Microscopy techniques in flavivirus research

The Flavivirus genus is composed of many medically important viruses that cause high morbidity and mortality, which include Dengue and West Nile viruses. Various molecular and biochemical techniques have been developed in the endeavour to study flaviviruses. However, microscopy techniques still have irreplaceable roles in the identification of novel virus pathogens and characterization of morphological changes in virus-infected cells. Fluorescence microscopy contributes greatly in understanding the fundamental viral protein localizations and virus–host protein interactions during infection. Electron microscopy remains the gold standard for visualizing ultra-structural features of virus particles and infected cells. New imaging techniques and combinatory applications are continuously being developed to push the limit of resolution and extract more quantitative data. Currently, correlative live cell imaging and high resolution three-dimensional imaging have already been achieved through the tandem use of optical and electron microscopy in analyzing biological specimens. Microscopy techniques are also used to measure protein binding affinities and determine the mobility pattern of proteins in cells. This chapter will consolidate on the applications of various well-established microscopy techniques in flavivirus research, and discuss how recently developed microscopy techniques can potentially help advance our understanding in these membrane viruses.     

Ultrastructural localization of the RNA of immunodeficiency viruses using electron microscopy in situ hybridization and in vitroinfected lymphocytes

Cells infected in vitro with immunodeficiency viruses have been examined by electron microscopy in situ hybridization (EM ISH) methods for localization of viral RNA. Techniques used for preparation of specimens and probes are described. Unambiguous positive results were obtained using a mixture of two or three single negative strand DNA oligonucleotides complementary to regions of the gag, env and nef genes, each 200-300 bases and labelled with dig-11-UTP. Positive strand probes were used as a negative control. Cells were fixed with a mixture of formaldehyde and glutaraldehyde, dehydrated in ethanol with progressive lowering of temperature and embedded in Lowicryl K4M or HM20 at -35 degrees C. Permeabilization or pre-treatment of sections with proteinase K was not essential. The hybridization mixture was applied for 3-4h at 37 degrees C and probe was visualized by direct immuno-staining with sheep anti-digoxigenin antibodies conjugated to 10nm gold. This method would be suitable for future studies of the pathogenesis of retroviral infections and as a basis for further development of the EM ISH technique. EM ISH of in vitro infections of immunodeficiency viruses has shown the location of viral RNA in immature and mature viruses and its relationship to multimerized Gag protein during viral budding. The label for RNA has also been found in the cytoplasm of infected cells; it was mainly located adjacent to the plasma membrane and unassociated with visible Gag proteins. This may indicate that viral RNA migrates to the plasma membrane independently of the Gag protein and may, in some instances, arrive at the plasma membrane prior to the Gag protein. Viral RNA has also been found in the nucleus of peripheral blood mononuclear cells (PBMC) that were showing no morphological evidence of infection. The RNA was typically located in the nucleolus and in peripheral dense chromatin. These cells, which displayed morphological features of macrophage lineage, may have been the initial cell type to be infected in the PBMC.     

Electron Microscopy in Polymer Science

In many fields of research in science, engineering, and medicine, electron microscopy as a method for directly imaging submicroscopic structures has become increasingly important in recent decades. Electron microscopy (EM) includes several different techniques: conventional transmission electron microscopy (TEM), high-resolution electron microscopy (HREM), highvoltage electron microscopy (HVEM), scanning electron microscopy (SEM), analytical electron microscopy (AEM), emission electron microscopy (EEM), and others. In the past the central aim of using electron microscopy was structure determination, but recently it has been of growing importance for also investigating different processes, i.e., changes in materials by interaction with several influential factors (e.g., heat, electric or mechanic fields, mechanical loading). Of particular interest is the study of the micromechanical processes of deformation and fracture. Therefore, electron microscopy is a very powerful tool for materials science. The present review reports on some capabilities and limitation of the application of electron microscopy to solid polymers. In Section II the techniques of electron microscopy are briefly reviewed, followed by a section that describes the main methods of specimen preparation of solid polymeric materials. In Section IV the results of several applications of electron microscopy are discussed to reveal the morphology as well as the micromechanical deformation processes of several polymeric materials.     

Ultrastructural types of alveolar macrophages in bronchoalveolar lavages from patients with pulmonary sarcoidosis

By routine applied quantitative BAL methods are particularly helpful for the diagnosis of pulmonary sarcoidosis. Here the morphology of the alveolar cells does not play a role. However, morphological and especially electron microscopic investigations might contribute to the clarification of the aetiology of this disease. In a prospective study we investigated the bronchoalveolar lavages (BALs) from 10 patients with recently histologically diagnosed, untreated pulmonary sarcoidosis. Commonly applied cytological and immunological BAL diagnostic techniques were accompanied by morphological investigations of alveolar cells, especially alveolar macrophages, using light and electron microscopy. All patients showed lymphocytic alveolitis with an increased number of CD4 positive lymphocytes as well as an increased CD4/CD8 ratio. A striking light microscopic finding was the great morphological variety of the alveolar macrophages. Electron microscopy revealed typical lymphocytes, neutrophils, and eosinophils as well as three different types of alveolar macrophages in all 10 patients: type I (approx. 30%) with a normal macrophage morphology, a vacuole-rich type II (approx. 30%) with myelin-like structures and type III (approx. 40%) with electron-dense inclusions. The occurrence of intracellular myelin figures in type II macrophages is a hint for increased phagocytotic processes of surfactant with or without its overproduction in the sense of a secondary alveolar proteinosis. Numerous electron-dense inclusions in type III also indicate an increased macrophage activity that leads to an increased release of cytokines, which in turn can trigger an inflammatory reaction.     

Computing methods for icosahedral and symmetry-mismatch reconstruction of viruses by cryo-electron microscopy

Three-dimensional(3 D) reconstruction of icosahedral viruses has played a crucial role in the development of cryoelectron microscopy single-particle reconstruction, with many cryo-electron microscopy techniques first established for structural studies of icosahedral viruses, owing to their high symmetry and large mass. This review summarizes the computational methods for icosahedral and symmetry-mismatch reconstruction of viruses, as well as the likely challenges and bottlenecks in virus reconstruction, such as symmetry mismatch reconstruction, contrast transformation function(CTF)correction, and particle distortion.     

3D reconstruction of the hemocyanin subunit dimer from the chiton Acanthochiton fascicularis

Procedures are presented for the purification of the subunit dimer from Acanthochiton fasicularis hemocyanin. Electron microscopy of negatively stained specimens revealed a uniform population of macromolecules possessing the characteristic "boat shape". A 3D reconstruction from this EM data generated a approximately 3 nm resolution model that correlates well with earlier data of the purported subunit dimer, extracted from the 3D reconstruction of the didecamer of Haliotis tuberculata hemocyanin type 1.     

Probing bacterial interactions: integrated approaches combining atomic force microscopy, electron microscopy and biophysical techniques

Recent developments in the application of Atomic Force Microscopy (AFM) and other biophysical techniques for the study of bacterial interactions and adhesion are discussed in the light of established biological and microscopic approaches. Whereas molecular-biological techniques combined with electron microscopy allow the identification and localization of surface constituents mediating bacterial interactions, with AFM it has become possible to actually measure the forces involved in bacterial interactions. Combined with the flexibility of AFM in probing various types of physical interactions, such as electrostatic interactions, specific ligand-receptor interactions and the elastic forces of deformation and extension of bacterial surface polymers and cell wall, this provides prospects for the elucidation of the biophysical mechanism of bacterial interaction. However, because of the biochemical and a biophysical complexity of the bacterial cell wall, integrated approaches combining AFM with electron microscopy and biophysical techniques are needed to elucidate the mechanism by which a bacterium interacts with a host or material surface. The literature on electron microscopy of the bacterial cell wall is reviewed, with particular emphasis on the staining of specific classes of cell-wall constituents. The application of AFM in the analysis of bacterial surfaces is discussed, including AFM operating modes, sample preparation methods and results obtained on various strains. For various bacterial strains, the integration of EM and AFM data is discussed. Various biophysical aspects of the analysis of bacterial surface structure and interactions are discussed, including the theory of colloidal interactions and Bell's theory of cell-to-cell adhesion. An overview is given of biophysical techniques used in the analysis of the properties of bacterial surfaces and bacterial surface constituents and their integration with AFM. Finally, we discuss recent progress in the understanding of the role of bacterial interactions in medicine within the framework of the techniques and concepts discussed in the paper.     

GFP immunogold staining, from light to electron microscopy, in mammalian cells

GFP has emerged as an important reporter for monitoring gene expression, protein localization, cell transformation and cell lineage. The development of GFP as a marker in many different biological systems has emphasized the need to image GFP at high resolution. GFP immunogold labeling with colloidal gold particles becomes essential for electron microscopy (EM) ultrastructural detection. Because of the small size, colloidal gold particles require silver enhancement, a procedure to increase the size of the particle as well as gold toning to stabilize the silver layer. GFP preembedding immunogold staining enables high quality cellular-ultrastructural EM analysis mainly for two reasons, on one hand it allows adequate fixation for EM analysis maintaining GFP antigenicity, on the other hand it also enables the epoxy resins inclusion after immunogold staining. Both of them help to preserve better the ultrastructure. However GFP immunogold staining presents some drawbacks, such as the progressive decrease in immunogold labeling with tissue depth. Special attention must be taken when using GFP-tagged protein, since the fusion could interfere with their localization and function. In this review we provide a detailed protocol of the GFP immunogold staining, their main applications for EM and possible troubles.     

Antibacterial activities of magnesium oxide (MgO) nanoparticles against foodborne pathogens

The antibacterial activities of magnesium oxide nanoparticles (MgO NP) alone or in combination with other antimicrobials (nisin and ZnO NP) against Escherichia coli O157:H7 and Salmonella Stanley were investigated. The results show that MgO NP have strong bactericidal activity against the pathogens, achieving more than 7 log reductions in bacterial counts. The antibacterial activity of MgO NP increased as the concentrations of MgO increased. A synergistic effect of MgO in combination with nisin was observed as well. However, the addition of ZnO NP to MgO NP did not enhance the antibacterial activity of MgO against both pathogens. Scanning electron microscopy was used to characterize the morphological changes of E. coli O157:H7 before and after antimicrobial treatments. It was revealed that MgO NP treatments distort and damage the cell membrane, resulting in a leakage of intracellular contents and eventually the death of bacterial cells. These results suggest that MgO NP alone or in combination with nisin could potentially be used as an effective antibacterial agent to enhance food safety.     

Electron plasma waves generation in suddenly created isotropicplasma

Electron plasma waves excitation in suddenly created isotropic plasma as a result of weak nonlinear interaction of linearly polarized plane electromagnetic (EM) wave and electrons has been considered. By the use of standard perturbation method the problem is solved in closed form for the case of a simple harmonic source EM wave. The appearance of the second harmonic and time independent modes have been demonstrated. The efficiency of excitation of these modes is possible to control by varying the frequency of the source wave     

Formation of two-dimensional crystals of icosahedral RNA viruses

The formation of 2D arrays of three small icosahedral RNA viruses with known 3D structures (tomato bushy stunt virus, turnip yellow mosaic virus and bromegrass mosaic virus) has been investigated to determine the role of each component of a negative staining solution containing ammonium molybdate and polyethylene glycol. Virion association was monitored by dynamic light scattering (DLS) and virus array formation was visualised by conventional transmission electron microscopy and cryo-electron microscopy after negative staining. The structural properties of viral arrays prepared in vitro were compared to those of microcrystals found in the leaves of infected plants. A novel form of macroscopic 3D crystals of turnip yellow mosaic virus has been grown in the negative staining solution. On the basis of the experimental results, the hypothesis is advanced that microscopic arrays might be planar crystallisation nuclei. The formation of 2D crystals and the enhancing effect of polyethylene glycol on the self-organisation of virions at the air/water interface are discussed. SYNOPSIS: The formation of 2D arrays of icosahedral viruses was investigated by spectroscopic and transmission electron microscopic methods.     

The use of trehalose in the preparation of specimens for molecular electron microscopy

Biological specimens have to be prepared for imaging in the electron microscope in a way that preserves their native structure. Two-dimensional (2D) protein crystals to be analyzed by electron crystallography are best preserved by sugar embedding. One of the sugars often used to embed 2D crystals is trehalose, a disaccharide used by many organisms for protection against stress conditions. Sugars such as trehalose can also be added to negative staining solutions used to prepare proteins and macromolecular complexes for structural studies by single-particle electron microscopy (EM). In this review, we describe trehalose and its characteristics that make it so well suited for preparation of EM specimens and we review specimen preparation methods with a focus on the use of trehalose.     

Microwave-assisted tissue processing for same-day EM-diagnosis of potential bioterrorism and clinical samples

The purpose of this study was to explore the turnaround times, section and image quality of a number of more "difficult" specimens destined for rapid diagnostic electron microscopy (EM) after microwave-assisted processing. The results were assessed and compared with those of conventionally processed samples. A variety of infectious agents, some with a potential for bioterrorism, and liver biopsies serving as an example for routine histopathology samples were studied. The samples represented virus-producing cell cultures (such as SARS-coronavirus, West Nile virus, Orthopox virus), bacteria suspensions (cultures of Escherichia coli and genetically knockout apathogenic Bacillus anthracis), suspensions of parasites (malaria Plasmodium falciparum, Leishmania major, Microsporidia cuniculi, Caenorhabditis elegans), and whole Drosophila melanogaster flies infected with microsporidia. Fresh liver samples and infected flies were fixed in Karnovsky-fixative by microwaving (20 min), all other samples were fixed in buffered glutaraldehyde or Karnovsky-fixative overnight or longer. Subsequently, all samples were divided to evaluate alternative processing protocols: one part of the sample was OsO4-postfixed, ethanol-dehydrated, Epon-infiltrated (overnight) in an automated tissue processor (LYNX, Leica), and polymerized at 60 degrees C for 48 h; in parallel the other part was microwave-assisted processed in the bench microwave device (REM, Milestone), including post-osmication and the resin block polymerization. The microwave-assisted processing protocol required at minimum 3 h 20 min: the respective epon resin blocks were uniformly polymerized allowing an easy sectioning of semi- and ultrathin sections. Sections collected on non-coated 200 mesh grids were stable in the electron beam and showed an excellent preservation of the ultrastructure and high contrast, thus allowing an easy, unequivocal and rapid assessment of specimens. Compared with conventional routine methods, microwave technology facilitates a significant reduction in sample processing time from days to hours without any loss in ultrastructural details. Microwave-assisted processing could, therefore, be a substantial benefit for the routine electron microscopic diagnostic workload. Due to its speed and robust performance it could be applied wherever a rapid electron microscopy diagnosis is required, e.g., if bioterrorism or emerging agents are suspected. Combining microwave technology with digital image acquisition, the 1-day diagnosis based on ultrathin section electron microscopy will become possible, with crucial or interesting findings being consulted or shared worldwide with experts using modern telemicroscopy tools via Internet.     

新型金属丝靶电子源实验研究

电子束在基础科学研究、工农业生产和医疗领域发挥了重要作用。提出了一种新型的电子源技术方案:高功率激光脉冲轰击金属丝靶,可以产生大量能量在百keV量级的热电子,一部分热电子在丝靶表面自生电磁场的作用下沿着丝靶运动,丝靶后方可以获得指向性良好的电子束。实验上成功在金、钨和铜丝靶后方获得了电子束团,测量了束团束斑、电荷量和能谱。铜丝靶单发实验收集到的电子束团总电荷量可达3 nC,能量分布在0～240 keV区间内,能谱在100 keV附近呈现峰值。提出了微波压缩方案,设计了2腔微波聚束腔,利用ASTRA对微波腔压缩过程进行了模拟计算。结果显示,可以将电荷量1 nC、长度55 ps的束团压缩至27 ps,满足后续微波加速器对电子源的要求。     

Multi-channel Heterodyne Radiometer on HT-7 Tokamak

The incoherent radiation emanating out of the tokamak plasmas gives vital information about the electron temperature. Electron cyclotron emission (ECE) is a powerful diagnostic tool for the measurement of electron temperature due to many advantages such as the high spatial and temporal resolutions. This paper presents the sixteen channel superheterodyne radiometer as an electron cyclotron emission diagnostic to measure the electron temperature on HT-7 tokamak.     

Structure of mammalian spermatozoa in respect to viability, fertility and cryopreservation

Morphological assessment of spermatozoa has a long history and it is generally accepted that specific morphologic structural deviations correlate with male sub- and infertility. Although many different and also new methods are used in semen analysis, light microscopy is still used for routine morphological evaluation. This paper gives an overview about the detailed structure of physiological mammalian spermatozoa as well as the most common morphological deviations in correlation to fertility. This should be the basis for explanation of problems resulting from semen cryopreservation. General aspects of semen cryopreservation should be regarded before to facilitate the understanding of methods and mechanisms.     

Recent advances in imaging of properties and growth of low dimensional structures for photonics and electronics by XPEEM

Spectroscopic Photoemission and Low Energy Electron Microscopy (SPELEEM) is a very powerful and diverse microscopy technique for the investigation of surfaces, interfaces, buried layers and nanoscale objects like nanoparticles and nanowires. The many significant results from photoemission Electron microscopy (PEEM) in recent years are linked with the exploitation of advanced light sources such as synchrotrons and new advanced laser systems. Combined also with low energy electron microscopy (LEEM) it allows a complementary chemical and structural analysis making LEEM/PEEM a versatile multitechnique instrument. To illustrate the extreme diversity, we give a summary of the recent studies with the SPELEEM installed at the soft X-ray beamline I311 at the MAXII synchrotron storage ring and a portable electrostatic PEEM used with ultra-fast XUV laser technology. The examples cover topics such as full-cone 3D band mapping by using the photoelectron diffraction mode of the microscope, growth mechanism and detailed doping profile of III–V nanowires, growth and intercalation of graphene on SiC substrates, droplet dynamics on GaP(1 1 1) surface, surface chemistry and control of nanostructure fabrication. Moreover, the first results of PEEM experiments using extreme ultraviolet attosecond pulse trains are discussed.     

Ultrastructural pathology of experimental autoimmune uveitis: A review

Experimental autoimmune uveitis (EAU), a disease induced in animals immunised at distant sites with retinal antigens, in conjunction with appropriate adjuvants, is a well recognised paradigm of various forms of human uveitis since many of the clinical and histopathological manifestations of the human disease can be closely mimicked in this model. Electron microscopy and other nonultrastructural morphological techniques have proven valuable tools in studying the pathology of EAU due to the highly organised and heterogeneous nature of the affected cells and tissues within the eye, namely the neural retina, retinak pigment epithelium and choroid. The role of various resident ocular immune cells including mast cells, dendritic cells, choroidal macrophages and retinal microglia in the induction and pathogenesis of EAU, is reviewed. In addition, the role of various effector cells in the inflammatory cell infiltrate, namely polymorphonuclear leukocytes, T cells and macrophages, and their interaction with retinal target cells is discussed. This review also considers the morphological changes which precede and accompany early inflammatory cell infiltration through the blood-ocular barrier which are of critical importance to initiation of the autoimmune process. In many ways EAU represents an ideal model for autoimmune diseases in general and especially for those involving the central nervous system. The precise microanatomical arrangement of the eye and its distinct blood-tissue barriers have allowed researchers studying EAU to precisely identify the target cells, pathways used by ingressing inflammatory cells and the nature of the effector cells in this disease.     

Morphological Distribution in Micro-Injected Polypropylene Parts in the Presence of β-Nucleating Agent

Micro-injection molding is attracting much attention nowadays. Characterization of the morphological distribution in parts prepared by micro-injection molding is thus of growing importance. The morphological features of micro-parts may strongly differ from those of the macro-parts prepared by conventional injection molding, resulting in specific physical properties. In the present study, β-nucleated isotactic polypropylene micro-parts (μPPB) with 200 μm thickness, as well as macro-parts (PPB) with 2000 μm thickness, were prepared. Polarized light microscopy (PLM), scanning electron microscopy (SEM), differential scanning calorimetry (DSC), and wide-angle X-ray diffraction (WAXD) were used to investigate their morphological features. The results show that the morphology distribution in μPPB had many differences from that of the PPB. The one-dimensional WAXD and DSC analysis showed similar results; the degree of crystallinity of the μPPB was higher than that of the PPB. However, the content of β-crystals of μPPB was lower than that of the PPB. This can be explained by the restraining effect for the formation of β-crystals in β-nucleated isotactic polypropylene (iPP) under the strong shear field. The through-the thickness-morphology of both μPPB and PPB exhibited a “skin-core” structure from PLM observations, but the former had a large fraction of shear layer in comparison to the latter implied. The SEM observations showed that the shear layer of μPPB consisted of a highly oriented shish-kebab structure, while that of the core layer consisted of deformed spherulites structure. The two-dimensional WAXD pattern of the core layer of PPB, showing full Debye rings, indicated an overall random orientation of the iPP chains, while the arcing indicated a pronounced orientation in the shear layer. The more pronounced arcing of the μPPB indicated a more pronounced orientation.     

Assessment of thermal and antimicrobial properties of PAN/Zn-Al layered double hydroxide nanocomposites

Polyacrylonitrile-based Zn–Al layered double hydroxide composites (PAN/LDH) have been synthesised with different LDH content by in situ polymerisation technique. The nanocomposites were systematically studied by Fourier transform infrared spectroscopy, X-ray diffraction (XRD), thermo gravimetric analysis (TGA), field emission scanning electron microscope (FE SEM), high resolution transmission electron microscopy (HRTEM), energy dispersive spectroscopy (EDS) and antibacterial activity measurement. The successful formation of exfoliated nanocomposite was inferred from the XRD patterns and HRTEM images. The thermal decomposition of PAN was enhanced upon nanocomposite (PAN/LDH) formation. The antimicrobial activity of PAN/LDH nanocomposites is evaluated for antibacterial activity against some clinically important bacterial pathogens and the bacterial growth is monitored at different percentage of LDH. The PAN/LDH composites displayed considerable antibacterial activity, on the contrary the virgin PAN did not possess any antibacterial activity. The likely electrostatic interaction among LDH layers with charged surface of bacterial cell is assumed to be responsible for antimicrobial activity. The prepared nanocomposite has appreciable thermal stability in combination with antibacterial activities by which the material is suitable for packaging and fabrication in textile application.