Electron microscopy, electron diffraction, and element analysis of wet biological specimens

Temperature controlled differentially pumped environmental chambers now allow more routine examination of wet specimens in the electron microscope. A sensitive test of their efficiency is the ability to provide high resolution electron diffraction patterns from wet, unfixed protein microcrystals. Fortunately, wet specimens can be prepared with only a few tens of nanometers thickness of remaining water, so extraneous electron scattering by liquid water can be kept to a minimum. It still remains to be determined whether microprobe analysis (X-ray or electron energy-loss spectroscopy) using wet specimens gives better element localization in cells than the current freezing methods. More extensive comparisons are also required of the ultrastructural preservation and visibility of macromolecules immersed in a thin layer of water vs immersion in a thin layer of amorphous ice. However, the recent introduction of commercial forms of the necessary equipment now make these comparisons more feasible.     

Direct observation of unstained wet biological samples by scanning-electron generation X-ray microscopy

Analytical tools of nanometre-scale resolution are indispensable in the fields of biology, physics and chemistry. One suitable tool, the soft X-ray microscope, provides high spatial resolution of visible light for wet specimens. For biological specimens, X-rays of water-window wavelength between carbon (284 eV; 4.3 nm) and oxygen (540 eV; 2.3 nm) absorption edges provide high-contrast imaging of biological samples in water. Among types of X-ray microscope, the transmission X-ray microscope using a synchrotron radiation source with diffractive zone plates offers the highest spatial resolution, approaching 15-10 nm. However, even higher resolution is required to measure proteins and protein complexes in biological specimens; therefore, a new type of X-ray microscope with higher resolution that uses a simple light source is desirable. Here we report a novel scanning-electron generation X-ray microscope (SGXM) that demonstrates direct imaging of unstained wet biological specimens. We deposited wet yeasts in the space between two silicon nitride (Si₃N₄) films. A scanning electron beam of accelerating voltage 5 keV and current 1.6 nA irradiates the titanium (Ti)-coated Si₃N₄ film, and the soft X-ray signal from it is detected by an X-ray photodiode (PD) placed below the sample. The SGXM can theoretically achieve better than 5 nm resolution. Our method can be utilized easily for various wet biological samples of bacteria, viruses, and protein complexes.     

Progress in applying the high-voltage electron microscope to biomedical research

This review attempts a physical definition of the technical problems and achievements in applying the high-voltage electron microscope (HVEM) to biological and medical research. It is hoped that the review will summarize for biologists, funding agencies, and institutions the achievements of the HVEM, its future prospects, and the main problem areas that still need to be explored. At present it is not known whether future HVEMs will favor the fixed beam or the scanning transmission electron microscopy (STEM) mode. The STEM mode offers reduced radiation damage as a result of more efficient electron detection and ease of manipulation of the collected signals by separating the elastic and inelastic signals. Energy filtration to remove the inelastic signal provides a means to enhance the contrast and improve the resolution for thick specimens. Several prototype STEM-mode HVEMs are now under development and it is expected that, in a few years, comparisons of fixed beam and STEM modes will be possible. The review discusses several HVEM instrument features that remain poorly developed. In the area of image recording a photographic emulsion has been designed to give optimized performance at an acceleration voltage of 1 MV. However, this remains unavailable commercially. Conversion of the HVEM electron image to a usable light image by phosphors etc., involves some difficulties, making it difficult to obtain good performance from TV systems. Since the HVEM is particularly useful for three-dimensional imaging, the further development of improved goniometers for stereo viewing and image reconstruction is important. The large volume available in the objective specimen volume and the increased penetration at high acceleration voltages make the HVEM particularly suitable for the application of environmental chambers in the microscopy and electron diffraction of thick wet specimens. An improved signal-to-noise ratio improves the prospects for elemental analysis at high acceleration voltages. When carefully carried out, improved resolution can be obtained in dark-field over that obtainable at 100 kV. Dark-field provides the easiest way to obtain high contrast on weakly stained or unstained objects. Its further improvement requires the use of specially thick and shaped beam stops and apertures that are not penetrated by the 1 MV beam. Recent HVEM studies of whole cells and microorganisms are reviewed. These studies already show that the former thin-section approach led to some incorrect ideas about the shape of some organelles and their three-dimensional relationships. This new information is proving important in helping to establish the function of fibrillar and membranous components of the cell. The most important limitation in examining thick sections is the large depth of field that causes excessive overlap of in-focus structures in stereo views of thick sections. In a few cases special specific heavy metal stains have been developed to overcome this problem, but an optical solution would be more generally applicable. Attempts are now being made to unscramble overlapped detail by applying the image reconstruction techniques of tomography and holography. It is concluded that even with existing techniques, the HVEM examination of thick sections provides a very useful improvement in sampling statistics and in three-dimensional imaging of cell structures over that obtainable by examining thin sections at a lower acceleration voltage (100 kV). Randomized author sequence.     

Chemical and ultrastructural investigations of Mycobacterium bovis BCG: implications for the molecular structure of the mycobacterial cell envelope

Abstract The mycobacterial cell wall visualized by transmission electron microscopy (TEM) of thin sections of resin-embedded specimens is generally believed to consist of an electron-dense peptidoglycan, an electron-transparent arabinogalactan-mycolate layer and an electron-dense outer layer (OL). In addition, a pseudocapsule known as the ‘electron-transparent zone’ (ETZ) has been observed after phagocytosis of mycobacteria by macrophages. TEM of thin sections of Mycobacterium bovis BCG, Tice^® substrain, revealed an OL bilayer, each of which measured 2–4 nm in diameter. The intermediate electron-transparent layer varied from 1 to about 250 nm in diameter and appears to be a previously observed oxygen-dependent amorphous integument that consists of hot water-extractable neutral polysaccharides, especially a recently characterized α-glucan, comprising about 12% of the dry cell weight. This and other recent studies of BCG have revealed cell-surface features that may provide a better understanding of the outer mycobacterial cell envelope.     

Factors that Influence the Formation and Stability of Thin,Cryo-EM Specimens

Poor consistency of the ice thickness from one area of a cryo-electron microscope (cryo-EM) specimen grid to another, from one grid to the next, and from one type of specimen to another, motivates a reconsideration of how to best prepare suitably thin specimens. Here we first review the three related topics of wetting, thinning, and stability against dewetting of aqueous films spread over a hydrophilic substrate. We then suggest that the importance of there being a surfactant monolayer at the air-water interface of thin, cryo-EM specimens has been largely underappreciated. In fact, a surfactant layer (of uncontrolled composition and surface pressure) can hardly be avoided during standard cryo-EM specimen preparation. We thus suggest that better control over the composition and properties of the surfactant layer may result in more reliable production of cryo-EM specimens with the desired thickness.     

Measurement of the unstained biological sample by a novel scanning electron generation X-ray microscope based on SEM

We introduced a novel X-ray microscope system based on scanning electron microscopy using thin film, which enables the measurement of unstained biological samples without damage. An unstained yeast sample was adsorbed under a titanium (Ti)-coated silicon nitride (Si₃N₄) film 90 nm thick. The X-ray signal from the film was detected by an X-ray photodiode (PD) placed below the sample. With an electron beam at 2.6 kV acceleration and 6.75 nA current, the yeast image is obtained using the X-ray PD. The image is created by soft X-rays from the Ti layer. The Ti layer is effective in generating the characteristic 2.7-nm wavelength X-rays by the irradiation of electrons. Furthermore, we investigated the electron trajectory and the generation of the characteristic X-rays within the Ti-coated Si₃N₄ film by Monte Carlo simulation. Our system can be easily utilized to observe various unstained biological samples of cells, bacteria, and viruses.     

Cross-beta order and diversity in nanocrystals of an amyloid-forming peptide

The seven-residue peptide GNNQQNY from the N-terminal region of the yeast prion protein Sup35, which forms amyloid fibers, colloidal aggregates and highly ordered nanocrystals, provides a model system for characterizing the elusively protean cross-beta conformation. Depending on preparative conditions, orthorhombic and monoclinic crystals with similar lath-shaped morphology have been obtained. Ultra high-resolution (<0.5A spacing) electron diffraction patterns from single nanocrystals show that the peptide chains pack in parallel cross-beta columns with approximately 4.86A axial spacing. Mosaic striations 20-50 nm wide observed by electron microscopy indicate lateral size-limiting crystal growth related to amyloid fiber formation. Frequently obtained orthorhombic forms, with apparent space group symmetry P2(1)2(1)2(1), have cell dimensions ranging from /a/=22.7-21.2A, /b/=39.9-39.3A, /c/=4.89-4.86A for wet to dried states. Electron diffraction data from single nanocrystals, recorded in tilt series of still frames, have been mapped in reciprocal space. However, reliable integrated intensities cannot be obtained from these series, and dynamical electron diffraction effects present problems in data analysis. The diversity of ordered structures formed under similar conditions has made it difficult to obtain reproducible X-ray diffraction data from powder specimens; and overlapping Bragg reflections in the powder patterns preclude separated structure factor measurements for these data. Model protofilaments, consisting of tightly paired, half-staggered beta strands related by a screw axis, can be fit in the crystal lattices, but model refinement will require accurate structure factor measurements. Nearly anhydrous packing of this hydrophilic peptide can account for the insolubility of the crystals, since the activation energy for rehydration may be extremely high. Water-excluding packing of paired cross-beta peptide segments in thin protofilaments may be characteristic of the wide variety of anomalously stable amyloid aggregates.     

Oviposition behaviour of the aphid parasitoid,Aphidius ervi: Are wet aphids recognized as host?

Females of the central European population of the aphid parasitoid, Aphidius ervi, did not attack wet pea aphids (Acyrthosiphon pisum) that were washed previously with water. After 1 hour, this phenomenon disappeared and A. ervi attacked washed hosts to the same degree as dry ones. Similarly, A. ervi attacked dead aphids killed in liquid nitrogen readily if they were dry but not if they were wet. This effect was also reversible and disappeared after 1 h. When A. ervi females were foraging on broad beans (Vicia faba), they laid significantly more eggs into dry aphids than into wet aphids. Resource utilization of wet aphids, however, was significantly lower in this design than in Petri dishes, due to a changed drop-off behaviour of the aphid. We conclude that females did not use visual cues for host recognition but instead relied on chemical cues. These cues may be covered by a thin water layer directly after aphids became wet. Our results also demonstrate the importance of abiotic factors for the estimation of the reproductive success of parasitoids in the field.     

Application of electron tomography to fungal ultrastructure studies

Access to structural information at the nanoscale enables fundamental insights into many complex biological systems. The development of the transmission electron microscope (TEM) has vastly increased our understanding of multiple biological systems. However, when attempting to visualize and understand the organizational and functional complexities that are typical of cells and tissues, the standard 2-D analyses that TEM affords often fall short. In recent years, high-resolution electron tomography methods, coupled with advances in specimen preparation and instrumentation and computational speed, have resulted in a revolution in the biological sciences. Electron tomography is analogous to medical computerized axial tomography (CAT-scan imaging) except at a far finer scale. It utilizes the TEM to assemble multiple projections of an object which are then combined for 3-D analyses. For biological specimens, tomography enables the highest 3-D resolution (5 nm spatial resolution) of internal structures in relatively thick slices of material (0.2-0.4 microm) without requiring the collection and alignment of large numbers of thin serial sections. Thus accurate and revealing 3-D reconstructions of complex cytoplasmic entities and architecture can be obtained. Electron tomography is now being applied to a variety of biological questions with great success. This review gives a brief introduction into cryopreservation and electron tomography relative to aspects of cytoplasmic organization in the hyphal tip of Aspergillus nidulans.     

Improved Performance and Stability of Inverted Organic Solar Cells with Sol–Gel Processed,Amorphous Mixed Metal Oxide Electron Extraction Layers Comprising Alkaline Earth Metals

Stability of organic photovoltaic devices (OPVs) is a limiting factor for their commercialization and still remains a major challenge whilst power conversion efficiencies are now approaching minimum requirements. The inverted organic solar cell (iOSC) architecture shows promising potential for improving significantly the cell's working lifetime. However, when solution processed ZnO is used as electron extraction layer, an undesirable light‐soaking step is commonly required before the device reaches a non‐permanent maximum performance. This work investigates the use of Sr and Ba doped ZnO films, ZnSrO and ZnBaO, formed by sol‐gel deposition using molecular alkoxide precursor solutions, as electron extraction layers in a model iOSCs with poly [3‐hexylthiophene] (P3HT): [6, 6]‐phenyl C60 butyl acid methyl ester (PCBM) as the active layer. We show that using these ternary oxides the light‐soaking step can be circumvented by preventing a dipole forming between the oxide and the active organic layer as supported by electroabsorption spectroscopy measurements of the device built‐in field. It is suggested that Sr or Ba doping results in suppression/reduction of the oxygen adsorption at mobile oxygen vacancy sites on the metal oxide surface. Like in thin film transistor (TFT) applications, where materials like InGaZnO are rapidly becoming an important technology, the use of amorphous, mixed metal oxides allows improving the performance and stability of interfacial charge extraction layers for organic solar cells.     

The mucous coat on gill lamellae of rainbow trout (Oncorhynchus mykiss)

The existence of a layer of mucus covering the gill lamellae of healthy rainbow trout (Oncorhynchus mykiss) was investigated. Using cryo-scanning electron microscopy, a smooth, undulating, thin layer was observed which completely covered gill filaments and lamellae, thereby obscuring epithelial microridges. After processing cryopreserved gill arches in glutaraldehyde for conventional scanning electron microscopy, the layer was no longer present and epithelial microridges were clearly visible. The identity of this layer was investigated using cryopreserved gills which were treated in one of two ways. First, gills were incubated with a rabbit antiserum to gill mucus, with normal rabbit serum, or with phosphate-buffered saline. Following fixation in glutaraldehyde and processing, only the gill tissue incubated with the mucus-specific antiserum was still covered with the smooth layer. The layer was also retained on the gills of fish anesthetized in a solution containing mucusspecific antiserum and then processes in glutaraldehyde for conventional scanning electron microscopy. The tenacious nature of the mucous layer was demonstrated by its stability following exposure to formalin and a cationic detergent. Second, the presence of this layer was confirmed on gill tissue which was cryopreserved, followed by freeze-substitution and vapor fixation, and then examined by transmission electron microscopy.     

Development of structure-less pollen wall inCeratophyllum demersum L. (Ceratophyllaceae)

Developmental process of structure-less exine is studied in a hydrophilous plant,Ceratophyllum demersum L., with electron microscopy. The plant shows a characteristic feature in tetrad formation. A callose wall is not synthesized and exine initiation does not occur during the tetrad stage. After release of microspores, a trilaminar layer with two electron-dense lines is formed in the surface of each microspore. The trilaminar layer develops to a thin structure-less exine that is considered to consist of only an endexine. The unusual exine would be an adaptive feature for submersed pollination in fresh water.     

Water-conducting properties of lipids during pollen hydration

Based on the authors’ previous work an attempt has been made to study water flow in the lipid matrix during pollen hydration. The present study has demonstrated that in the presence of small amounts of water, the type of lipids used defined the time of hydration of pollen in vivo on the stigma and in vitro. Several approaches were used including cryo‐scanning electron microscopy, magnetic resonance imaging and Fourier transform infrared microspectroscopic imaging, with the purpose of detecting very small amounts of water. The results show that no water is detectable in the lipid matrix. It was observed and concluded that the water for pollen hydration accumulates as a thin layer at the contact side between pollen and stigma, during the normal process of pollination in plant species with a wet stigma. However, using the same species deprived of the stigma by cell ablation, it was shown that the layer of water observed in wild‐type plants is not necessary for pollen hydration.     

Structure, Cytochemistry, and Locomotion of Haemogregarina sp. from Rana berlandieri

Intra- and extracellular gametocytes of Haemogregarina sp. from Rana berlandieri were studied by light and electron microscopy. Locomotion in free gametocytes appears to be related to series of horizontal “peristaltic” waves of constriction, passing from anterior to posterior along the body. Intracellular gametocytes lie within a vacuole in the erythrocyte cytoplasm. The pellicle of the parasite consists of a trilaminar plasmalemma and an inner electron dense layer, beneath which lies a ring of 80 microtubules. The inner dense layer becomes thickened and modified in the apical region, to form a cap-like structure. The gametocytes contain a prominent nucleus, several mitochondria, and many granular inclusions. One type of inclusion consists of elliptical, electron-dense, profeinaceous bodies scattered throughout the cytoplasm, while other inclusions are larger and electron-opaque, polysaccharide in nature, and occur predominantly in the pre- and post-nuclear regions. In the electron microscope, pronounced pellicular folds were observed in longitudinally sectioned extracellular gametocytes. These folds are thought to represent the waves of constriction seen in motile specimens by light microscopy. The mechanism of movement of the parasite is discussed and compared with that in haemosporidian ookinetes, as well as in gregarines.     

Structure of the cell surface of the yeast Candida tropicalis and its relation to hydrocarbon transport

The surface structure of the hypdrocarbon-utilizing yeast Candida tropicalis was investigated by scanning and transmission electron microscopy (SEM and TEM respectively). The sample preparation technique was based on a rapid cryofixation without any addition of cryoprotectants. In subsequently freeze-dried samples the surface structure was analysed by scanning electron microscopy. Thin sections were prepared from freeze substituted samples. Both techniques revealed hair-like structures at the surface of hydrocarbon-grown cells. The hairy surface structure of the cells was less expressed in glucose-grown cells and it was absent completely after proteolytic digestion of the cells. When cells were incubated with hexadecane prior to cyryofixation a contrast-rich region occured in the hair fringe of thin sections as revealed by TEM. Since these structures were characteristic for hexadecane-grown cells and could not be detected in glucose-grown or proteasetreated cells it was concluded that they originate from hexadecane adhering to the cell surface and are functionally related to hexadecane transport. The structure of the surface and its relation to hydrocarbon transport are discussed in view of earlier results on the chemical composition of the surface layer of the cell wall.Abbreviations SEM Scanning electron microscopy - TEM transmission electron microscopy     

藏马鸡卵壳的扫描电镜观察

利用扫描电镜对我国特有珍禽──藏马鸡的卵壳进行了超微结构观察。电镜下显示：藏马鸡卵壳从内向外由壳膜层、锥体层、海绵层和表层等组成。壳膜层内层致密、含少量纤维,外层为纵横交错成网状的纤维结构,锥体层由许多乳头状突起密集排列组成,海绵层为似沉积岩层的层状结构,表层在卵壳最外面,上由具保护性的透明蛋白质薄膜覆盖。与同属的褐马鸡的卵壳进行比较,其超微结构存在差异。     

Morphological differences in the capsular polysaccharide of nitrogen-fixing Azotobacter chroococcum B-8 as a function of iron and molybdenum starvation

Abstract Azotobacter chroococcum B-8 was nutritionally stressed for iron (Fe) and molybdenum (Mo) by successive transfers on Fe-deficient and Mo-deficient solid-plating media and was subsequently observed with transmission electron microscopy after capsular staining in ruthenium red. Under Fe- and Mo-sufficient conditions, the capsule appeared to be condensed into a thin, electron-dense layer. Under iron stress, and to a lesser extent molybdenum stress, the capsule appeared more diffuse and extensive. This apparent diffusion was irreversible upon the addition of iron before processing. Thus there seems to be a correlation between capsule diffusion and metal starvation, as well as a correlation between capsule extent and metal starvation.     

CHANGES IN WATER ECONOMY IN RELATION TO ANATOMICAL AND MORPHOLOGICAL CHARACTERISTICS DURING THALLUS DEVELOPMENT INPARMELIA ACETABULUM

The influence on uptake and water loss of the structural changes experienced by Parmelia acetabulum during thallus development were investigated. Small specimens were characterized by flat lobes and a thin thallus and cortex. Large specimens appeared strongly rugose, imbricate and irregularly folded, and had a significantly thicker cortex and medulla than small thalli. Maximum water storage capacity did not differ between large and small thalli, although water retention was much higher in large thalli, presumably due to the interaction of structural characteristics and a higher boundary layer resistance. This translated into a longer duration of the period of thallus hydration in large thalli compared to small thalli. Incubation of thalli in water-vapour-saturated atmospheres induced full recovery of photosynthetic electron transport to the values before desiccation in small thalli but only induced a partial recovery in large thalli. The close correlation between photosynthetic electron transport and net photosynthesis during desiccation found in this species suggested that carbon-fixation activity could be regained to a larger extent by incubation of thalli in water vapour in small compared to large thalli. The higher ability for water vapour uptake of small thalli might allow them to efficiently use small amounts of intermittently available water or periods of high relative humidity. The significance of this differential ability to utilize water is discussed with regard to the known ecological preferences of the species.     

The extracellular matrix of the cuticle of Gordius panigettensis (Gordioiidae,Nematomorpha): observations by TEM,SEM and AFM

The cuticle of Gordius panigettensis (Sciacchitano, 1955) was studied by scanning electron microscopy (SEM), transmission electron microscopy (TEM) and atomic force microscopy (AFM). The cuticle is composed of 30-50 compact layers. The number of the layers is higher in the central part of the animal's body and decreases at the extremities. Each layer is composed of parallel tightly packed fibres approximately 640 nm in diameter and of indefinite length. The fibres run strictly parallel within each layer, while in adjoining layers they run at a variable angle from 45 degrees in the central body to 90 degrees in the extremities. Each fibre shows a barely detectable filamentous inner structure and is enveloped in a thin highly regular net formed by hexagonal meshes. Our results suggested that these fibres should be proteinaceous although non-collagenous. Thinner radial fibres run among the large fibres and across all the layers and span the whole thickness of the cuticle from the epithelial layer located deep underneath the large fibres up to the epicuticle on the external surface of the animal.