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.     

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.     

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.     

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.     

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.     

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.     

Structure and function of skin in the pelagic sea snake,Hydrophis platurus

We describe and interpret the functional morphology of skin of the Yellow-bellied sea snake, Hydrophis platurus. This is the only pelagic sea snake, and its integument differs from what is known for other species of snakes. In gross appearance, the scales of H. platurus consist of non-overlapping, polygonal knobs with flattened outer surfaces bearing presumptive filamentous sensillae. The deep recesses between scales (‘hinge’) entrap and wick water over the body surface, with mean retention of 5.1 g/cm of skin surface, similar to that determined previously for the roughened, spiny skin of marine file snakes, Acrochordus granulatus. This feature possibly serves to maintain the skin wet when the dorsal body protrudes above water while floating on calm oceanic slicks where they forage. In contrast with other snakes, including three species of amphibious, semi-marine sea kraits (Laticauda spp.), the outer corneous β-protein layer consists of a syncytium that is thinner than seen in most other species. The subjacent α-layer is also thin, and lipid droplets and lamellar bodies are seen among the immature, cornifying α-cells. A characteristic mesos layer, comprising the water permeability barrier, is either absent or very thin. These features are possibly related to (1) permeability requirements for cutaneous gas exchange, (2) reduced gradient for water efflux compared with terrestrial environments, (3) less need for physical protection in water compared with terrestrial ground environments, and (4) increased frequency of ecdysis thought to be an anti-fouling mechanism. The lipogenic features of the α-layer possibly compensate for the reduced or absent mesos layer, or produce layers of cells that comprise what functionally might be termed a mesos layer, but where the organization of barrier lipids nonetheless appears less robust than what is characteristically seen in squamates.     

Beam-induced motion of vitrified specimen on holey carbon film

The contrast observed in images of frozen-hydrated biological specimens prepared for electron cryo-microscopy falls significantly short of theoretical predictions. In addition to limits imposed by the current instrumentation, it is widely acknowledged that motion of the specimen during its exposure to the electron beam leads to significant blurring in the recorded images. We have studied the amount and direction of motion of virus particles suspended in thin vitrified ice layers across holes in perforated carbon films using exposure series. Our data show that the particle motion is correlated within patches of 0.3-0.5 μm, indicating that the whole ice layer is moving in a drum-like motion, with accompanying particle rotations of up to a few degrees. Support films with smaller holes, as well as lower electron dose rates tend to reduce beam-induced specimen motion, consistent with a mechanical effect. Finally, analysis of movies showing changes in the specimen during beam exposure show that the specimen moves significantly more at the start of an exposure than towards its end. We show how alignment and averaging of movie frames can be used to restore high-resolution detail in images affected by beam-induced motion.     

ELECTRON MICROSCOPIC OBSERVATION OF SPECIMENS UNDER CONTROLLED GAS PRESSURE

A technique for encasing specimens in a thin gas layer during their observation in the Siemens Elmiskop I is described. All gases can be employed at pressures up to one atmosphere. Destruction of specimens can occur in the beam; all organic specimens are particularly liable to decompose. The conditions under which this can be avoided are given. A useful application of the technique allows one to prevent specimens from drying out, as they normally do in vacuum. A further application uses the controlled removal of carbon for thinning organic layers and for selective etching of organic materials.     

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.     

On the distribution of the Fire-bellied Toad,Bombina bombina,in Turkey

Abstract

The Lake of Köyceg¯iz (Köyceg¯iz Gölü) in southwest Turkey is influenced by several external factors such as sulfuric springs, :Mediterranean seawater and a relatively strong changing wind. These give rise to an exceptional hydrochemistry and hydrophysics, which are reflected in the phyto and zooplankton. The complicated layer structure of the lake is determined more by chemical gradients than by temperature. The water body is divided in two layers of differing hydrology. The upper layer is subject to full circulation while the lower one is strictly stratified by chemical gradients caused by the sulfuric sources and an influx of-Mediterranean water. The lake can be classified as meromictic. Subsurface freshwater springs may well influence the system, but to a lesser degree. Phyto- and zooplankton are characterized by a limited number of species and a low biomass. ’This is due to a very thin euphotic zone as well as the brackish character of the lake water.     

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.     

Dry under water: Comparative morphology and functional aspects of air‐retaining insect surfaces

Superhydrophobic surfaces prevent certain body parts of semiaquatic and aquatic insects from getting wet while submerged in water. The air layer on these surfaces can serve the insects as a physical gill. Using scanning electron microscopy, we investigated the morphology of air‐retaining surfaces in five insect species with different levels of adaptation to aquatic habitats. We found surfaces with either large and sparse hairs (setae), small and dense hairs (microtrichia), or hierarchically structured surfaces with both types of hairs. The structural parameters and air‐film persistence of these surfaces were compared. Air‐film persistence varied between 2 days in the beetle Galerucella nymphaea possessing only sparse setae and more than 120 days in the bugs Notonecta glauca and Ilyocoris cimicoides possessing dense microtrichia (up to 6.6 × 10⁶ microtrichia per millimeter square). From our results, we conclude that the density of the surface structures is the most important factor that affects the persistence of air films. Combinations of setae and microtrichia are not decisive for the overall persistence of the air film but might provide a thick air store for a short time and a thin but mechanically more stable air film for a long time. Thus, we assume that a dense cover of microtrichia acts as a “backup system” preventing wetting of the body surface in case the air–water interface is pressed toward the surface. Our findings might be beneficial for the development of biomimetic surfaces for long‐term air retention and drag reduction under water. In addition, the biological functions of the different air retention capabilities are discussed. J. Morphol., 2011. © 2011 Wiley‐Liss, Inc.     

Applications of high voltage electron microscopy to botanical ultrastructure

Although high voltage electron microscopes have been in general use over the past decade microscopists have tended to ignore the contribution their use could make to the study of plant ultrastructure. The majority of biological high voltage research has been restricted to the fields of zoology and bio-medicine.The high voltage electron microscope (HVEM) has several advantages over the conventional transmission electron microscope (CTEM) when applied to biological specimens. These include increased penetrating power of the electron beam, reduced chromatic abberation in thick specimens, and both reduced beam heating and ionization damage. All these factors permit the observation of thick sections, whole cells and hydrated specimens. Most botanical HVEM research has been restricted to the study of thick sectioned material. Various staining techniques have been applied to overcome the decrease in image contrast at high accelerating voltages, but the commonest have been modifications of lead and uranium stains previously developed for thin sections. Selective staining can simplify the mass of information in a thick specimen thus specific structures may be studied against an unstained background. Acidified phosphotungstic acid can be used to stain the plasma membrane and osmium impregnation will selectively stain many of the cytoplasmic membranes in a variety of specimens. Other techniques for the selective localization of cell components, such as enzyme cytochemistry and autoradiography have yet to be fully exploited by high voltage electron microscopists.Interpretation of the great quantity of information in a thick specimen can be facilitated by tilting the specimen and producing stereo pairs. Quantitative depth information can be extracted from stereo pairs by the use of measuring mirror stereoscopes or by direct measurement from each member of a stereo pair. Serial thick sectioning has been employed as an alternative to prolonged serial thin sectioning to aid in the reconstruction of large specimens.Stereo images can be viewed in a variety of ways with lenticular pocket stereoscopes, reflecting mirror stereoscopes, prismatic spectacles, polarized spectacles when projected onto a non depolarizing screen or presented on TV monitors.     

Superstructures of wet inactive chromatin and the chromosome surface

Superpacking of chromatin and the surface features of metaphase chromosomes have been studied by SiO replication of wet, unstained, and unfixed specimens in an exceedingly thin (≤ 1 nm) aqueous layer, keeping them wet. Hydrophilic Formvar substrates allow controlled thinning of the aqueous layer covering the wet specimens. Whole mounts of chromatin and chromosomes were prepared by applying a microsurface spreading method to swollen nuclei and mitotic cells at metaphase. The highest level of nucleosome folding of the inactive chromatin in chicken erythrocytes and rat liver nuclei is basically a second-order superhelical organization (width 150–200 nm, pitch distance 50–150 nm) of the elementary nucleosome filament. In unfavorable environments (as determined by ionic agents, fixative, and dehydrating agents) this superstructure collapses into chains of superbeads and beads. Formalin (10%) apparently attacks at discrete sites of chromatin, which are then separated into superbeads. The latter consist of 4–6 nucleosomes and seemingly correspond to successive turns of an original solenoidal coil (width 30–35 nm), which forms the superhelical organization. When this organization is unfolded, eg, in 1–2 mM EDTA, DNAse-sensitive filaments (diameter 1.7 nm) are seen to be wrapped around the nucleosomes. The wet chromosomes in each metaphase spread are held to each other by smooth microtubular fibers, 20–30 nm in diameter. Before they enter into a chromsome, these fibers branch into 9–13 protofilaments, each 5 nm wide. The chromosome surface contains a dense distribution of subunits about 10–25 nm in diameter. This size distribution corresponds to that of nucleosomes and their superbeads. Distinct from this beaded chromosome surface are several smooth, 23–30-nm-diameter fibers, which are longitudinal at the centromere and seem to continue into the chromatid structure. The surface replicas of dried chromosomes do not show these features, which are revealed only in wet chromosomes.     

Sporangia containing Scylaspora from the Lower Devonian of the Welsh Borderland

ABSTRACT. Bulk maceration of Early Devonian (Lochkovian) deposits from the Welsh Borderland has yielded eight specimens of a new type of sporangium characterized by its elongate shape and distinctive spores. The specimens have been examined using scanning electron, transmission electron and light microscopy. The elongate sporangia occur isolated and are fragmented to varying degrees. They contain trilete spores that possess a proximal surface with shallow murornate ornament and a distal surface that is laevigate. The spores belong to the dispersed spore genus Scylaspora , and this is the first report of such spores in situ . Ultrastructural studies demonstrate that the spore walls are bilayered with a lamellate inner layer overlain by an essentially homogeneous outer layer. There is little or no associated extra-exosporal material. To date this is the earliest reported example of lamellate wall ultrastructure in trilete spores. Models of spore wall development are suggested in the light of evidence provided by spore wall ultrastructure. Detailed comparisons of the characters preserved in the fossils (morphological, anatomical and ultrastructural), with those in other fossil and extant plants, currently shed little light on the phylogenetic relationships of these specimens, primarily due to the paucity of comparable data. It is proposed that the plant is probably of vascular status, but in the absence of evidence for vascular tissue, it must be classified as rhyniophytoid.     

Cytoskeletal network underlying the human erythrocyte membrane. Thin-section electron microscopy

A filamentous network underlying the human erythrocyte membranes can be clearly visualized in situ by electron microscopy of thin sections of specimens fixed with tannic acid-glutaraldehyde. The network is composed of two layers: the first, a layer of vertical components with granular appearance, which are seen to be directly associated with the membrane proper, and the second, a horizontally disposed, anastomosing meshwork of filamentous components, approximately 9 nm in thickness, which are attached to the vertical components. The diameter and appearance of the filamentous components are similar to those of purified spectrin. EDTA treatment (0.1 mM, pH 8.0), which was used to extract spectrin and actin, resulted in the disappearance of the filamentous meshwork, leaving only the granular components.     

Analysis of the spatial variability of maize root density

Water absorption by and seedling emergence of barley (Hordeum vulgare) seeds was studied in a two layer drying out system. Seeds were placed 3 cm below surface in sandy loam (Typic Ustochrept) soil having 4 or 7g.100g^–1 water underlain by wet (10g.100g^–1) layer 2, 4 or 6cm below seed. The study was carried out at 18°, 23°, 28° and 33°C with and without a thin liquid-flow barrier placed on top of the wet layer.Water absorption by seed and coefficient of rate of emergence showed parabalic relation with temperature and strong soil-water × temperature interactions. Liquid-flow barrier considerably reduced the seed water absorption, percent emergence and coefficient of rate of emergence showing thereby that liquid flow was the principal mode of upward water transport from the wet soil to the seed. Influence of both the wet soil and the liquid-flow barrier was detectable up to about 8 cm; shorter the distance greater the effect. It is concluded that in a drying out seed-zone, in addition to wetness of the soil surrounding the seed the wetness of the soil several cm below the seed is also crucial for seedling emergence. Also indicated that the optimum temperatures in drying out seed-zones are different from those in the absence of evaporation.     

Araldite as an Embedding Medium for Electron Microscopy

Epoxy resins are suitable media for embedding for electron microscopy, as they set uniformly with virtually no shrinkage. A mixture of araldite epoxy resins has been developed which is soluble in ethanol, and which yields a block of the required hardness for thin sectioning. The critical modifications to the conventional mixtures are the choice of a plasticized resin in conjunction with an aliphatic anhydride as the hardener. The hardness of the final block can be varied by incorporating additional plasticizer, and the rate of setting can be controlled by the use of an amine accelerator. The properties of the araldite mixture can be varied quite widely by adjusting the proportions of the various constituents. The procedure for embedding biological specimens is similar to that employed with methacrylates, although longer soaking times are recommended to ensure the complete penetration of the more viscous epoxy resin. An improvement in the preservation of the fine structure of a variety of specimens has already been reported, and a typical electron microgram illustrates the present paper.