Microscopical methods promote the understanding of the integument biology of Rhinella ornata

In Rhinella ornata, the integument of different body regions was investigated using low-vacuum scanning electron and light microscopy through histochemical and immunohistochemical methods, and revealed the basic structure found in other anurans. Keratinocytes formed the keratinized squamous stratified epithelium, and flask cells occurred among the epidermal superficial layer. Just below the epidermis, the dermis was subdivided into a spongious dermis and a compact dermis. Mixed and granular glands were located in the spongious dermis, and myoepithelial cells surrounded their secretory portions. The Eberth-Katschenko (EK) layer occurred as basophilic areas between the spongious and compact dermis and throughout the spongious dermis. A series of alternating layers of bundles of collagenous fibers characterized the compact dermis, being firmly attached to a thin hypodermis. Regarding the morphological features, each integument region revealed distinct structural aspects. The dorsal integument was rougher than ventral, having conical tubercles and spines upon the verrucae. In the ventral and subgular regions, the slightly elevations are visualized, tubercles were absent but some spines occurred. The pelvic integument had a non-keratinized epidermis with an irregular profile and small poorly developed elevations. In addition, the EK layer was absent, and the presence of numerous blood vessels suggests its participation in water absorption and distribution. In the parotoid gland, the epidermis was similar to the dorsal integument; except that the reticular dermis was present, and contained three exocrine glandular types. Expression of cytokeratin was evident in the basal and intermediary layer, but absent in the cornified layer. Flask cells showed strong cytokeratin labeling. Consequently, the integument had the basic morphology, but exhibits regional characteristics, which may be related to the physiology of each surface.     

Dermal collagen organization in Bufo ictericus and in Rana catesbeiana integument (Anuran, Amphibian) under the evaluation of laser confocal microscopy

Collagen structural organization plays an important role in the mechanical property of the vertebrate integument. Bufo ictericus and Rana catesbeiana integument was investigated by light microscopy and laser confocal microscopy. Collagenous elements of the dermis were statistical analyzed. The integument is formed by the keratinized squamous stratified epidermis supported by the dermis that is subdivided into the spongious layer with a loose arrangement, and the compact layer formed by collagenous fibers arranged compactly in a criss-crossed manner. Thick collagenous columns have a perpendicular trajectory, and are formed by the assembling of alternating collagenous lamellae in both animals. Short intercolumns of collagenous fibrils connecting collagenous lamellae obliquely or transversally are observed in R. catesbeiana dorsal integument. The present study provides evidences that B. ictericus and R. catesbeiana integument has well-organized compact dermis, constituted by collagenous lamellae in a plywood manner. The integument organization is in contrast to the literature in some aspects. This dermal arrangement is important to the biomechanical property of both anuran integuments.     

Hyaluronan in the epidermal and the dermal extracellular matrix: its role in cutaneous hydric balance and integrity of anuran integument

New researches have revealed that hyaluronan (HA) is not a passive molecule. HA has being pointed out to participate in many processes, such as cell interactions, proliferating and migrating cell events and function as hydrate agent. The present study was undertaken to localize HA in Bufo ictericus integument providing a better understating for the role of cutaneous HA. Paraffinized sections were stained with haematoxylin-eosin and with 1% Alcian blue 8GX at pH 1.0 and 2.5. Alcianophilic reaction was visualized in both spongious dermis and Eberth-Katschenko layer. The mucus cells of mucus glands were also stained with AB methods. Besides these histological techniques, the localization of HA was performed using the FITC-labeled HA probe labeled with fluorescein isothiocyanate. In the extracellular matrix of spongious dermis, the reaction for HA was evident, being less intense in hypodermis and in pericellular keratinocyte matrix of the cornified tubercles regions. Thus, since HA was localized in the pericellular epidermal matrix and in the spongious dermis of anuran integument, it plays an important role in hydric balance, and is essential for integument integrity and functionality.     

Morphological characterization of the anuran integument of the Proceratophrys and Odontophrynus genera (Amphibia, Anuran, Leptodactylidae)

The morphological characteristics of the leptodactylid integument of Proceratophrys and Odontophrynus genera were investigated by means of stereoscopic, low vacuum scanning electron and light microscopy. The integument surface of Proceratophrys boiei, Proceratophrys laticeps and Proceratophrys appendiculata exhibited several projections, while the integument of Odontophrynus americanus had rounded elevations with smooth profile. Light microscopic observations showed the basic integument morphology for all anurans, i.e., an epidermis and a dermis, which is subdivided into a spongious layer and a compact layer. The epidermis is formed by basal, intermediary and cornified layers. However, in Proceratophrys genus the cornified layer had an irregular outline, while in O. americanus the external surface was smooth. In the spongious dermis, mucous and venom exocrine glands were observed, but in O. americanus an exclusive glandular type with apocrine secretory pattern was identified. The integument morphology showed peculiar characteristics that may be helpful for genus distinction. Thus, morphological methods may be considered as an efficient means to characterize and to differentiate anuran genera.     

Structure of the sexually dimorphic gland of Cycloramphus fuliginosus (Amphibia, Anura, Cycloramphidae)

Cycloramphus fuliginosus males (Amphibia, Leptodactylidae) have discoid glandular elevations on the abdominal inferior body region which are considered a characteristic of genus. In this work, this sexually dimorphic gland of C. fuliginosus was investigated by low vacuum scanning electron microscopy, light microscopy, and transmission electron microscopy. In this cycloramphid, the covered integument exhibits the basic structure: the epidermis, a keratinized squamous stratified epithelium with flask cells, and the dermis. The dermis is subdivided into spongious and compact dermal layers. In the iliac gland region, an aggregate of small mucous glands and larger syncytial tubuloalveolar glands occur in the spongious dermis, and they do not go beyond the Eberth-Katschenko layer limit. The adenocytes of the mucous gland produce neutral glycoproteins, in contrast to the larger tubuloalveolar glands that elaborate a proteinaceous secretion. Myoepithelial cells surround the alveoli, and play an important role in the secretion extrusion. The final secretion, elaborated by the iliac gland, is a mixture of mucus and protein. Both secretions are important to the cycloramphid biology, and may act as pheromone and/or as chemical parental care.     

Structural aspects of the Eberth-Katschenko layer of Bufo ictericus integument: histochemical characterization and biochemical analysis of the cutaneous calcium (Amphibian, Bufonidae)

Anuran, such as Brazilian toad Bufo ictericus Spix, 1824, possess a calcified dermal layer called the Eberth-Katschenko (EK) layer located between the stratum spongiosum and the stratum compactum. It has been regarded as a protective barrier against desiccation or involved in storage and mobilization of calcium, or a barrier for the interchange of substances between the animal's internal and external environment. The B. ictericus integument was removed from the ventral and dorsal regions and examined by light microscopy, using histochemical techniques, and also submitted to biochemical calcium analysis. The intermediate layer is strongly basophilic and metachromatic. In the EK layer Alcian blue at different critical electrolytic concentration revealed hyaluronic acid, which coexists with the dermal calcium. We conclude that the EK layer of B. ictericus is a discontinuous layer and is formed of hyaluronic acid and a sulfated GAG different from chondroitin sulfate, heparin and heparan sulfate or keratan sulfate. The amount of cutaneous calcium is independent of region and sex. GAGs may play an important role in dermal hydric balance, protecting the animal against desiccation, and hyaluronic acid is probably able to anchor dermal calcium. Thus, all compounds of the EK layer are essential for integument integrity and functionality.     

Ultrastructure of the skin melanophores and iridophores in paddlefish, Polyodon spathula

The ultrastructure of melanophores and iridophores of Polyodon spathula has been examined by transmission electron microscopy. In the skin, two types of chromatophores, melanophores and iridophores were founded. Melanophores were localized both in epidermis and dermis. Epidermal melanophores were present on the dorsal region of the trunk, sides, outer surface of the operculum and rostrum. Iridophores were founded in the dermis from ventral skin. The cytoplasm of iridophores is filled with reflecting platelets with variable orientation. The length of the long axis of the platelets varies from 1 to 2.10 microm.     

Histochemistry of skin glands of Trachycephalus aff. venulosus Laurenti, 1768 (Anura, Hylidae)

The skin of the adult tree frog, Trachycephalus aff. venulosus, was investigated by light microscopy. Histochemistry was carried out using various stain techniques and showed the basic integument morphology of the skin with squamous epithelium in the epidermis and connective tissue in the dermis, subdivided into spongy and compact layers. The integument observed contains two types of gland; the mucous glands and granular glands. The mucous glands are small and located in the upper layer of the stratum spongiosum of the connective tissue and contain neutral and acid sulfated glycoproteins. The granular glands are large and form a syncytial secretory compartment within the acinus, which is surrounded by smooth muscle cells. Histochemical analyses demonstrated the presence of collagen proteins in both types of gland. An unusual finding was the different densities of the granules in the granular glands. The morphological characteristics of the integument in Trachycephalus aff. venulosus was also compared with others species of Anura.     

Ultrastructure and morphometry of the egg of Psorophora albigenu Lutz, 1908 (Diptera: Culicidae)

Eggs of Psorophora albigenu were analyzed by light and scanning electron microscopy. Eggs were laid singly and were black and elliptical in outline. A length of approximately 586.4 ± 10.83 μm, a width of 172.3 ± 8.09 μm (n=30), and an egg index (length/width ratio) of 3.33 were observed. The anterior extremity tapered abruptly from a width of 64.9 μm, while such tapering was more gradual at the posterior extremity from a width of 77.4 μm. The ventral surface of the chorionic coating presented cells with tubular aspect containing tubercles in rows with a density of 9-16 tubercles per cell. These tubercles presented two different sizes, with the largest measuring 7.87 ± 01.58 μm and the smallest measuring 0.71 ± 0.53 μm (n=30) in longitudinal diameter. The micropyle orifice was very evident, with a diameter of 3.36 μm. The morphometric characteristics of Ps. albigenu obtained in this study were distinct from those of other species of the Psorophora genus, including Psorophora varipes which has been considered for a long time to be synonymous with Ps. albigenu.     

Mouthparts of Megaselia scalaris (Loew) (Diptera: Phoridae)

Mouthparts of adult Megaselia scalaris (Loew), a fly species of medical importance, were examined using scanning electron microscopy. Sexual dimorphism of the labellum was observed in the mouthpart structures of this species. The labella of males were clothed with a dense covering of microtrichia, but these were found to be entirely absent from the labella of females. Aside from this difference, trichoid and conical sensilla that are most likely used as taste or contact chemoreceptors appear on the labellum and labrum of both sexes. In addition, five pairs of sharply pointed teeth at the ventral surface of the labellum is another feature that is shared by the two sexes. A plausible feeding mechanism for this fly is also advanced.     

Glycosaminoglycans and glycoconjugates in the adult anuran integument (Lithobates catesbeianus)

Glycosaminoglycans (GAGs) from the integument of Lithobates catesbeianus were biochemically characterized and histochemically localized. Moreover, carbohydrate distribution was investigated using conventional and lectin histochemistry at light microscopy. Hyaluronan (HA), dermatan sulfate (DS) and a heparanoid were found in the integument. Sulfated and carboxylated GAGs were visualized in the Eberth-Katschenko (EK) layer, in the mucous glands, in the hypodermis as well as in the mast cells. Furthermore, glucose and galactose were identified in the integument through thin layer chromatography (TLC) assays. N-Acetyl-β-glucosamine residues were identified in the mucous glandular cells, between the corneum and spinosum strata, in the subepidermal region, and in the EK layer. N-Acetyl-galactosamine residues were evident in the EK layer, corresponding to a residue of the dermatan sulfate chain, which may be related to the collagenous fiber arrangement. These glycoconjugates occurred as secretory glandular products and as dermal structural elements. Moreover, HA and DS are the predominant GAGs in the L. catesbeianus integument. Considering the importance of glycoconjugates, they play a significant role to the integrity of the skin, providing mechanical support for integument cells. In addition, they are important to the water regulation mechanisms, since L. catesbeianus is preferably aquatic.     

A new structural model of bivalve ligament from Solen grandis

It is widely accepted that the structure of bivalve ligament contains two layers: outer organic lamellar and inner aragonite fibrous layer. However, by using scanning electron microscopy (SEM), X-ray diffraction (XRD), and infrared spectroscopy (FTIR), we found an additional protein fibrous layer in the Solen grandis ligament. The ligament consists of three layers: an outer layer (OL), middle layer (ML), and inner layer (IL). OL consists of lamellar protein, oriented normal to dorsal edge. ML has unique spring-like protein fibers (ca. 120 nm in diameter) structure, stretching continuously from the left to right valve. Interestingly, IL comprises of aragonite fibers (ca. 100 nm in diameter) oriented posteriorly and inclined at an angle about 26° to dorsal edge at dorsal part; then they turn gradually to ventral part, showing striking zigzag structure vertical to ventral surface (VS). The ligament presents a new three-layered structural model different from previous reports; it may inspire not only in materials synthesis and mechanical design, but also in bivalve classification and evolution.     

Test Objects with a Rectangular Profile for SEM: 2. Certification of the Groove Width

A procedure for certifying the width of relief grooves with a rectangular profile in silicon is described. It is comprised of two operations. The first operation consists in measuring the thickness of a silicon-oxide film on a single-crystal silicon substrate by ellipsometry. The second operation consists in determining the change in the width of grooves after restoring the native silicon oxide on the surface of the groove side walls. The procedure for measuring the thickness of the native silicon–oxide layer by scanning electron microscopy is described.     

Interaction between laser beam and target in pulsed laser deposition: laser fluence and ambient gas effects

The most prominent phenomena on the target surface induced by laser irradiation in pulsed laser deposition is the formation of conical morphologies in the irradiated area. The conical morphologies formed under different laser fluences and the ambient oxygen pressures in KrF laser ablation of Pb(Zr_0.53Ti_0.47)O₃ were studied in detail by using scanning electron microscopy. The results indicate that, depending on the melting extent of the irradiated surface, there are two kinds of cones: one type with well-defined cone tip and cone body and the other having only a cone tip. Pb is very deficient in cone tip. The gas ambient pressure plays a remarkable role in the laser-target interaction. These results are interpreted by employing the impurity shielding mechanism and the surface instability mechanism.     

不同微槽结构绝缘子真空沿面闪络特性

针对单极性脉冲电压下不同微槽结构绝缘子真空沿面闪络特性开展实验研究。根据二次电子运动特性,设计了多种微槽宽度,对比了微槽结构和平面结构的绝缘子耐压特性差异。槽宽为1mm的微槽样品耐压水平与平面结构样品耐压水平相当,槽宽小于1mm的样品组耐压水平均高于平面结构样品,最高电压提高倍数约为1.4,说明一定尺寸范围内的微槽设计将提高绝缘子真空耐压水平。通过电场强度计算分析微槽结构对二次电子运动的影响过程可知,较大的微槽宽度可使电子限制在槽内运动,较小的微槽宽度将抑制初始电子的发展,最终二者都能达到抑制闪络的目的。通过显微镜观测各组样品表面特征,材料表面微观缺陷将可能降低材料耐压水平。     

External morphology of the mouthparts of the whitebacked planthopper Sogatella furcifera (Hemiptera: Delphacidae), with special reference to the sensilla

The whitebacked planthopper, Sogatella furcifera (Horváth), is a major pest of rice in Asia, with the adults and larvae sucking the phloem sap and affecting plant growth. The ultrastructural morphology of the mouthparts, with distribution of sensilla located on the maxillae and labium in adult S. furcifera was observed using scanning electron microscopy and illustrated, with a brief discussion of their taxonomic and putative functional significance. The gross mouthparts comprising an uppermost small cone-shaped labrum, a three-segmented labium with a deep longitudinal groove on the anterior side, and a stylet fascicle consisting of two mandibular and two maxillary stylets, are consistent with the conventional piercing–sucking type of mouth parts found in Hemiptera. The mandibles bear 5 teeth on the external convex region on the distal extremity. Smooth maxillary stylets, interlocked to form a larger food canal and a smaller salivary canal, are asymmetrical only in the internal position of longitudinal carinae and grooves. Two dendritic canals are also found in each maxilla and one in each mandible. The cross-section of the stylet fascicle is oval. The labial tip is a rosette shape. Two types of sensilla trichoides, two types of uniporous peg sensilla, two pairs of sensilla basiconica and a pair of subapical labial sensory organs have been detected at different locations on the labium, specifically the labial tip is divided into dorsal paired sensory fields with 9 pairs of uniporous peg sensilla and 1 pairs of dome-shaped sensilla, and ventral paired sensory fields with 2 pairs of sensilla basiconica.     

Microanatomy and ultrastructure of outer mantle epidermis of the cuttlefish,Sepia esculenta (Cephalopoda: Sepiidae)

This study describes the ultrastructural characteristics of external epidermis of mantle of Sepia esculenta using light and electron microscopy. The epidermis was thicker on the ventral surface than on the dorsal surface, with a higher secretory cell distribution on the ventral surface than on the dorsal surface. The epidermis was a single layer composed of epithelial cells, secretory cells, ciliated cells and neuroglial cells. Epithelial cells were columnar with well-developed microvilli on the free surface, and the microvilli were covered with glycocalyx. The epithelial cells were connected to the neighboring cells by tight junctions and membrane interdigitations of the apico-frontal surface. Well-developed microfilaments were arranged in a vertical direction in the cortical cytoplasm. The secretory cells were categorized into three types (A, B and C) in accordance with the light microscopical characteristics and ultrastructures of the secretory granules. The distribution of these cells was in the following order: Type A > Type B > Type C. SEM observation revealed that the secretory pore size of the Type A secretory cells was approximately 8.6 μm × 12.2 μm. Cytoplasm displayed a red color as the result of Masson's trichrome stain and H–E stain, and contained polygonal granules of approximately 1.2 μm² with a high electron density. The secretory pore size of the Type B secretory cells was approximately 10.1 μm × 12.1 μm. As the results of AB–PAS (pH 2.5) and AF–AB (pH 2.5) reactions, the cytoplasm displayed a red color. The cells contained membrane bounded secretory granules with very low electron density. The secretory pore of the Type C secretory cells was circular shape, and approximately 5.5 μm × 5.5 μm. Cytoplasm was found to be homogeneous under H–E stain and Masson's trichrome stain, and displayed a red color. As the result of AB–PAS (pH 2.5) reaction, the cytoplasm displayed a red color. The electron density of the secretory substance was the highest among the three types of secretory cells. The ciliated cells had a ciliary tuft on the free surface and were distributed throughout the mantle with the exception of the adhesive organs. Neuroglial cells were connected to the basal membrane, epithelial cells, secretory cells and nerve fibers through cytoplasmic process, and contained neurosecretory granules with high electron density within the cytoplasm.     

Terahertz surface plasmon-polariton propagation and focusing on periodically corrugated metal wires

In this Letter, we show how the dispersion relation of surface plasmon polaritons (SPPs) propagating along a perfectly conducting wire can be tailored by corrugating its surface with a periodic array of radial grooves. In this way, highly localized SPPs can be sustained in the terahertz region of the electromagnetic spectrum. Importantly, the propagation characteristics of these spoof SPPs can be controlled by the surface geometry, opening the way to important applications such as energy concentration on cylindrical wires and superfocusing using conical structures.     

Remnant grooves on alumina surfaces

Thermal grooving of grain boundaries in alumina has been studied using a combination of visible-light microscopy (VLM) and atomic-force microscopy (AFM). The partial angles of grooves that develop during heat treatments at 1650 °C were estimated by AFM. The observation of remnant grooves is key to determining the magnitude of the movement of a grain boundary. Imaging using VLM and AFM enabled the smoothing of remnant grooves to be observed through a progression of heat treatments, which enables the anisotropic nature of the surface diffusivity of alumina to be monitored.     

Impact of ice structure on ultrafast electron dynamics in D2O clusters on Cu(111)

The structure of D2O clusters on a Cu(111) surface and the femtosecond dynamics of photoexcited excess electrons are investigated by low-temperature scanning tunneling microscopy and two-photon photoemission spectroscopy. Two types of amorphous ice clusters, porous and compact, which exhibit characteristic differences in electron dynamics, are identified. By titration with Xe we show that in both structures solvated electrons preferentially bind on the cluster surface.