Electron-transparent Material Research Articles

Cell wall dynamics under conditions of diffuse growth in the thick-walled cortical tissue (prosoplectenchyma) of Ramalina usnea

AbstractA recent field study indicated that thalli of the beard lichen Ramalina usnea undergo diffuse (“intercalary”) growth throughout their length. We examined thallus sections with TEM to better understand how the highly thickened cell walls of the prosoplectenchymatous cortex behave under conditions of continued expansion. Cell protoplasts were surrounded by massive accumulations of structured electron-dense wall layers interspersed with amorphous, electron-transparent substances, visible as concentric rings in transverse section. Nearest the protoplast, electron-dense wall layers were distinct and more or less alternated with irregular deposits of electron-transparent material. With increasing distance from the protoplast, the electron-dense wall layers were increasingly disrupted and intermixed among the electron-transparent materials. New cell branches grew through the accumulated wall materials, interrupting the layers they penetrated while producing their own concentric wall layers. The differing amounts of cell wall material accumulated was further indication of the different relative ages of such neighbouring cells. These observations suggest that cell walls are disrupted by diffuse tissue expansion and continually replaced by new walls and wall materials deposited to their interior at the interface with the protoplast. This pattern of development, documented previously in R. menziesii and U. longissima, suggests that component cells of lichen prosoplectenchyma behave quite differently from those of diffusely expanding filaments studied in non-lichen-forming fungi, where a single, discrete cell wall is maintained throughout growth.

A new nature of microporous architecture with hierarchical porosity and membrane template via high strain rate collision

This paper presents the formation of an unusual porous structure at Al/Al interface joined by magnetic pulse welding. The porous structure consists of a hierarchical microporous architecture with pore size of less than 2 µm that represents more than 80% over the whole area, in which 38% of them are sub-micron size pores. It also exhibits ultra-thin wall, sufficiently thin enough to behave as an electron-transparent material with a wall thickness of 50 nm. The formation of this porous structure is attributed to a cavitation process of a molten material in three stages including, (1) nucleation, (2) growth and coalescence and (3) solidification. Further analysis of this cavitation process using a dedicated numerical simulation reveals that the nucleation and growth of pores may arise from vaporization governed by a rapid isothermal and isochoric expansion of liquid metal due to the high rate of depressurization. This result explains the potential mechanism of coalescence for the creation of the open porosity. The difference between cavitation onset and highly developed porous structure is attributed to the difference in depressurization during various welding conditions. Welding performed with high intense collision enables to have large impact pressure, therefore those welding conditions provide higher depressurization gap and severe fluid rupture due to significantly large inherent surface tension of the fluid. A fast solidification at a high cooling rate of 10 °C/ns enables to freeze the micropores and resulting with the solidified porous architecture.

Observation of a new B2 structured phase in Ti-15Mo (wt%)

The formation of non-equilibrium and transient phases in metastable beta titanium alloys during low temperature thermal treatments is currently of great interest, as they provide a potential method of controlling the size and distribution of the equilibrium alpha phase and, hence, the resulting mechanical properties. Here, for the first time, we report on the formation of a new, B2 structured phase in the Ti-Mo system. The phase was observed in electron transparent material during in situ, and following ex situ, heat treatment at 300 °C. The B2 phase was enriched in Mo compared to the surrounding matrix material and formed in regular arrays of approximately square cross-section particles interspersed by thin beta channels. Electron diffraction indicated that the lattice parameter of this new phase was smaller than that of the parent phase, leading to significant strain in the beta channels. Critically, the B2 phase was only observed in material that had been electro-polished prior to heat treatment, and, therefore, it is hypothesised that this phase forms as a result of the preparation method and thin foil effects.

Reproductive biology in Anophelinae mosquitoes (Diptera, Culicidae): Fine structure of the female accessory gland

The morphology and ultrastructure of female accessory reproductive glands of Anopheles maculipennis s.s., Anopheles labranchiae and Anopheles stephensi were investigated by light and electron microscopy. The reproductive system in these species is characterized by two ovaries, two lateral oviducts, a single spermatheca and a single accessory gland. The gland is globular and has a thin duct which empties into the vagina, near the opening of the spermathecal duct. Significant growth of the accessory reproductive gland is observed immediately after blood meal, but not at subsequent digestion steps. At ultrastructural level, the gland consists of functional glandular units belonging to type 3 ectodermal glands. The secretory cells are elongated and goblet shaped, with most of their cytoplasm and large nucleus in the basal part, close to the basement lamella. Finely fibrous electron-transparent material occupies the secretory cavity that is in contact with the end of a short efferent duct (ductule) emerging from the gland duct. The present study is the first detailed description of female accessory gland ultrastructure in Anophelinae and provides insights into the gland's functional role in the reproductive biology of these insects.

Occurrence of endospores within conidia and hyphal cells of morphologically atypical isolates of Ophiostoma guerci (Georg.) Nannf.

Ultrastructural observations of two <i>Ophiostoma querci</i> isolates presented in this paper showed endospores within hyphal cells and <i>Sporothrix</i> conidia. The endospores were apparently included in a matrix of an electron transparent material or were associated with unidentified granules scattered throught cytoplasm of enclosing cells. The endospores may be liberated by breakdown of the enclosing cell. When free they were observed giving rise to hyphae.

Large Area Plan-View Transmission Electron Microscopy Sample Preparation for Direct-Bonded Interfaces

A focused ion beam (FIB) sample preparation technique is developed to produce large areas of electron transparent material for plan-view transmission electron microscopy measurements from direct-bonded interfaces without thinning the bonded wafers. A FIB cut allows plan-view sample creation from the bonded interface region, and therefore rapid characterization of the defect density as a function of bonding conditions, substrate miscut, or pre-bonding surface treatments. Uniform electron transparent plan-view areas are extracted from the interface region to facilitate observation of the interface reconfiguration. To demonstrate the technique, the electrically active interfaces of a series of bonded III-V structures is studied as a function of substrate miscut as well as in-plane misorientation. Distinct differences in the interface reconstruction are observed and the interface reconstruction produces screw and edge dislocation networks at the III-V interfaces. The degree of twist and tilt is confirmed with both cross section electron microscopy measurements as well as in-plane x-ray diffraction measurements.

Characterization of LPCVD amorphous silicon carbide (a-SiC) as material for electron transparent windows

We investigate on the physical and chemical properties of non-stoichiometric amorphous silicon carbide (a-SiCx) thin layers deposited by low-pressure chemical vapor deposition (LPCVD). Characterizations are specifically tailored to understand the suitability of a-SiCx as electron transparent window material. The structure, composition, continuity, intrinsic stress, etch-rates and roughness of the layers are determined. The a-SiCx layers are highly uniform and continuous, showing tensile intrinsic stress and high chemical inertness. The layer with the lowest roughness (0.22 nm) and intrinsic stress (0.7 GPa) is employed to fabricate electron transparent windows of 16 nm for use in a Transmission Electron Microscope (TEM). The resulting windows are highly transparent, enabling the acquisition of TEM images down to the resolution limit of the microscope (0.12 nm). Moreover, the a-SiCx windows show a resistance to electron beam damage which is up to 9 times higher than that of the commonly employed low-stress LPCVD silicon nitride (SiNx). Therefore, the proposed a-SiCx is particularly suited for TEM experiments where high chemical inertness and/or long exposures to the TEM electron beam are required. In addition, the a-SiCx layer could advantageously be employed in the design of microelectromechanical systems (MEMS), especially those operating in harsh environments.

Antheridial dehiscence in ferns

We investigated the mechanism of antheridial dehiscence in ferns for the first time using fluorescence microscopy as well as scanning and transmission electron microscopy. The mechanism leading to antheridial dehiscence in Polystichum setiferum, Asplenium trichomanes and A. onopteris was found to depend on the different cellulose contents of the inner and outer walls of the ring cells detected with calcofluor white stain and the Thiery test. The extremely low cellulose content of the ring cell walls facing spermatozoids made them less mechanically resilient than external wall cells. When the ring cells absorbed water they expanded only into the antheridial cavity, pushing the gametes against the cap cell, which detached from the ring cell below and enabled spermatozoid release. The newly released spermatozoids were spherical bodies covered in cellulose fibrils. The significance of cellulose fibrils could be to isolate the gametes from each other, to reinforce the electron transparent material and to protect the gamete from pressure created by the ring cells during release.

Structure of male accessory glands of Bolivarius siculus (fischer) (Orthoptera, Tettigoniidae) and protein analysis of their secretions

In Tettigoniidae (Orthoptera), male reproductive accessory glands are involved in the construction of a two-part spermatophore; one part, the spermatophylax, is devoid of sperm and considered a nuptial gift. The morphology, ultrastructure, and secretion protein content of the male reproductive accessory glands from Bolivarius siculus were investigated. Two main groups of gland tubules open into the ejaculatory duct: the "first-order" glands, a number of large anterior tubules, and the "second-order" glands, smaller and more numerous tubules positioned posteriorly. Along with a further subdivision of the gland tubules, we here describe for the first time an additional gland group, the intermediate tubules, which open between first and second-order glands. The mesoderm-derived epithelium of all glands is a single layer of microvillated cells, which can be either flattened or cylindric in the proximal or distal region of the same gland. Epithelial cells, very rich in RER and Golgi systems, produce secretions of both electron-dense granules and globules or electron-transparent material, discharged into the gland lumen by apocrine or merocrine mechanisms, respectively. With one exception, a unique electrophoresis protein profile was displayed by each of the gland types, paralleling their unique morphologies. To assess the contribution of different types of accessory glands to the construction of the spermatophore, the protein patterns of the gland secretions were compared with those of the extracts from the two parts of the spermatophore. All samples showed bands distributed in a wide range of molecular weight, including proteins of very low molecular mass. However, one major high molecular weight protein band (>180 kDa) is seen exclusively in extracts from the first-order glands, and corresponds to an important protein component of the spermatophylax.

The morphology of ultrafine particles on and near major freeways

The morphology of ultrafine particles (UFPs; diameter < 100 nm) collected on and near major Los Angeles freeways in April 2006 is reported. Samples were size selected with a differential mobility analyzer, collected by a nanometer aerosol sampler, and analyzed using a transmission electron microscope. Typical observed morphologies included aggregates, spheres, irregularly shaped particles, and particles with multiple inclusions. For freeway aerosols with 50 nm electrical mobility diameter, most (>90%) electron-opaque particles were surrounded by an electron-transparent material. This suggests that much of these particles were heterogeneously internally mixed. The fraction of UFPs in a given morphology class collected on and at increasing downwind distance from the I-405 freeway was compared. The fraction of aggregates measured 90 m downwind of I-405 was significantly less than the fraction measured on the freeway ( p-value < 0.01). Because aggregates are a primary aerosol (directly emitted), this may indicate that secondary aerosol (formed in the atmosphere) becomes more prevalent with increasing distance from the freeway. The fraction of particles with multiple inclusions measured 90 m downwind of I-405 was significantly greater than the fraction measured on the freeway ( p-value < 0.01). The increase in the number of particles with multiple inclusions with increasing distance from the freeway suggests that dilution does not prevent particles from colliding and merging which may alter the particle size distribution.

Fine structure of the spermatheca and of the accessory glands inOrchesella villosa(Collembola, Hexapoda)

The spermatheca and the accessory glands of the collembolan Orchesella villosa are described for the first time. Both organs exhibit ultrastructural differences, according to the time of the intermolt in which the specimens were observed. A thick cuticular layer lines the epithelial cells of the accessory glands. In the reproductive phase, they are involved in secretory activity; a moderately dense secretion found in the apical cell region opens into the gland lumen. Cells with an extracellular cistern are intermingled with the secretory cells. These cells could be involved in fluid secretion, with the secretory product opening into the cistern which is filled with an electron-transparent material. After the reproductive phase, the gland lumen becomes filled with a dense secretion. The accessory gland secretion may play a protective role towards the eggs. The spermatheca is located between the accessory glands; its epithelium is lined by a thin cuticle forming spine-like projections into the lumen and consists of cells provided with an extracellular cistern. Secretory cells, similar to those seen in the accessory glands, are missing. Cells with a cistern could be involved in the production of a fluid secretion determining sperm unrolling and sperm motility.

Ultrastructure of the early stages of Colletotrichum acutatum infection of strawberry tissues

The early stages of the infection of attached leaves and petioles of strawberry (Fragaria ×ananassa Duch. 'Camarosa') by Colletotrichum acutatum Simmonds were studied using scanning and transmission electron microscopy. Pre-penetration events of these tissues were similar, but the production of secondary conidia (microcyclic conidiation) was detected only on leaves. At the ultrastructural level, different stages of maturation of appressoria were observed and described. In young appressoria, the cell wall was composed of two layers and the plasma membrane displayed a wavy appearance. In the following stage, the appressorium developed a third electron-transparent layer between the cell wall and the plasma membrane. This new electron-transparent material was especially visible in the region of the appressorium near the cuticle. The plasma membrane of this appressorium showed a smooth appearance. Afterwards, a penetration peg emerged through the pore penetrating the cuticle and reached the epidermal wall where it enlarged to form an intramural infection vesicle. Both structures of infection, the penetration peg and the intramural infection vesicle, produced during the early phases of infection of strawbery tissues by C. acutatum, have not been previously reported and confirm that its invasion strategy is that of a subcuticular intramural pathogen. Once the infection was well established, abundant subcuticular and intramural hyphae were produced on petioles, causing severe degradation of the host cell walls. Occasionally, the cuticle appeared disrupted in those regions where the host walls were very degraded and dilated. Differences between colonization of petioles and leaves were observed.Key words: Colletotrichum acutatum, Fragaria ×ananassa, infection vesicle, invasion strategy, penetration peg.

Biogenic particles in the surface microlayer and overlaying atmosphere in the central Arctic Ocean during summer

Transmission electron microscopy photographs of airborne particles are compared with those of particles found inthe surface microlayer of the open water between ice floes in the central Arctic Ocean in summer. The similarity inmorphology, physical properties, X-ray spectra and a chemical reaction of the numerous aggregates and their buildingblocks predominantly smaller than 70 nm diameter, and of bacteria and other micro-organisms found in both, stronglysuggests that the airborne particles were ejected from the water by bursting bubbles. The shape of the size distribution ofaggregates in the air is very similar to that in the water, each with a well-defined Aitken mode but shifted towards smallersizes. Diffuse electron-transparent material joining and surrounding the heat resistant and non-hygroscopic particulatesin both the air and water is shown to have properties consistent with the exopolymer secretions (EPS) of microalgae andbacteria in the water. EPS are highly surface-active, highly hydrated molecules that can spontaneously assemble intogels. They are broken down by ultraviolet light or acidification. These properties provide an explanation for the differentresistance to dehydration of bacteria from air and water samples when subjected to a vacuum, and the apparent absenceof sea salt on airborne bacteria and aggregates. The difference in size distribution between the air and water samples isalso explained. The role of EPS and particulate matter from the open lead surface microlayer in the production of theairborne Aitken mode particles and cloud condensation nuclei is examined and concluded to be very important.

Which morphological forms of the fungus Aureobasidium pullulans are responsible for pullulan production?

Attempts were made to clarify the precise location and possible site of production of the α-glucan pullulan in different morphological forms of the fungus Aureobasidium pullulans. Gold-conjugated pullulanase was used as the specific probe for this purpose. No cell wall pullulan-like material was detected by transmission electron microscopy (TEM) in any morphological form of this fungus, although intracellular electron transparent material bound this probe. When silver enhancement of this gold-conjugated pullulanase probe was used, the data strongly suggested that only swollen cells and chlamydospores, and neither hyphae nor unicellular blastospores, often held responsible for pullulan formation, appeared to produce pullulan-like material.

Electrolytic preparation of metallic thin foils with large electron-transparent regions

This paper presents a successful technique for the preparation of thin foils for examination in the transmission electron microscope with large regions of electron-transparent material. The technique is also demonstrated to achieve similar large, thin areas from the cross-section of very thin materials down to a sheet thickness of approximately 180 μm.

Aluminium Effects on Microtubule Organization in Dividing Root-Tip Cells of Triticum turgidum. II. Cytokinetic Cells

were examined, using tubulin immunolabeling and electron microscopy. In cells, which at the beginning of the treatment were at a transitional stage between anaphase and telophase, the transformation of the interzonal microtubule (Mt) system into a phagmoplast was delayed. In cells treated at a telophase/ cytokinetic stage, the lateral phragmoplast expansion towards the cell periphery was delayed or inhibited. Besides, in cells entering telophase through an abnormal mitosis, Al inhibited phragmoplast formation and induced the organization of atypical tubulin bundles. The latter formed a network around the reassembling polyploid nucleus. The Al-effects resulted in the disturbance of cytokinesis and the formation of binuclear or polyploid cells, which lacked typical Mts. Instead of them, the post-telophase cells displayed atypical tubulin aggregations. In addition, Al affects cell plate development. Dividing cells, encompassing early interphase daughter nuclei, contained incomplete, atypical cell plates. The latter were quite thick, wavy and perforated, showing large “islands”, which contained electron transparent material. In some cells, the atypical cell plates gave rise to incomplete daughter walls, but in some others they were dismantled. The aberrant cell plates as well as the young daughter cell walls fluoresced intensely after aniline blue staining, an observation suggesting that they contain significant quantities of callose. The above findings combined with those derived from the study of the Al-effects on the mitotic spindle show that Mt cytoskeleton is a target site of Al toxicity in dividing cells.

Spermiogenesis and spermatozoon ultrastructure of Polydora neocaeca (Polychaeta: Spionidae) from Rhode Island

Summary The ultrastructure of spermatozoa and spermiogenesis in Polydora neocaeca is described. Most morphological features and measurements of cellular components of the spermatozoa are similar to previously studied members of the genus Polydora. However, early spermatids contain finely granular chromatin which becomes finely fibrillar in late spermatids and uniformly electron-dense in mature spermatozoa. Fibrillar chromatin morphology has not been reported within the Spionidae and may represent a species-specific trait or a previously unrecognized transient developmental process. Spermatozoa of Polydora neoceaca exhibit an elongate, cylindrical nucleus and a bullet-shaped acrosome that contains channels of electron transparent material. No microtubules are associated with nuclear elongation. The basal body of the axoneme occupies a short implantation fossa in the nucleus. The middlepiece contains 6–10 mitochondria closely aligned with the axoneme. Platelets, presumably carbohydrate storage bodies, are found along the nucleus and middlepiece. Additional electron-dense granules occur in the cytoplasm of the middlepiece.

Ultrastructural features of spermatocytes and spermatozoids in the fern Phyllitis scolopendrium (L.) Newm. subsp. scolopendrium

Phyllitis scolopendrium Newm. subsp. scolo-pendrium spermatozoids are cells 10 µm long in the form of spirals with about four turns. Their chromatin is partly honeycomb-shaped and partly highly condensed. The nuclear envelope over the latter has a regular, thin intermembrane space crossed by fibers that are probably involved in connecting the chromatin with elements of the microtubular ribbon. The cytoplasm is traversed by long cistern-shaped folds of the plasma membrane, believed to be involved in a late process of cell simplification through segregation and detachment of parts of the cytoplasm. The spermatozoids are embedded in 1–1.5 µm thick amorphous electron-transparent material containing cellulose fibrils. These fibrils are considered a network connected to the original spermatocyte wall and elements of elastic support for the amorphous material. The different polysaccharide composition of the inner and outer parts of the walls causes changes in the size and shape of the ring cells, so that the spermatozoids are pushed against and past the cap cell. The gametes are released through limited laceration of the cap cell. The laceration is due to the generally weak substructure of the cell wall. A light microscope sequence of spermatozoid release and scanning electron microscope features of newly released spermatozoids are shown.

Spermiogenesis, sperm structure and spermatozeugmata in the gymnolaematous bryozoan Electra pilosai (Bryozoa, Gymnolaemata)

Summary The spermatozoa of the gymnolaematous bryozoan Electra pilosa are thread-like cells about 350 μm long. The head region is needle-shaped, consisting of a long thread-like nucleus tapering anteriorly. No acrosorne is present. The midpiece consists of an axoneme surrounded by two long mitochondrial derivatives and two thin, nonmembrane-bounded granular rods alternating with the mitochondria. The two mitochondrial derivatives are of unequal size. The larger one is provided with lamellae of alternating electron-dense and electron-transparent material. Both mitochondria have a crescent form when seen in transverse section. In the tail region the axoneme is surrounded by a tube-like sheath composed of a thick cell membrane. An annulus is located at the junction between midpiece and tail. The flagellum is of the 9+2 type and in the tail region the number of microtubules is reduced. The nine peripheral components of the axoneme continue as single microtubules. Spermatozoa of Electra are tightly arranged in unencapsulated aggregates of c. 30 (32) cells capable of active swimming. Sperm heads and midpiece regions have a regular parallel orientation in the spermatozeugmata and are probably connected with each other by mucus. The presence of spermatozeugmata is indicative of internal fertilization in Electra. Ent-aquasperm of the type found in Electra pilosa are probably the most common type of gymnolaemate sperm. Sperm structure supports the hypothesis that Stenolaemata and Gymnolaemata are more closely related with each other than either of them is with the Phylactolaemata.

Colonization of beech leaves ( Fagus sylvatica) by the endophyte Discula umbrinella (teleomorph: Apiognomonia errabunda)

Kinetics of the colonization process of beech leaves by Discula umbrinella have been studied. D. umbrinella can either form an appressorium at the end of the germ-tube or penetrate subcuticularly. Penetration through the host cell wall is achieved by a comparatively fine penetration hypha that pierces the cuticular membrane and the epidermal cell wall; the extracellular matrix usually present around both external fungal hyphae and appressoria is absent in subcuticular and subepidermal hyphae. Cuticular and epidermal cell-wall degradation was evident around the penetration hypha. Subcuticularly the fungus was found in the reticulate part of the cuticle, underneath the amorphous layer of the cuticular membrane. The epidermal cell wall underneath the subcuticular hyphae was at least partly digested, and the cytoplasm contained electron-dense compounds that may be of phenolic origin and are probably related to the infection process. At this stage the parenchyma cells were morphologically still intact. At later stages of infection D. umbrinella grew through the epidermal cell wall into the parenchyma and colonized mainly the intercellular spaces of the mesophyll, although intracellular growth was occasionally seen. Transmission electron microscopy studies of appressoria and subcuticular hyphae suggest that enzymic activities could be mainly responsible for fungal penetration. Enzymic digestion of cuticle and epidermis cell walls is also suggested by the presence of electron-transparent material in beech leaf sections, which results probably from the digestion of the cell wall in the vicinity of penetrating hyphae. These results indicate that endophytes penetrate into their host in ways similar to those described for biotrophic and hemibiotrophic pathogenic fungi. Under in vitro conditions, D. umbrinella is able to colonize host tissues, causing conspicuous necroses. The time between infection and onset of the first symptoms varied from 48 h to several days, depending on the infection conditions. During the latent period the fungus is mainly located at the subcuticular or subepidermal level.