands alternately along different crystal planes, where the principal cleavage plane is the (1 1 1) crystal plane. The NM maps showed no sharp plastic deformation around the microcrack, but discrete edge dislocations can be found only near the crack tip.     

The formation of zona radiata in Pseudosciaena crocea revealed by light and transmission electron microscopy

The egg envelope is an essential structure occurring during oogenesis. It plays an important role during the process of fertilization in the large yellow croaker Pseudosciaena crocea. Elucidation of egg envelope formation helps us to understand fertilization mechanisms in teleosts. In the present work, we studied the formation of egg envelope in P. crocea by light microscopy, as well as by transmission and scanning electron microscopy. Four layers exist outside the oocyte plasmalemma, i.e., theca cell layer, basal membrane, granulosa cell layer and zona radiata. According to our observation, zona radiata is a multilaminar structure just like the same structure reported in teleosts, but the origin of this structure is a little different. Before it is formed, a peripheral space filled with different density of vesicles is the place where zona radiata is formed. Zona radiata (Z1) is secreted only by oocyte itself, it belongs to the primary envelope; zona radiata 2 (Z2) and zona radiata 3 (Z3) belong to the secondary envelope, because the two layers are formed after granulosa cells appear, and microvilli participate this process. It is very interesting that Z2 and Z3 are situated between Z1 and the granulosa cell first, but they translocate to the other side of Z1. This microanatomy difference may due to the participation of microvilli. The new finding about egg envelope formation in P. crocea will help us to do further investigation on fertilization mechanisms and will make artificial breeding possible which may contribute to the resource recovery of this species.     

Secondary electron microscopy and transmission electron microscopy studies of carbon nanotubes in C-Ni films

Carbon nanotubes films have been studied with SEM and TEM. The studied films were obtained using a two step method: PVD process and CVD process. Strongly defected and curled carbon nanotubes containing Ni nanoparticles formed the film with thickness of about 300–400 nm. Observed carbon nanotubes were of lengths from 100 nm to 300 nm and did not stick to each other.     

Bidder's organ of Bufo ictericus: a light and electron microscopy analysis

Male toads of the Bufonidae Family have rudimentary ovaries designated Bidder's organs, and if the testes are removed this organ develops into a functional ovary, representing a morphological strategy for the reproduction of the species. The Bidder's organ of Bufo ictericus was examined using routine and histochemical techniques by light microscopy and transmission electron microscopy. Each Bidder's organ presented a typical ovarian morphology, being composed of a cortex and a medulla. Bidderian follicles in different stages of development were visualized in the cortex, where they are better developed. The germ cells exhibit a large oocyte with a round-shaped nucleus. The Bidderian follicles are supported by a loose net of reticular fibers. In the medullar region, collagen fibers were immersed in the matrix rich in blood vessels that also contained a small quantity of neutral glycoproteins rich in hexose and/or sialic acid and carboxylated polymers with a characteristic distribution of glycosaminoglycans. The oocyte and the follicular cells were separated by a narrow space containing microvilli. The oocyte exhibit a well developed smooth endoplasmic reticulum, a poorly developed Golgi apparatus, and occasional lysosomes. Concentric cisternal complexes are often visualized; however, their morphological significance remains unclear. The peroxisomes display a fine granular matrix without a crystalline core, with a weak 3,3′-diaminobenzidine-reaction. Intimate association between peroxisomes, peroxisomes and lipid inclusions was observed in the oocyte, suggesting its participation in yolk metabolism.     

Application of automated crystallography for transmission electron microscopy in the study of grain-boundary segregation

Analytical Electron Microscopy (AEM) has brought significant progress in the study of grain-boundary segregation. Using X-ray energy-dispersive spectrometry (XEDS) in the AEM, elemental segregation information can be related to the crystallographic character of the same boundary via conventional Transmission Electron Microscope (TEM) diffraction techniques. While significant efforts have been made to improve XEDS analysis of sub-nanometer segregation layers, the methods for crystallographic characterization of grain boundaries have remained the same for several decades and labor-intensive processes. Recently, a method termed Automated Crystallography for TEM (ACT) was developed, which automates crystallographic characterization of grains under TEM observation. In the present work, we combine ACT and X-ray mapping via EDS in AEM for the study of Sb grain-boundary segregation in a rapidly solidified Cu-0.08 wt % Sb alloy. In contrast with previous reports, a large degree of anisotropy in Sb segregation level between different boundaries is found. ACT results suggest that one of the several grain boundaries observed with no detectable Sb segregation is very close to a Sigma 3 coincidence-site lattice structure. The reason for the observed anisotropy in the present alloy is discussed, based upon McLean's theory of segregation.     

原位透射电子显微学进展及应用

文章简要介绍了近年来原位透射电子显微学的进展,并指出,原位透射电子显微技术的发展使得在纳米、原子层次观察样品在力、热、电、磁作用下以及化学反应过程中的微结构演化成为可能。通过研究物质在外界环境作用下的微结构演化规律,揭示其原子结构与物理化学性质的相关性,指导其设计合成和微结构调控,促进新物质的探索和深层次物质结构研究,为解决凝聚态物理学中的具体问题提供了直接、准确和详细的方法。     

原位透射电子显微学进展及应用

文章简要介绍了近年来原位透射电子显微学的进展,并指出,原位透射电子显微技术的发展使得在纳米、原子层次观察样品在力、热、电、磁作用下以及化学反应过程中的微结构演化成为可能。通过研究物质在外界环境作用下的微结构演化规律,揭示其原子结构与物理化学性质的相关性,指导其设计合成和微结构调控,促进新物质的探索和深层次物质结构研究,为解决凝聚态物理学中的具体问题提供了直接、准确和详细的方法。     

High-resolution transmission electron microscopy study of crystallography and morphology of TiC precipitates in tempered steel

In spite of the significance of NaCl-type transition-metal carbides in steels, their crystallography and morphology have not been understood on an atomic scale. High-resolution electron microscopy was employed in the present work to examine the crystallography and morphology of TiC particles that precipitated in the quenched and tempered 0.05?wt%?C–0.20?wt%?Ti–2.0?wt%?Ni steel. Plate-like TiC precipitates with a thickness ranging from a couple of atomic layers to 20?nm were observed in steels by tempering at 550, 600 and 800°C. It was found that the Baker–Nutting orientation relationship is always satisfied with the ferrite matrix within about 5° for both the nanosized and the coarse TiC particles. The habit plane of the TiC precipitate is the (100) ferrite plane. A moderate tendency for the faceting of lateral interfaces on the {001} and {011} ferrite planes was found. The presence of interfacial misfit dislocations was revealed by examining the excess lattice fringes terminating at the interfaces between the TiC platelet and the ferrite matrix. The location and number of the excess lattice fringes terminating at the broad plane and the lateral interface were consistent with the mismatch in the atomic arrangement between the Baker–Nutting orientation relationship related TiC and ferrite.     

Atomic force microscopy and transmission electron microscopy of biocompatible magnetic fluids: A comparative analysis

The present study provides a comparative analysis of the size dispersity of magnetic nanoparticles (MNPs) within magnetic fluids as obtained from atomic force microscopy (AFM) and transmission electron microscopy (TEM). Whereas the mean particle diameter obtained from the AFM data presented a reduction of about 34% as compared to the value obtained from the TEM data, the standard deviation obtained from the AFM data is twice the value found from the TEM data. Similarities and differences in the size dispersity parameters are discussed in terms of sample preparation and tip characteristics. A two-dimensional mode for the deposition of the MNPs on top of the mica substrate is discussed as well.     

The application of graphene as a sample support in transmission electron microscopy

Transmission electron microscopy has witnessed rampant development and surging point resolution over the past few years. The improved imaging performance of modern electron microscopes shifts the bottleneck for image contrast and resolution to sample preparation. Hence, it is increasingly being realized that the full potential of electron microscopy will only be realized with the optimization of current sample preparation techniques. Perhaps the most recognized issues are background signal and noise contributed by sample supports, sample charging and instability. Graphene provides supports of single atom thickness, extreme physical stability, periodic structure, and ballistic electrical conductivity. As an increasing number of applications adapting graphene to their benefit emerge, we discuss the unique capabilities afforded by the use of graphene as a sample support for electron microscopy.     

Application of transmission electron microscopy for studying phase relations in the bismuth-rich region of the Ba-Bi-O system

The crystal structure and cation composition of oxides of the Ba-Bi-O system in the composition range 80–100 mol % BiO_1.5 have been investigated by the methods of transmission electron microscopy. Ordered phases of the compositions Ba: Bi = 2: 9, 1: 6, and 1: 15 with a rhombohedral structure have been revealed. In the range of the compositions Ba: Bi from 1: 36 to 1: 46, phases with triclinic, monoclinic, and cubic structures have been found. The monoclinic and cubic phases have structures similar to the structures of the α and γ modifications of bismuth oxide Bi₂O₃, respectively. Phase formation was found to be dependent on the following parameters: annealing temperature, partial oxygen pressure, oxygen content in the initial pairs of reacting components (BaO₂-Bi₂O₃, Ba(NO₃)₂-Bi₂O₃, BaCO₃-Bi₂O₃), and crucible material (alundum or platinum).     

Scanning and transmission electron microscopy and X-ray analysisof leaf salt glands of Limoniastrum guyonianum Boiss. under NaCl salinity

Leaf salt glands of Limoniastrum guyonianum were examined by scanning and transmission electron microscopes and energy dispersive X-ray analysis (EDAX) system, after growing for three months on sandy soil with or without 300 mM NaCl. Results showed that salt glands were irregularly scattered on both leaf sides and sunk under the epidermal level. Salt excretion occurred in both conditions and is mainly composed of calcium and magnesium in control plants, and essentially sodium and chloride in plants subjected to salt treatment. A salt gland is comprised of collecting, accumulating, and central compartments, and is made up of total thirty-two cells. The collecting cells were characterized by large central vacuoles. Accumulating cells contain numerous, large, and unshaped vacuoles and rudimentary chloroplasts. The central compartment was comprised of four basal cells and each one is surmounted by an apical cell. The basal cells are granulated, containing large nucleus, numerous mitochondria, endoplasmic reticulum, ribosomes, polyribosomes, and small vacuoles or vesicles. Equally, the apical cells are rich in organelles. Application of 300 mM NaCl to the culture medium increased vacuoles number and size, and organelles density especially the mitochondria which suggests energy requirement for ions transport. The reduction in size and number of vacuoles toward the interior of salt glands of treated plants and the fusion of the smallest ones with the plasma membrane substantiate the implication of such vacuoles in salt excretion process. The current study which is the first report on L. guyonianum salt gland has provided an in-depth understanding on structure–function relationship in the multicellular salt glands.     

Combined field ion microscopy and transmission electron microscopy of heavy ion damage in tungsten

Abstract

A field ion microscopy (FIM) and transmission electron microscopy (TEM) investigation of radiation damage in tungsten after heavy ion bombardment has been carried out. Field ion specimens of tungsten were irradiated with 180–230 keV Xe⁺ ions. The irradiation doses were varied between 4 × 10¹¹ and 4 × 10¹² ions/cm². The irradiated specimens were examined in FIM. Experiments combining both TEM and FIM were performed in order to compare the results obtainable by these two methods. The distribution of defects visible by TEM was inhomogeneous. The influence of the imaging field in FIM on the defects visible in TEM is discussed.     

An electron diffraction and high-resolution transmission electron microscopy study of defect structure in silicon doped with transition metal impurities

Features of defect formation upon the decomposition of a supersaturated solid solution of a transition metal in silicon were studied. Zinc was used as the transition metal impurity. The silicon was doped with zinc by high-temperature diffusion annealing with subsequent quenching. Microstructures of this material were studied by electron diffraction and high-resolution X-ray transmission electron microscopy combined with X-ray energy-dispersion microanalysis. It was established that the studied material was a quite perfect single crystal but contained chaotically distributed dislocations.     

One-step synthesis of a suspended ultrathin graphene oxide film: Application in transmission electron microscopy

Ultrathin graphene films find their use as advantageous support for nano- and biomaterials investigations. Thin film causes a very slight deterioration to measured signals, thus providing more details of the object's structure at nanoscale. The ultimate thinness of graphene works in the best way for this purpose. However, obtaining suspended thin film of a large-area, which is convenient for applications, is often a relatively complicated and time-consuming task. Here we present a one-step 1-min technique for synthesis of an extremely thin (about 1–2 nm) continuous film suspended over cells of a conventional copper grid (50–400 μm mesh). This technique enables us to acquire a large-area film which is water-resistant, stable in organic solvents and can act as a support when studying nanoparticles or biomaterials. Moreover, the very mechanism of the film formation can be interesting from the point of view of other applications of ultrathin graphene oxide papers.