ALBERT

Beschreibung: The mammalian skin has developed sensory structures (mechanoreceptors) that are responsible for different modalities of mechanosensitivity like touch, vibration, and pressure sensation. These specialized sensory organs are anatomically and functionally connected to a special subset of sensory neurons called mechanosensory neurons, which electrophysiologically correspond with Aβ fibers. Although mechanosensory neurons and cutaneous mechanoreceptors are rather well known, the biology of the sense of touch still remains poorly understood. Basically, the process of mechanosensitivity requires the conversion of a mechanical stimulus into an electrical signal through the activation of ion channels that gate in response to mechanical stimuli. These ion channels belong primarily to the family of the degenerin/epithelium sodium channels, especially the subfamily acid-sensing ion channels, and to the family of transient receptor potential channels. This review compiles the current knowledge on the occurrence of putative mechanoproteins in mechanosensory neurons and mechanoreceptors, as well as the involvement of these proteins on the biology of touch. Furthermore, we include a section about what the knock-out mice for mechanoproteins are teaching us. Finally, the possibilities for mechanotransduction in mechanoreceptors, and the common involvement of the ion channels, extracellular membrane, and cytoskeleton, are revisited. Microsc. Res. Tech., 2012. © 2012 Wiley Periodicals, Inc.

Beschreibung: The present study was designed to evaluate the anticarcinogenic potential of Azadirachta indica against N -nitrosodiethylamine (NDEA)-induced hepatocarcinogenesis. Further, the associated histopathological and ultrastructural changes were also analyzed. Hepatic cancer model was developed by the intraperitoneal administration of NDEA to mice at weekly intervals, in successive increasing doses, for a period of 8 weeks. Aqueous A. indica leaf extract (AAILE) was administered orally at a dosage of 100 μg/g body weight thrice a week till termination of the study. A relationship between histopathological grading and chemopreventive effect of A. indica had been established at various stages of carcinogenesis. Anticancer activity of A. indica was evaluated in terms of tumor incidence, tumor multiplicity, and survival rate. A significant reduction in tumor incidence (33%), tumor multiplicity (42%), and increase in survival (34%) was observed upon administration of AAILE to NDEA-abused mice. Transmission and scanning electron microscopic investigations showed severe alterations in organelle organization, cellular arrangement, degree of differentiation, cellular metabolism, and morphology of the hepatocytes. These changes appeared to be distinctly delayed upon AAILE supplementation. The results suggest A. indica may have anticancer potential against NDEA-induced hepatic cancer. Microsc. Res. Tech., 2012. © 2011 Wiley Periodicals, Inc.

Beschreibung: The main purpose of this article was to describe the morphology of mesenchymal stem cells (MSCs) differentiated in vitro towards osteogenic and chondrogenic lineages and to focus on the ultrastructural features associated with these processes. Human mononuclear cells (hMNC) were isolated, expanded, and analyzed for the expression of specific cell surface markers to demonstrate their stem cell characteristics. Human mononuclear cells were differentiated in vitro in an osteogenic and in a chondrogenic sense for 7, 14, 21, and 28 days. Subsequently, they were processed using electron microscopic analysis (FEISEM). Alizarin red and alcian blue staining were carried out to demonstrate the deposition of mineral salts and proteoglycans in the extracellular matrix. Undifferentiated MSCs showed a cell surface covered by filopodia and ondulopodia. During differentiation, the MSCs changed their shape from a round to a fibroblastic-like shape. At the end of the differentiation, several filaments with a parallel orientation in the osteogenic samples as well as a network organization in the chondrogenic samples were detected in the extracellular spaces. This study demonstrated that there are morphological features associated with the undifferentiated and differentiated states of the MSCs, which could be utilized as new parameters for identifying and classifying these cells. Microsc. Res. Tech., 2012. © 2011 Wiley Periodicals, Inc.

Beschreibung: The aim of this in vitro study was to assess the agreement among four techniques used as gold standard for the validation of methods for occlusal caries detection. Sixty-five human permanent molars were selected and one site in each occlusal surface was chosen as the test site. The teeth were cut and prepared according to each technique: stereomicroscopy without coloring (1), dye enhancement with rhodamine B (2) and fuchsine/acetic light green (3), and semi-quantitative microradiography (4). Digital photographs from each prepared tooth were assessed by three examiners for caries extension. Weighted kappa, as well as Friedman's test with multiple comparisons, was performed to compare all techniques and verify statistical significant differences. Results : kappa values varied from 0.62 to 0.78, the latter being found by both dye enhancement methods. Friedman's test showed statistical significant difference ( P 〈 0.001) and multiple comparison identified these differences among all techniques, except between both dye enhancement methods (rhodamine B and fuchsine/acetic light green). Cross-tabulation showed that the stereomicroscopy overscored the lesions. Both dye enhancement methods showed a good agreement, while stereomicroscopy overscored the lesions. Furthermore, the outcome of caries diagnostic tests may be influenced by the validation method applied. Dye enhancement methods seem to be reliable as gold standard methods. Microsc. Res. Tech., 2012. © 2011 Wiley Periodicals, Inc.

Beschreibung: Spinal ganglion (SG) neurons are subdivided, on the basis of their cytoplasmic aspect at light and electron microscopy, into dark (D) and light (L) neurons. Numerous efforts have been made to find specific markers able to identify D and L neuronal cytotypes. The isolectin B4 (IB4), utilized to identify nonpeptidergic D neurons in mice, unfortunately, has not proved as effective in other species. The 200-kDa neurofilament protein (NF200) is considered as a typical marker of L neurons in the rat, cat, and chick. The aim of this study was to analyze the histological, morphometric, and neurochemical characteristic of NF200-immunoreactive (IR) horse SG neurons, to better characterize them morphologically and functionally. NF200-IR neurons showed two levels (strong and weak) of staining intensity. Most (84%) strongly stained NF200-IR neurons corresponded to L neurons, and showed similar bimodality as in the size distribution study, which seems to indicate a third population of neurons, in addition to the two populations (small and large) previously identified. In triple-staining experiments where NF200 was colocalized with IB4, substance P (SP), and neuronal nitric oxide synthase (nNOS) neuronal markers, most NF200-IR neurons were single stained. On the contrary, most IB4-, SP-, and nNOS-stained neurons were triple labeled and almost equally subdivided between strong and weak NF200-IR with the latter being always smaller in size than strong NF200-IR neurons. In conclusion, horse SG neurons display significant morphometric and neurochemical differences compared with those of rodents. Microsc. Res. Tech., 2012. © 2011 Wiley Periodicals, Inc.

Beschreibung: Rhipicephalus sanguineus is a widely distributed tick species that has adapted to the urban environment, and the dog is its main host. This species is also known as a vector and reservoir of diseases caused by bacteria, protozoa, and viruses. Currently, acaricides of synthetic chemical origin have been widely and indiscriminately used, leading to the development of resistance to these products by ticks and causing damage to the environment. Thus, these issues have made it necessary to seek other forms of controlling these ectoparasites. R. sanguineus was artificially infested in host New Zealand White rabbits, which were divided into four treatment groups: control (CG1 and CG2) and treatment (TG1 and TG2) groups. TG1 and TG2 hosts were provided with feed supplemented with esters of ricinoleic acid from castor oil at a concentration of 5 g/kg of feed for 7 and 15 days. Afterward, the ovaries of the female ticks were removed for analysis by transmission electron microscopy. The results showed ultrastructural changes in the somatic and germ cells of ovaries from TG1 and TG2 females, particularly with respect to chorion deposition, a protective membrane of the oocyte, as well as in the transport process of vitellogenic materials via the hemolymph and pedicel cells. Moreover, the mitochondria were less electron-dense and had cristae that were more disorganized than the mitochondria from CG1 and CG2 individuals. Thus, this study demonstrated the action of esters on the ovaries of R. sanguineus , signaling the prospect of a way to control this ectoparasite without affecting nontarget organisms or the environment. Microsc. Res. Tech., 2012. © 2011 Wiley Periodicals, Inc.

Beschreibung: The aim of this study was to evaluate the effects of the 5.25% sodium hypochlorite (NaOCl), 2% chlorhexidine (CHX), and MTAD solutions on the surface of gutta-percha and Resilon cones by using atomic force microscopy (AFM). Accessory cones were washed and dried. The cones were randomly divided into six groups: gutta-percha immersed in NaOCl, CHX, and MTAD, and Resilon immersed in NaOCl, CHX, and MTAD. AFM images of the same area were made in different periods of time. JPK™ Image Processing Software was used to evaluate the images. The parameters used to evaluate the changes were RMS and line profiles. No statistically significant change was observed in the RMS values. The line profiles detected changes only for gutta-percha surfaces after immersion in NaOCl and MTAD solutions. In conclusion, 5.25% NaOCl and MTAD are associated with local changes in surface roughness of gutta-percha cones. No change was observed when 2% CHX was used. The use of all tested solutions did not produce any changes on Resilon surface. Microsc. Res. Tech., 2012. © 2012 Wiley Periodicals, Inc.

Beschreibung: This study evaluated the presence of residues after removal of calcium hydroxide [Ca(OH) 2 ] associated with different vehicles. Thirty single-rooted teeth were instrumented to a master apical file #25 using 2.5% NaOCl as main irrigant and 17% trisodium EDTA (ethylenediaminetetraacetic acid) as final agent irrigant. Then, the root canals were dressed with Ca(OH) 2 associated with silicone oil (Group 1), 2% chlorhexidine gluconate (Group 2), or propylene glycol (Group 3). After coronal sealing, all teeth were kept in a moist environment at room temperature. After 7 days, the teeth were reopened and medicaments were removed using 5 mL of saline solution and instrumentation with master apical file followed by new irrigation with 5 mL of 2.5% NaOCl. Subsequently, teeth were split longitudinally and assessed by scanning electron microscopy. The wall cleanliness of the cervical and apical thirds of the roots were evaluated and scored by three blinded examiners. Statistical analysis was performed using Kruskal–Wallis and Wilcoxon tests at 5% level of significance. All roots had residues of Ca(OH) 2 on the canal walls. All experimental groups had similar results ( P 〉 0.05) regardless of the third evaluated. There was significant difference between the apical and cervical thirds only in Group 3 ( P 〈 0.05). Association of different vehicles to Ca(OH) 2 does not influence the persistence of residues on the root canal walls. Microsc. Res. Tech. 2012. © 2012 Wiley Periodicals, Inc.

Beschreibung: In this study, healing of cutaneous wounds in Labeo rohita using scanning electron microscope is reported. Wound area could be divided into three regions. Immediately after infliction of wound, edges retract exposing underlying tissues in wound gap (Region I). Simultaneously, at region close to wound edge (Region II), mucous goblet cell openings are observed with copious mucous secretions. Within 1 h, Region I gets covered by mucous secretions, and epidermis at edges starts migrating. Opposing fronts gradually advance and by 4–6 h come in contact to epithelialize wound gap. Zone of contact of fronts is demarcated by epidermal ridge, which is relatively prominent at 8 h. It gradually diminishes and is not distinguished at 24 h and afterward. At 1–4 h, microridges on epithelial cell surfaces appear irregularly arranged, widely spaced, short with abrupt ends at Region I; relatively extensive at Region II; and similar to those in controls at region surrounding Region II (Region III). At 12 h and afterward, microridges appear similar to those in controls at Regions I and II. At 1–2 h, isolated swollen epithelial cells, often in process of detachment and exfoliation at surface, are observed at Regions I and II. Such cells are infrequent at 8 h and afterward. Region I covered by migrated epidermis appears trough like at 4 h to 2 days, level of which gradually rises and at Day 4, surface of epidermis appears at a level similar to that at Regions II and III. Changes have been associated with the imbalance of osmotic homeostasis due to disruption of barrier between internal and external environment of skin. Microsc. Res. Tech. 2012. © 2012 Wiley Periodicals, Inc.

Beschreibung: The Brazilian africanized Apis mellifera is currently considered as one of the most important pollinators threatened by the use of insecticides due to its frequent exposition to their toxic action while foraging in the crops it pollinated. Among the insecticides, the most used in the control of insect pragues has as active agent the pyriproxyfen, analogous to the juvenile hormone (JH). Unfortunately the insecticides used in agriculture affect not only the target insects but also beneficial nontarget ones as bees compromising therefore, the growth rate of their colonies at the boundaries of crop fields. Workers that forage for provisions in contaminated areas can introduce contaminated pollen or/and nectar inside the beehives. As analogous to JH the insecticide pyriproxyfen acts in the bee's larval growth and differentiation during pupation or metamorphosis timing. The flighty muscle is not present in the larvae wingless organisms, but differentiates during pupation/metamorphosis. This work aimed to investigate the effect of pyriproxyfen insecticide on differentiation of such musculature in workers of Brazilian africanized honey bees fed with artificial diet containing the pesticide. The results show that the bees fed with contaminated diet, independent of the insecticide concentration used, show a delay in flight muscle differentiation when compared to the control. Microsc. Res. Tech. 2012. © 2012 Wiley Periodicals, Inc.

Beschreibung: In this study, we report ghrelin and leptin immunoreactive (ir) cells distribution in the gastrointestinal tract and blood ghrelin and leptin levels in rainbow trout ( Oncorhynchus mykiss ) and Murray cod ( Maccullochella peelii peelii ) fed diets with different fatty acid compositions. Juvenile rainbow trout and Murray cod were fed five iso-energetic experimental diets containing fish oil (FO) or one of the following vegetable oils (VO): olive oil (OO), sunflower oil (SO), linseed oil (LO), and palm oil (PO); as the added dietary lipid source. The presence and distribution of both ghrelin and leptin ir cells in the gastrointestinal tract were affected by the inclusion of VO. Ghrelin ir cells were found in the gastric glands of rainbow trout and in the mioenteric plexuses of the stomach of Murray cod fed FO. Ghrelin ir cells were localized in the mucosa of the intestine of rainbow trout and Murray cod fed VO. Leptin ir cells were more abundant in the epithelial lining of the mucosa folds and in the glands of the stomach of rainbow trout fed VO. Leptin immunoreactivity was detected in the gastric mioenteric plexus of Murray cod fed FO. No differences were found both in ghrelin and leptin levels in blood plasma or in the growth rates of rainbow trout and Murray cod fed the different experimental diets. These observations suggest that dietary fatty acids play a role in the peripheral feeding regulation. Microsc. Res. Tech., 2011. © 2011 Wiley Periodicals, Inc.

Beschreibung: Phytoliths are an important component for interpreting the ancient botanical record. However, phytoliths can be altered through heating, either as the result of such activities as firing ceramics, clay molds use for casting metal or in hearths. Phytoliths can also be altered through heating as the result of creating comparative sample from living plants. By heating phytoliths at graduated intervals it was found that different types of phytoliths lost their diagnostic morphological characteristics at significantly different temperatures. The phytoliths used in this study are derived from economically important plants to Chinese archaeology and culture. Given the consistent results of the alteration of different type of phytoliths at specific temperatures it should eventually be possible to use phytolith alterations as a proxy measure of the original firing temperature of ancient objects and features. Microsc. Res. Tech. 2012. © 2012 Wiley Periodicals, Inc.

Beschreibung: In this study, we investigated the effects of contaminated water on the blood parameters of the fish Prochilodus lineatus exposed during 7 and 20 days. Blood was collected with heparinized syringes, and blood smears were prepared and stained with Leishman stain. Slides were examined and photographed using a Leica light microscope. Total white blood cell counts and differential counts of thrombocytes and leukocytes were obtained for statistical analysis. The group exposed to water samples from Lago Azul exhibited an increase in the number of leukocytes and in the total number of white blood cells, suggesting that the chemical contaminants in this environment were acting similarly to antigens in this fish species, causing the proliferation of defense cells. In the group exposed to detergent during 20 days, the number of thrombocytes decreased. These results suggest that the variations in the number of leukocytes were indicators of environmental pollution and that biodegradable detergents may, after a certain time of exposure, affect vital functions in fish, such as coagulation and prevention of infections, which directly involves thrombocytes. Microsc. Res. Tech., 2012. © 2011 Wiley Periodicals, Inc.

Beschreibung: This study aimed to evaluate the in vivo remineralization of acid-etched enamel in non-brushing areas as influenced by fluoridated orthodontic adhesive and toothpaste. One hundred and twenty teeth from 30 volunteers were selected. The teeth were assigned to four treatments: no treatment (negative control); 37% phosphoric acid-etching (PAE) (positive control); PAE + resin-modified glass ionomer cement (RMGIC); and, PAE + composite resin. Patients brushed teeth with fluoridated ( n = 15) or non-fluoridated ( n = 15) toothpastes, so that etched enamel was protected with screens and it was not in contact with the brush bristles. Remineralization was evaluated by means of laser fluorescence (LF), environmental scanning electronic microscopy, and energy dispersive spectrometry after extraction. The LF means were compared by means of Wilcoxon and Mann Whitney tests. Environmental scanning electron microscopy scores were compared among the groups using a Kruskal Wallis test, whereas the Ca/P ratio was evaluated by means of an Analysis of Variance with subparcels (treatments) and Tukey's post-hoc test. There were no statistically significant differences between the tooth pastes and between the orthodontic adhesives evaluated. Most teeth presented only partial enamel remineralization. Therefore, t he fluoride released by the RMGIC was not enough to cause increased crystal regrowth in the acid-etched enamel. The use of fluoridated toothpaste did not provide positive additional effect. Microsc. Res. Tech., 2012. © 2012 Wiley Periodicals, Inc.

Beschreibung: The adipose fin is small, nonpared, and usually located medially between the dorsal and caudal fin. Its taxonomic occurrence is very restrict; thus, it represents an important trace for taxon distinction. As it does not play a known vital physiological roll and it is easily removed, it is commonly used in marking and recapture studies. The present study characterizes the adipose fin of Prochilodus lineatus , as it is poorly explored by the literature. The adipose fin consists basically of a loose connective core, covered by a stratified epithelium supported by collagen fibers. At the epithelium, pigmented cells and alarm substance cells are found. Despite the name, adipocytes or lipid droplets are not observed on the structure of the fin. Microsc. Res. Tech., 2012. © 2011 Wiley Periodicals, Inc.

Beschreibung: In this article, we propose a new shape from focus (SFF) method to estimate 3D shape of microscopic objects using surface orientation cue of each object patch. Most of the SFF algorithms compute the focus value of a pixel from the information of neighboring pixels lying on the same image frame based on an assumption that the small object patch corresponding to the small neighborhood of a pixel is a plane parallel to the focal plane. However, this assumption fails in the optics with limited depth of field where the neighboring pixels of an image have different degree of focus. To overcome this problem, we try to search the surface orientation of the small object patch corresponding to each pixel in the image sequence. Searching of the surface orientation is done indirectly by principal component analysis. Then, the focus value of each pixel is computed from the neighboring pixels lying on the surface perpendicular to the corresponding surface orientation. Experimental results on synthetic and real microscopic objects show that the proposed method produces more accurate 3D shape in comparison to the existing techniques. Microsc. Res. Tech. 2012. © 2011 Wiley Periodicals, Inc.

Beschreibung: The effects of atopic dermatitis (AD) on scalp hair properties, such as morphology and water content, were investigated using atomic force microscopy (AFM) and thermogravimetric analyzer. Hairs from lesional and nonlesional scalp regions of eight patients with AD were investigated. The severity of the disease, which was evaluated using the SCORing Atopic Dermatitis index, was 48.75 (range, 40–80). Hairs from 15 normal adults were also examined as controls. The surface images were taken in an area of 20 × 20 μm 2 with 512 × 512 pixels and a scan speed of 0.8 line/s. AD affected the cuticle structures and scales of scalp hair. The edges of cuticles were torn and collapsed, and the scales were very thick. The water contents of both types of AD hair were less than the control: 12% ± 0.7%, 11.7% ± 0.4%, and 13% ± 0.8% for lesional AD hair, nonlesional AD hair, and control hair, respectively. The scalp hair of patients with AD can be characterized by thick and globular scale patterns. The hair of patients with AD has less water content than normal hair showing a good agreement with the property of skin having AD. Microsc. Res. Tech., 2012. © 2011 Wiley Periodicals, Inc.

Beschreibung: OBJECTIVE: Bond strengths of composite resin to enamel using self-etch adhesive (SEA) Clearfil SE bond system on intact enamel and enamel pre-etched with phosphoric acid were compared. The objective was to determine if the pre-etching would increase the bond strengths of the SEA systems to intact enamel and to evaluate the effect of pre-etching on bond formation of self-etch adhesives on intact enamel. MATERIALS AND METHOD: Labial surfaces of 40 caries free permanent upper central and lateral incisors were cleaned, sectioned of their roots. All specimens were mounted on acrylic block and divided randomly into four groups. In two groups the application of self-etch adhesive, Clearfil SE bond was carried as per manufacturer's instructions, composite cylinders were built, whereas in the other two groups, 37% phosphoric acid etching was done before the application of self-etching adhesives. Then the resin tags were analyzed using scanning electron microscope and shear bond strength was measured using Instron universal testing machine. RESULTS: When phosphoric acid was used, there was significant increase in the depth of penetration of resin tags and in the Shear Bond Strength of composite to enamel. CONCLUSION: The results indicate that out of both treatment groups, pre-etching the intact enamel with 37% phosphoric acid resulted in formation of longer resin tags and higher depth of penetration of resin tags of the Clearfil SE bond, and attaining higher bond strength of the Clearfil SE bond to intact enamel. Microsc. Res. Tech., 2012. © 2011 Wiley Periodicals, Inc.

Beschreibung: The tip is one of the critical factors to improve the efficiency in picking up individual DNA molecules from solid substrates based on atomic force microscope (AFM) nanomanipulation. We found that wearing AFM tips on certain solid substrates in advance to nanomanipulation operation would largely improve the pickup efficiency, which was ascribed to the increasing affinity of the tip to the DNA molecules along with the increase of the tip radius after wearing. It was demonstrated that bare mica was superior to APTES-modified mica to keep the tip clean while wearing, which was crucial for DNA pickup during AFM nanomanipulation. Microsc. Res. Tech., 2012. © 2011 Wiley Periodicals, Inc.

Beschreibung: This study evaluated by scanning electron microscopy the interface between root dentin and resin cements, with or without additional application of a dual-cured one-step self-etching dentin adhesive. In addition, the push-out bond strength of these materials to dentin was evaluated. Root canals of 50 maxillary human canines were subjected to biomechanical preparation and randomly assigned into five groups ( n = 10) according to the obturation employed: I. AH Plus/gutta-percha; II. Endo-REZ/gutta-percha; III. Epiphany SE/Resilon; IV. Endo-REZ/gutta-percha + adhesive; and V. Epiphany SE/Resilon + adhesive. After obturation, two cross sections of 1.0 mm of each third of the root were obtained and analyzed by SEM and push-out. Data were analyzed by Kruskal–Wallis, Two-way ANOVA and Tukey's HSD tests (α = 5%). Significant differences occurred between scores for gap parameters and tags; greater tag formation resulted for Epiphany SE ( P 〈 0.05). There were also significant differences between groups for the bond strength. AH Plus (1.24 ± 0.70) showed higher values ( P 〈 0.05) compared to groups II (0,17 ± 0.19), III (0.10 ± 0.06), IV (0.09 ± 0.08), and V (0.06 ± 0.03), which were statistically similar ( P 〉 0.05). There was no significant difference between the root thirds ( P 〉 0.05). It was concluded that the use of a self-etching adhesive system did not improve the adhesion of resin-based sealers to dentin and that AH Plus showed better bond strength when compared to other cements. Microsc. Res. Tech., 2012. © 2011 Wiley Periodicals, Inc.

Beschreibung: The aim of this study was to evaluate the presence of porosities inside the glass ionomer cement (GIC) after different techniques of material insertion. MATERIAL AND METHOD: Specimens were prepared with high-viscosity GIC Ketac Molar Easymix and divided into three groups according to the insertion method: spatula (PI), Centrix injector (CI), and low-cost syringe (LCS). The specimens were fractured and observed with scanning electronic microscopy to quantitatively evaluate porosity inside the material using Image J Software. RESULTS: Statistical analysis, ANOVA application, and Tukey test to significance level of 5%, revealed that there was no statistical difference between the groups. CONCLUSION: Although the use of LCS has not decreased the porosity of the material, this insertion method is easy, accessible, and low cost, which makes it a viable alternative of use in the ART technique and in others bucal health programs. Microsc. Res. Tech., 2012. © 2012 Wiley Periodicals, Inc.

Beschreibung: The European sea bass, a member of the Moronidae family, is a food fish, considered one of the first models for the intensive breeding in salt water. It has nowadays an important and increasing presence in the international fishing markets. Sea basses are carnivorous, feeding on little fishes and invertebrates. Considering the important role of the tongue during the intraoral transport and the swallowing of food, scarce data are present in literature about its morphology. The aim of this study was to analyze the morphology of the tongue by means of scanning electron and light microscopy. Adult sea basses were obtained from the aquarium of the Sicilian Center of Experimental Ichthyiopathology of the University of Messina. The fishes were anaesthetized with MS 222 and the heads were then quickly removed and processed for the paraffin embedding and SEM processing. Three different tongue regions could be distinguished: an apex, a body, and a root. Scanning electron and light microscopy showed the presence of numerous canine-like teeth, surrounded by taste buds and numerous fungiform and conical papillae. The teeth were curved and their tips were posteriorly oriented. The results confirm, in teleosts too, the fundamental role of the tongue in the mechanics of food ingestion. Moreover, the presence of taste buds demonstrates the interaction of food processing and taste. These data could be a potential source to identify new and better methods of nutrition in the breeding of this fish. Microsc. Res. Tech., 2012. © 2011 Wiley Periodicals, Inc.

Beschreibung: In Digelasinus diversipes, spermatozoa are maintained in bundles, with 74 spermatozoa on average, in the seminal vesicle. These spermatozoa are very short (20 μm) and consist of a head and flagellum. The head includes an acrosome (perforatorium covered by the acrosomal vesicle) and a nucleus. A regular electron-lucent region separates the acrosomal vesicle from the perforatorium, which is inserted parallel to the anterior ending of the nucleus. The small flagellum is composed of two symmetrical mitochondrial derivatives, a centriolar adjunct, an axoneme (9 + 9 + 2), and two accessory bodies. The centriolar adjunct begins above the posterior end of the nucleus and ends covering the anterior tip of two mitochondrial derivatives. In the terminal region of the axoneme, the central microtubules terminate first. The presence of a subacrosomal space, a short mitochondrial derivative diameter, and a short spermatodesm is the ultrastructure characteristics of spermatozoa shared by all “symphyta” species. Differences in the insertion of the perforatorium into the nucleus and the position of the centriolar adjunct distinguish Dielocerinae and the Arginae studied previously. The number of spermatozoa per cyst is variable. Furthermore, additional characteristics that had not been described for “symphyta” were also found, such as the number of follicles per testis. Microsc. Res. Tech., 2012. © 2011 Wiley Periodicals, Inc.

Beschreibung: The morphology and ultrastructure of the antennae and antennal sensilla of seven male Dendrolimus species and a male Trabala vishnou gigantina (Yang) (Lepidoptera: Lasiocampidae) were examined by light microscope and scanning electron microscope. Six morphological types of antennal sensilla were identified: Sensilla trichodea, Sensilla chaetica, Sensilla styloconica, Sensilla coeloconica, Böhm bristles, and foot-like sensilla. Six of the Dendrolimus moths and Trabala vishnou gigantina Yang share the same antennal sensilla type, as do various geographic populations of the same species. The exception, Dendrolimus spectabilis Butler, has foot-like sensilla. However, the antennal sensilla subtypes were significantly different among species and/or populations. There were no remarkable differences in the width of the scape, pedicel, subflagellum, and the side-branches between the eight male species studied. However, we observed significant differences in the number of flagellomere and the length of scape, pedicel, subflagellums, and side-branches. The length and basal diameter of various types of antennal sensilla did not vary significantly among Dendrolimus moths. Beyond that, there were no differences among populations of the same kind of species. Hierarchical cluster analysis found two clusters: the first contained D. punctata punctata (Walker), D. punctata wenshanensis (Tsai et Liu), D. tabulaeformis (Tsai et Liu), and D. spectabilis and D. superans (Butler), and the second contained D. grisea (Moore) and D. kikuchii kikuchii (Matsumura). Trabala vishnou gigantina was placed separately from the two clusters. We conclude that D. punctata wenshanensis, D. tabulaeformis , and D. spectabilis are geographic subspecies of D. punctata punctata . Microsc. Res. Tech. 2012. © 2012 Wiley Periodicals, Inc.

Beschreibung: The effects of short-term genistein exposure on ovarian folliculogenesis in immature rats were examined stereologically. To determine whether genistein acts as an estrogen agonist or antagonist, the results were compared with the effects of 17α-ethynylestradiol. Immature female rats received 50 mg/kg/bw of genistein in dimethyl sulfoxide subcutaneously daily for three consecutive days from 18 to 20 days. The second group was injected with 1 μg/kg/bw of 17α-ethynylestradiol in olive oil in the same schedule. Each group had a corresponding control. Genistein increased ovary and ovarian stroma volumes by 18.50% ( P 〈 0.05) and 53.40% ( P 〈 0.05), respectively, and changed the parenchyma to stroma ratio in favor of stroma. Genistein induced decreases in the number of primordial (by 17.23%; P 〈 0.05), primary (16.62%; P 〈 0.05), and secondary follicles (12.29%: P 〈 0.05), whereas the number of atretic secondary follicles increased (5.10-fold; P 〈 0.05). The number of healthy large follicles was raised by 27.3% ( P 〈 0.05), accompanied by 35.64% more atretic large follicles ( P 〈 0.05). Similarly to genistein, estradiol changed the parenchyma to stroma ratio in favor of stroma, and reduced the number of primordial follicles, but the number of primary follicles was elevated. There were more healthy and atretic small and large follicles. In conclusion, genistein acted as an estrogen antagonist and had an inhibitory effect on the initial phase of folliculogenesis. In the other phases, genistein acted as an estrogen agonist, stimulating transition from the preantral to antral stage of folliculogenesis, and altering the ratio of follicular parenchyma and ovarian stroma in favor of stroma. Microsc. Res. Tech. 2012. © 2012 Wiley Periodicals, Inc.

Beschreibung: This study quantitatively examined the short and mid-long term effects of radiofrequency (RF) treatment on the normal dermal collagen fibrils of live rabbits. Effects were evaluated by histology and scanning probe microscopy analysis of dermal tissues treated using three RF energy levels (10, 20, and 30 W) and either a single- or multiple-pass procedure. Progressive changes in the morphology of rabbit dermal collagen fibrils were investigated over a 30-day post-treatment period. All RF-treated groups, except for the low-energy group (10 W), displayed more prominent inflammatory responses compared to the control. This inflammatory response was more prominent a day after treatment. Dermal tissues 30-days after RF treatment exhibited prominent myofibroblast activity associated with collagen contractile activity during wound healing in addition to chronic inflammation. A decrease in the morphology of dermal collagen fibrils after RF treatment continued until seven days postoperatively. The collagen fibril diameter increased to near baseline at 30 days postoperatively. Low-energy and multi-pass treatments resulted in greater collagen fibril contraction and recovery at the nanostructural level at 30 days postoperatively than did a single high-energy treatment. Microsc. Res. Tech. 2012. © 2012 Wiley Periodicals, Inc.

Beschreibung: Ionization damage (radiolysis) and knock-on displacement are compared in terms of scattering cross section and stopping power, for thin organic specimens exposed to the electrons in a TEM. Based on stopping power, which includes secondary processes, radiolysis is found to be predominant for all incident energies (10–300 keV), even in materials containing hydrogen. For conducting inorganic specimens, knock-on displacement is the only damage mechanism but an electron dose exceeding 1000 C cm −2 is usually required. Ways of experimentally determining the damage mechanism (with a view to minimizing damage) are discussed. Microsc. Res. Tech., 2012. © 2012 Wiley Periodicals, Inc.

Beschreibung: Morphology of all larval instars and puparium of Dohrniphora cornuta (Bigot), a most common phorid fly species indoors in China, is presented using scanning electron microscopy. The first instar larva was composed of 12 segments, each of segments 3–11 with six slender tubercles situated dorsally, dorsolaterally, and laterally in transverse row. These tubercles divided into two segments, of which the basal one was smooth, and the brush-shaped distal one was comprised of a cluster of fine spines. Antennae and maxillary palp complex were visible. Two slits could be seen at the posterior spiracle. Besides the presence of anterior spiracle, the tubercles of second instar became stouter than those of first instar and were covered by numerous long bristles from the base to top. The posterior spiracle contained four slits. Third larval instar was similar to second instar. The bubble membrane comprised of ≈120 globules with a pointed tip on their top presented at the segment 5 of third instar larvae. Puparia showed a retracted cephalic region and a pair of distinct pupal respiratory horns on the dorsum. The respiratory horns were long and bore numerous branches from base to apex. The apex of branch with two longitudinal slits was relatively broad and curled dorsally. Microsc. Res. Tech. © 2012 Wiley Periodicals, Inc.

Beschreibung: The aim of this study was to evaluate the effects of sodium hypochlorite (NaOCl), chlorhexidine digluconate (CHX), and mixture of tetracycline, citric acid, and detergent (MTAD) solutions on the surfaces of nickel titanium (NiTi) rotary files with scanning electron microscopy (SEM) and atomic force microscopy (AFM). Irrigation solutions including 2.5% NaOCl, 5% NaOCl, 2% CHX, and MTAD were used. Four new ProTaper rotary NiTi files (F3) were immersed in each four irrigation solutions for 10 min, separately. One untreated NiTi file was used as a control specimen. The surface analysis was performed with SEM and AFM. The surface analysis in AFM was performed on 12 different regions located between first and second millimeters from the tip. SEM images were taken with different magnifications. No surface alteration was recorded under SEM evaluation. AFM revealed that the root mean square (RMS) values of all treated specimens were statistically higher than the control sample ( P 〈 0.05). The sample which was treated with CHX showed lowest RMS values in test groups. All tested solutions caused surface alterations. CHX demonstrated limited surface alterations when compared to the other tested solutions. Microsc. Res. Tech. © 2012 Wiley Periodicals, Inc.

Beschreibung: Asceptic loosening remains the primary cause for failure of joint implant. The active role of fibroblasts in mediating asceptic loosening is however not well documented. In this study the initial interactions of fibroblasts with metal particles was studied by evaluating changes in the cytoskeletal structure and cytokine level. Murine L929 fibroblasts cultured with cobalt chromium particles were observed by phase contrast and scanning electron microscopy (SEM). Changes in the cytoskeletal rearrangement of F-actin and α-actinin focal adhesion plaques were studied by confocal microscopy. Expression of the proinflammatory cytokines IL-6 and IL-1α were analyzed by ELISA. The role of actin filaments and microtubules in particle uptake were determined at low temperature and in presence of colchicine and cytochalasin B. Phase contrast and SEM studies reveal that the metal particles adhere to the fibroblasts. The cellular cytoplasm was observed to grow over the particles and is suggestive of particle uptake. Confocal microscopy shows the presence of voids within the F-actin cytoskeletal framework corresponding to areas occupied by the metal particles, indicating the possible uptake of these particles. Aggregates of α-actinin into patches at the cell surface were also noted. Adherence and uptake of particles did not occur at low temperature and in presence of cytochalasin B, indicating that it is an active energy-dependent process involving actin filaments. Changes in the levels of cytokine IL-6 and IL-1α were not observed suggesting the role of other cytokine molecules in mediating the inflammatory response to wear debri by fibroblasts. Microsc. Res. Tech. 2012. © 2012 Wiley Periodicals, Inc.

Beschreibung: The aim of this study was to analyze the rat temporomandibular joint (TMJ) synovial membrane at different ages using light, scanning, and transmission electron microscopy. Under light microscopic analysis, the TMJ structures were observed such as condyle, capsule, disk, the synovial membrane collagen type, and cells distribution. In the scanning electron microscopy, the synovial membrane surface exhibited a smooth aspect in young animals and there was an increase with ageing in the number of folds. The transmission electron microscopic analysis showed more synoviocytes in the synovial layer in the young group and still a great number of vesicles and cisterns dilation of rough endoplasmic reticulum in the aged group. In the three groups, a dense layer of collagen fibers in the synovial layer and cytoplasmic extensions were clearly seen. It was possible to conclude that synovial membrane structures in aged group showed alterations contributing to the decrease in joint lubrication and in the sliding between disk and joint surfaces. These characteristic will reflect in biomechanics of chewing, and may cause the TMJ disorders, currently observed in clinical processes. Microsc. Res. Tech. © 2012 Wiley Periodicals, Inc.

Beschreibung: Although in-office bleaching has been proven successful for bleaching teeth, controversy exists from morphological alterations in enamel morphology due to mineral loss and tooth sensitivity. This preliminary study aimed to evaluate the efficacy of a novel in-office tooth bleaching technique modified with a casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) paste (MI paste—MI) and its effect on the enamel morphology and tooth sensitivity. Three patients received a 35% hydrogen peroxide (Whiteness HP—HP) dental bleaching system. HP was prepared and applied on the teeth on one of the hemiarches, whilst teeth on the other hemiarch were bleached with a mixture of HP and MI. Tooth color, epoxy resin replicas, and sensitivity levels were evaluated in the upper incisors. The results were analyzed descriptively. Right and left incisors showed similar color change after bleaching. Incisors bleached with the mixture of HP and MI presented unaltered enamel surfaces and lower sensitivity levels. The currently tested tooth bleaching technique did not reduce the gel effectiveness while decreasing hypersensitivity levels and protecting the enamel against surface alterations caused by the high-concentration bleaching peroxide tested. The concomitant use of MI Paste and high-concentration hydrogen peroxide might be a successful method for decreasing tooth sensitivity and limiting changes in the enamel morphology during in-office bleaching. Microsc. Res. Tech. 2012. © 2012 Wiley Periodicals, Inc.

Beschreibung: This study describes the very first assessment of the expression and localization of translationally controlled tumor protein (TCTP) in adult rat urinary organs. TCTP expression levels in kidneys, urinary bladder, and urethra were evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) and Western blotting, and its cellular localization was examined immunohistochemically in paraffin sections of various urinary organs. TCTP was found in all urinary organs. Its expression was high in the urethra and low in the bladder. TCTP was localized in glomerular podocytes, epithelium of proximal and distal renal tubules, in the loop of Henle, and in the transitional epithelium of the bladder and urethra, mostly in basal cell layers). The subcellular localization of TCTP in these urinary organs was cytoplasmic. These findings suggest that TCTP may be involved in urine formation and excretion. Microsc. Res. Tech. 2012. © 2012 Wiley Periodicals, Inc.

Beschreibung: In this work, NIH ImageJ plugins for extended depth-from-focus reconstructions (EDFR) based on spatial domain operations were compared and tested for usage optimization. Also, some preprocessing solutions for light microscopy image stacks were evaluated, suggesting a general routine for the ImageJ user to get reliable elevation maps from grayscale image stacks. Two reflected light microscope image stacks were used to test the EDFR plugins: one bright-field image stack for the fracture of carbon-epoxy composite and its darkfield corresponding stack at same ( x , y , z ) spatial coordinates. Image quality analysis consisted of the comparison of signal-to-noise ratio and resolution parameters with the consistence of elevation maps, based on roughness and fractal measurements. Darkfield illumination contributed to enhance the homogeneity of images in stack and resulting height maps, reducing the influence of digital image processing choices on the dispersion of topographic measurements. The subtract background filter, as a preprocessing tool, contributed to produce sharper focused images. In general, the increasing of kernel size for EDFR spatial domain-based solutions will produce smooth height maps. Finally, this work has the main objective to establish suitable guidelines to generate elevation maps by light microscopy. Microsc. Res. Tech. 2012. © 2012 Wiley Periodicals, Inc.

Beschreibung: Many relevant questions in biology and medicine require both topography and chemical information with high spatial resolution. Several biological events that occur at the nanometer scale level need to be investigated in physiological conditions. In this regard Atomic Force Microscopy (AFM) is one of the most powerful tools for label-free nanoscale characterization of biological samples in liquid environment. Recently, the coupling of Raman spectroscopy to scanning probe microscopies has opened new perspectives on this subject; however, the coupling of quality AFM spectroscopy with Raman spectroscopy in the same probe is not trivial. In this work we report about the AFM capabilities of an advanced high-resolution probe that has been previously nanofabricated by our group for coupling with Raman spectroscopy applications. We investigate its use for liquid AFM measurements on biological model samples like lipid bilayers, amyloid fibrils, and titin proteins. We demonstrate topography resolution down to nanometer level, force measurement and stable imaging capability. We also discuss about its potential as nanoscale chemical probe in liquid phase. Microsc. Res. Tech., 2012. © 2012 Wiley Periodicals, Inc.

Beschreibung: Due to depth of focus constraints, the acquisition of a single 2-D completely in-focus image of 3-D objects characterized by a relevant depth dimension is not possible with a standard light microscope. Since the Seventies numerous methods have been proposed to overcome this problem, mainly through different fusion processing techniques to extend the microscope's depth of focus. However, given a specific application, it is very difficult to know which method yields the best results because there are no validated approaches or tested metrics that are suitable for real world cases typically lacking in a reference ground truth. Although the Universal Quality Index (UQI) is widely used to evaluate output quality in image processing, it requires a reference ground truth. Some UQI extensions have been proposed to evaluate the output of fusion methods without a ground truth, but sufficient analyses have not been carried out to confirm their equivalence to the standard UQI in terms of (evaluation) performance. We propose a new method to extend the microscope's depth of focus and, using synthetic stacks of images with ground truth attached, show that it is superior to state-of-the-art methods. We also demonstrate that the output of metrics proposed as UQI extensions is different from that of the UQI. Finally, we validate a new approach to evaluate extended depth of focus methods using real world stacks of slices, as per the UQI, but without the need for a reference ground truth. Microsc. Res. Tech. © 2012 Wiley Periodicals, Inc.

Beschreibung: The gilthead seabream is a food fish, one of the most frequently used in aquaculture. In the species of commercial interest, feeding in captivity is very important and this process is strictly related to the morphological characteristics of the oral cavity. The aim of this study is, using the standard procedures for light and scanning electron microscopy, to analyze the morphology of the tongue dorsal surface to show if relationships are present between the tongue morphology and the nutritional habits and choices of this farmed species. The main characteristic of the gilthead seabream oral cavity floor is the presence of an apical pouch, with, probably, a protective role mainly for the apical, free part of the tongue. Three zones, like in other teleosts, an apex, a body and a root, can be clearly distinguished. In the pouch foliate-like papillae were observed, while the whole tongue is characterized by the presence of two types of papillae, respectively with a fungiform and cylindroid aspect, both randomly distributed throughout the whole dorsal surface of the tongue. Scattered and numerous taste buds, with the typical pear-onion shape, together with small and numerous taste pores are also present, distributed throughout the tongue surface. Our results demonstrate that in the gilthead seabream important mechanic and sensory roles are carried out by specific anatomical structures. Our anatomical data could give, together with further biochemical and physiological data, an important support with the aim of improving the nutrition of aquaculture species. Microsc. Res. Tech. © 2012 Wiley Periodicals, Inc.

Beschreibung: Pyrethroids such as permethrin are synthetic compounds widely used in the agriculture of many countries to combat plagues and in domestic products, such as acaricides. Not so long ago these chemicals were characterized as non-toxic for non-target organisms; however, recent studies have showed that these compounds could present toxic potential for many organisms. In this sense, this study presents genotoxic and mutagenic potential of permethrin administered intraperitoneally in mice under artificial conditions by the use of micronucleus assay in the peripheral blood of these animals. The mice were divided into five groups: group I = negative control (distilled water), group II = positive control (cyclophosphamide), group III = 30% of permethrin LD 50 (96 mg/kg), group IV = 50% of permethrin LD 50 (160 mg/kg), and group V = 80% of permethrin LD 50 (256 mg/kg). The peripheral blood was collected 24, 48, and 72 h after treatment. Results showed that all the tested permethrin dosages presented genotoxic and mutagenic effects 24 h after treatment, which would contradict the classification of this chemical product as moderately toxic, i.e., unable to cause damages to the cell DNA. Microsc. Res. Tech. © 2012 Wiley Periodicals, Inc.

Beschreibung: Salvia lachnostachys Benth., Lamiaceae, is a endemic species from southern Brazil. The essential oil of its leaves and flowers is mainly constituted by aliphatic compounds, such as dodecanoic acid, with sesquiterpenes as minor constituents. This work evaluated the morphology, anatomy, microchemistry, and phytochemistry of S. lachnostachys to provide advanced knowledge of Brazilian plants with medicinal potential. Light and scanning electron microscopy techniques were used in the anatomical and microchemical studies. Compounds were isolated by chromatographic techniques, identified by analysis of their NMR spectra and compared with published data. S. lachnostachys can be distinguished from other related species mainly by its petiolate leaves, terminal inflorescence, persistent bracts, and villous-glandular corolla. The stem and leaves of S . lachnostachys display anatomical characteristics common to the family Lamiaceae. However, this species can be distinguished from other family members by the morphology and the presence of eglandular and glandular trichomes, as well as the organization of the vascular bundles of the petiole. The phytochemical results revealed that S. lacnostachys produces oleanolic and ursolic acids in addition to the diterpene fruticuline A, which is a rare compound, previously found only in Salvia fruticulosa Benth. and S. corrugata Vahl. Ursolic and oleanolic acids are bioactive triterpenes that exhibit antiatherosclerotic, anticancer, antihypertensive, antinflammatory, antileukemic, antimutagenic, antioxidant, antiproliferative, and antiviral activities, and fruticuline A has antibacterial activity. Microsc. Res. Tech. © 2012 Wiley Periodicals, Inc.

Beschreibung: Because of the increasing medical-veterinary importance of ticks, the development of alternative control methods, less aggressive to the host and the environment has become the target of several researches. In this sense, the present study analyzed the action of different concentrations (5, 10, and 20%) of andiroba seed oil ( Carapa guianensis ) on the reproductive system of Rhipicephalus sanguineus females, through histochemical techniques and the quantification of the reproductive efficiency index. The results showed that andiroba oil is a potent natural agent, able to cause several changes in the oocytes of this species, impairing the reproductive success, once this natural product induces great physiological changes in the oocytes in all development stages, such as drastic reduction in proteins, polysaccharides, and lipids in these cells, and these components are essential for the viability of the embryo. In addition, it was observed that this product stimulate the oviposition, mainly at the concentration of 20%. This higher production of eggs represents a defense mechanism developed by the organism in order to ensure the reproductive success of the species, even in the presence of the toxic agent. However, the results obtained suggested that the laid eggs would not be viable, due to the great changes undergone by the oocytes. Thus, the present study showed that the use of this vegetal product would be an alternative way to control the ticks, bringing benefits similar to the ones obtained through the use of synthetic acaricides; however, with less damage to nontarget organisms and the environment as well. Microsc. Res. Tech., 2012. © 2012 Wiley Periodicals, Inc.

Beschreibung: The antennal morphology and sensilla ultrastructure of Tomicus yunnanensis , T. minor , and T. brevipilosus were studied by scanning electron microscopy and transmission electron microscopy. Eight common sensilla types were recorded: (1) sensilla trichodea ( S.tr .) types 1 and 2 were located on the club and were innervated by five and eight dendrites, respectively; (2) sensilla chaetica ( S.ch .) types 1 and 2 had no dendrites in the sensilla lymph lumen; (3) sensilla basiconica ( S.b .), top-protuberated S.b . and fluted cones ( Fl.c .) occurred on the club; and Böhm bristles (B.b.) occurred on the funicle. S.b . were the most abundant and were innervated by 10–14 dendritic branches. Top-protuberated S.b ., a new sensilla type, were innervated by one dendrite. Fl.c . were innervated by five dendrites. Only three sensilla furcatea were visible on the antennae of female T. yunnanensis . The possible functions of these sensilla are discussed in relation to their morphology and ultrastructure. No statistical differences between sexes were found in the size and numbers of each sensilla type. Although the three species had similar antennal morphology and sensilla type, sensilla on sub-segments 3 and 4 of the antennal club of T. minor were much sparser than those of T. yunnanensis or T. brevipilosus . Concerning the antennae of T. yunnanensis , there were more S.tr . type 2, S.ch . type 2 and S.b. and the size of S.tr . type 1 and S.b . were significantly greater than those of T. minor . Microsc. Res. Tech., 2012. © 2012 Wiley Periodicals, Inc.

Beschreibung: The polyphagous planthopper, Kallitaxila granulata (Stål) (Hemiptera: Fulgoromorpha: Tropiduchidae), has been recently introduced into southeastern China, the Philippine islands, and Hawaii, where it has done significant damage to agricultural and forest ecosystems. The external morphology of the heads of adult male and female K. granulata was examined using scanning electron microscopy. Seven types of sensilla were reported: trichoid sensilla and campaniform sensilla on the antennal pedicel, antennal scape and maxilla; plate organs on the antennal pedicel; coeloconic sensilla in Bourgoin's organ on the expanded flagellar base; ampullaceal sensilla on the antennal pedicel; wavy-pit sensilla on the antennal pedicel and antennal scape; and coin-shaped sensilla on each lateral side of the labium. Evans' organ was described as placoid sensilla sunk into shallow cuticular cavities below the antennae. The external morphology, distribution, and abundance of sensilla located on antennae, maxillae, and labium in K. granulata were illustrated, with a brief discussion of their taxonomic, phylogenetic, and putative functional significance. Microsc. Res. Tech., 2012. © 2012 Wiley Periodicals, Inc.

Beschreibung: In this article, we described the ultrastructure of the sensilla on the antenna, eyes, mouthparts, wings, legs, and external genitalia of female and male Trichogramma dendrolimi using scanning electron microscopy (SEM). The antenna possessed the most sensilla types. We found 13 types of sensilla on female antenna, which were trichoid sensilla (TS) type 1-4, chaetica sensilla (ChS) type 1-2, campaniform sensilla (CaS), falcate sensilla, placoid sensilla (PS) type 1-2, basiconic capitate peg sensilla (BCPS) type 1, coeloconic sensilla (CoS), and styloconic sensilla. Ten types of sensilla were found on the male antenna, some were the same as that on female T. dendrolimi antenna, such as TS types 1 and 3, CaS, PS type 1, BCPS type 1, and CoS, but TS types 5 and 6, ChS type 3, and BCPS 2 were specific to male T. dendrolimi antenna. The leg possesses eight types of sensilla and a kind of tympana structure. Four types of TSs were found on the wings. On the mouthparts, sensilla on the maxillary and labial palps were unique, including two TSs and one ChS. The ovipositor possesses three types of sensilla, and the copulatory organ possesses two types. The eyes had only one kind of TS. Furthermore, external morphology of antenna and external genitalia revealed distinct sexual dimorphisms. According to their morphology, the possible functions of these sensilla were discussed. These results may further our understanding of the sensory mechanisms of T. dendrolimi in response to infochemicals within the environment. Microsc. Res. Tech., 2012. © 2012 Wiley Periodicals, Inc.

Beschreibung: The antennal sensilla of both genders of macropterous and brachypterous adults of the small brown planthopper, Laodelphax striatellus (Fallén) (Hemiptera: Delphacidae) were examined using light and scanning electron microscopy. Scanning electron microscopy revealed seven types of antennal sensilla in adult L. striatellus which were not evenly distributed on all antennal segments. Sensilla chaetica, a sensillum campaniformium and a Böhm bristle were found on the scape. Sensilla chaetica, sensilla trichodea, sensilla placodea which always present as plaque organs, sensilla basiconica and a sensillum campaniformium were present on the pedicel. Three sensilla basiconica and one sensillum coeloconicum containing two sensory pegs were located on the swollen sensory region of the basal flagellum. Pores observed on the surface of s. trichodea and s. placodea suggest these organs probably play a role in olfaction, whereas the aporous s. chaetica with flexible sockets probably function as mechanoreceptors. The aporous s. basiconica with inflexible sockets are probable to be thermo-hygroreceptors while the Böhm bristle and s. campaniformia may act as antennal proprioceptors. The function of s. coeloconicum remains uncertain. The numerical dominance of antennal olfactory receptors suggests olfaction is an important function of the antenna in L. striatellus . Although a small degree of sexual/wing dimorphism was observed in the numbers of sensilla and in the length and width of antennae and antennal segments, the basic shape and structure of the antennae and antennal sensilla did not differ between the gender or wing form in L. striatellus . Microsc. Res. Tech. 2012. © 2012 Wiley Periodicals, Inc.

Beschreibung: This article presents the atypical formations in the structure of the corrosion crust and in the partially mineralized metallic core, which resulted during the underground stay of a bronze shield, dated between the 1st century B.C. and the 1st century A.D. For our study, we choose a representative fragment from the rim of the shield, which was analyzed by optical microscopy and by electron microscopy coupled with energy-dispersive X-ray spectroscopy, to study its morphology, its composition, and the location of chemical compounds on the surface and inside the bulk formed during the underground stay, by processes of chemical and physical alteration, assisted by contamination with structural elements from the site. Those processes, by monolithization and mineralization formed a series of structures consisting of congruent elements and phases with a complex composition. Those formations, defined as surface effects generated by exogenous factors and endogenous factors inside the bulk, are frequently found in ancient bronze objects (such as the exterior flat mole formations and the Liesegang effect in the stratigraphic structure of the bulk). Some of those structures have atypical characteristics as regards their structure, composition, and formation mechanism, which may be used in archeometry. Moreover, that includes the object in the category of special cases, in terms of artifact evolution during underground stay and of the atypical formations resulted from the action of pedological and environmental factors. Microsc. Res. Tech. 2012. © 2012 Wiley Periodicals, Inc.

Beschreibung: It is known that pre-eclampsia affects the structure of the umbilical cord including changes in diameter and wall thickness. In this work, the morphological changes of umbilical cords associated with pre-eclampsia were investigated using scanning electron microscopy (SEM) and atomic force microscopy (AFM). The SEM images showed the overall structural changes in the umbilical cord, and the AFM imaged the surface of the cord in the nanometer range. The amount of Wharton's jelly was reduced in the cords of pre-eclampsia patients and it was holed along the boundary. Compared to a normal pregnancy, the surface of a pre-eclampsia cord was relatively smooth. In all components (Wharton's jelly, veins, and arteries), the values for surface roughness, Sa (average value of the roughness), Sq (root mean square), and Sz (peak to peak value), were smaller than those of the control ( P 〈 0.05). Especially, the values for Sa of veins were ∼fourfold less than those of the controls ( P 〈 0.05). In pre-eclamptic cords, the amount of elastin in veins was increased while that of the artery was decreased. Microsc. Res. Tech. 2012. © 2012 Wiley Periodicals, Inc.

Beschreibung: The aim of this study was to evaluate the persistence of methacrylate-based cement residues on the dentin, after dentin surface cleaning with ethanol or acetone, with or without previous application of a dentin adhesive. Forty bovine crown fragments were obtained and the dentin surface was washed with 1.0 mL of 2.5% sodium hypochlorite (NaOCl), followed by 0.1 mL of 17% ethylenediaminetetraacetic acid application for 3 min, and final irrigation with 2.5% NaOCl. The specimens were air dried and resin-based cement was rubbed onto the dentine surface with a microbrush applicator. In 20 specimens, previously to cement, a dentin adhesive was applied in all surfaces. After 15 min, the surface was scrubbed with a cotton pellet and moistened with ethanol or acetone, compounding the following groups: G1—99.5% ethanol and G2—acetone, without previous use of dentin adhesive; G3—99.5% ethanol and G4—acetone, with previous use of dentin adhesive. The dentin surface was scrubbed until the cement residues could not be visually detected. Sections were then processed for scanning electron microscopy and evaluated at 500× magnification and scores were attributed to each image according to the area covered by residual sealer, and data were subjected to Kruskal–Wallis at 5% significance. The lower residue presence was observed in G3 ( P = 0.005). All surface presented cement residues when acetone was used as cleaning solution ( P = 0.0005). The cleaning solutions were unable to completely remove the cement residues from both surfaces. The ethanol used after previous application of the dentin adhesive promoted the lower presence of residues. Microsc. Res. Tech. 2012. © 2012 Wiley Periodicals, Inc.

Beschreibung: In this work, we studied the embryology of mice of 12, 14, and 18 days of gestation by gross observation, light microscopy, and scanning electron microscopy. Grossly, the embryos of 12 days were observed in C-shaped region of the brain, eye pigmentation of the retina, first, second, and third pharyngeal arches gill pit nasal region on the fourth ventricle brain, cervical curvature, heart, liver, limb bud thoracic, spinal cord, tail, umbilical cord, and place of the mesonephric ridge. Microscopically, the liver, cardiovascular system and spinal cord were observed. In the embryo of 14 days, we observed structures that make up the liver and heart. At 18 days of gestation fetuses, it was noted the presence of eyes, mouth, and nose in the cephalic region, chest and pelvic region with the presence of well-developed limbs, umbilical cord, and placenta. Scanning electron microscopy in 18 days of gestation fetuses evidenced head, eyes closed eyelids, nose, vibrissae, forelimb, heart, lung, kidney, liver, small bowel, diaphragm, and part of the spine. The results obtained in this work describe the internal and external morphology of mice, provided by an integration of techniques and review of the morphological knowledge of the embryonic development of this species, as this animal is of great importance to scientific studies. Microsc. Res. Tech. 2012. © 2012 Wiley Periodicals, Inc.

Beschreibung: A novel method for authenticating pollen grains in bright-field microscopic images is presented in this work. The usage of this new method is clear in many application fields such as bee-keeping sector, where laboratory experts need to identify fraudulent bee pollen samples against local known pollen types. Our system is based on image processing and one-class classification to reject unknown pollen grain objects. The latter classification technique allows us to tackle the major difficulty of the problem, the existence of many possible fraudulent pollen types, and the impossibility of modeling all of them. Different one-class classification paradigms are compared to study the most suitable technique for solving the problem. In addition, feature selection algorithms are applied to reduce the complexity and increase the accuracy of the models. For each local pollen type, a one-class classifier is trained and aggregated into a multiclassifier model. This multiclassification scheme combines the output of all the one-class classifiers in a unique final response. The proposed method is validated by authenticating pollen grains belonging to different Spanish bee pollen types. The overall accuracy of the system on classifying fraudulent microscopic pollen grain objects is 92.3%. The system is able to rapidly reject pollen grains, which belong to nonlocal pollen types, reducing the laboratory work and effort. The number of possible applications of this authentication method in the microscopy research field is unlimited. Microsc. Res. Tech. 2012. © 2012 Wiley Periodicals, Inc.

Beschreibung: Whole-mount immunohistochemistry (whole-mount IH) of the seminiferous tubule is widely used to investigate the self-renewal and differentiation of spermatogonial stem cells (SSCs). Examination of the length of spermatogonial cysts is critical for tracing SSCs lineage by using Whole-mount IH. However, it is difficult for antibody molecules to penetrate into the depth of seminiferous epithelium because its thickness and the tight peritubular myoid and basement membrane outside. Here, we developed a free-floating immunofluorescent procedure of mouse seminiferous tubules using regular incubation time and normal antibody concentration. Microscopic results showed that undifferentiated spermatogonia were positively labeled by promyelocytic leukemia zinc finger protein, E-cadherin, and glial cell line-derived neurotrophic factor family receptor alpha 1, respectively. Spermatogonial cysts in varied length were revealed clearly and spermatogonia subpopulations including A single (A s ), A paired (A pr ), and A aligned (A al ) were distinguished in lower background images. This method provides us an alternate simple way to trace the lineage of individual SSCs and show their three-dimensional locations and distributions within their niches anatomically in next step. Microsc. Res. Tech. 2012. © 2012 Wiley Periodicals, Inc.

Beschreibung: Morphology of all larval instars and puparium of Diplonevra peregrina , a most common phorid fly species indoors in China, is presented using scanning electron microscopy. The first instar larva was composed of 12 segments, each of segments 3–11 with six spicate tubercles situated dorsally, dorsolaterally, and laterally in transverse row. The dorsal tubercles were much longer than the laterals and dorsolaterals. Antennae and maxillary palp complex were visible. The caudal segment was margined by six long, stout tubercles covered by numerous long bristles at the base through the apex. Two slits could be seen at the posterior spiracle. Besides the presence of anterior spiracle, the tubercles of second instar became more stout than those of first instar and were covered by numerous long bristles from the base to top. The posterior spiracle contained four slits. Third larval instar was similar to second instar. The bubble membrane comprised of clusters of small spines presented at the segment 5 of third instar larvae. Puparia showed a retracted cephalic region and a pair of pupal respiratory horns on the dorsum. Microsc. Res. Tech. 2012. © 2012 Wiley Periodicals, Inc.

Beschreibung: This study reports the effects on micromorphology and temperature rise in human dentin using different frequencies of Er:YAG laser. Sixty human dentin fragments were randomly assigned into two groups ( n = 30): carious or sound dentin. Both groups were divided into three subgroups ( n = 10), according to the Er:YAG laser frequency used: 4, 6, or 10 Hz (energy: 200 mJ; irradiation distance: 12 mm; and irradiation time: 20 s). A thermocouple adapted to the tooth fragment recorded the initial temperature value (°C); then, the temperature was measured after the end of the irradiation (20 s). Morphological analysis was performed using images obtained with scanning electron microscope. There was no difference between the temperatures obtained with 4 and 6 Hz; the highest temperatures were achieved with 10 Hz. No difference was observed between carious and sound dentin. Morphological analyses revealed that all frequencies promoted irregular surface in sound dentin, being observed more selectively ablation especially in intertubular dentin with tubule protrusion. The caries dentin presented flat surface for all frequencies used. Both substrates revealed absence of any signs of thermal damage. It may be concluded that the parameters used in this study are capable to remove caries lesion, having acceptable limits of temperature rise and no significant morphological alterations on dentin surface. Microsc. Res. Tech. 2012. © 2012 Wiley Periodicals, Inc.

Beschreibung: Stem cells are present in the adult tissues of most diverse species. Bone marrow is recognized to be the most exploited site to obtain stem cells and cell progenitors. The objective of the present study was to characterize hematopoietic progenitor (HP) morphology and analyze the performance of adherent cell progenitors (ACPs) cultivated in vitro from black-rumped agouti bone marrow ( Dasyprocta prymnolopha ). Bone marrow aspirates were obtained from tibia crest and used to prepare histological slides and identify cell morphology. Cells were also scattered on culture plates for later isolation, expansion, and quantification. Smears obtained from bone marrow demonstrated HPs at different stages of maturity. In culture, these cells showed fibroblastoid morphology and a strong tendency to form colonies, demonstrated by the presence of cell aggregates, cytoplasmic elongations lying side by side. An 80% cell confluence was observed at 18 days in culture and progressive reduction in the percentage of nonadherent mononuclear cells. After eight passes, a mean cell viability of 96.07% was observed, from a pool of 1.6 × 10 7 cells (ACP). Thirteen 25-cm 2 culture bottles were trypsinized, resuspended in freezing medium, stored in 14 criotubes at a concentration of 1 × 10 6 cells per milliliter, and placed in liquid nitrogen at −196°C. Agouti bone marrow demonstrated high plasticity, moreover different HP lines, and a population of adherent cells demonstrated morphology similar to mesenchymal stem cells in culture. Microsc. Res. Tech. 2012. © 2012 Wiley Periodicals, Inc.

Beschreibung: Introduction: The corneal tissue was processed in fixatives and embedded in resin for transmission electron microscopy to observe the ultrastructure of the collagen fibrils (CFs). The effect of these processing methods on the CF diameter and the interfibrillar spacing was studied. Methods: Four normal human corneal buttons were used for this study. A part of each cornea was fixed in 2.5% glutaraldehyde containing cuprolinic blue in sodium acetate buffer and embedded in spurr's resin (SpurrCB). A second part of each cornea was fixed in 2.5% glutaraldehyde + osmium tetroxide and embedded spurr's resin (SpurrOsm). The third part of each cornea was fixed in paraformaldehyde (4%) and embedded in LR White at 4°C (LRWhite). Ultrathin sections were stained with uranyl acetate and lead citrate. Results: In the tissue, fixed in SpurrCB, the diameter was 38.4 ± 5.9 nm and spacing between CF was 52.5 ± 5.3 nm. In the tissue fixed in SpurrOsm, the diameter was 28.37 ± 5.84 nm and spacing between CF was 45 ± 4.57 nm. In the tissue fixed in LR White, the CF diameter was 24 ± 2.3 nm and spacing between CF was 39.0 ± 4.2 nm. The diameters and interfibrillar spacing of the tissue processed by SpurrCB, SpurrOsm, and LRWhite were significantly different ( P 〈 0.001) from one another. Conclusion: Our study shows that there is a variation in the CF diameter and spacing depending on the method of fixation and embedding resins used. This needs to be considered when comparative studies using different methods are done. Microsc. Res. Tech. 2012. © 2012 Wiley Periodicals, Inc.

Beschreibung: Simultaneous optical phase and retardation measurement of a birefringent specimen is demonstrated independently of a priori knowledge of the optic axis orientation. The two-dimensional retardation distribution in both magnitude and angle of the fast axis orientation is uniquely determined from transverse phase images recorded with a bright field transmission microscope using light polarized at a minimum of three different polarization orientations. This approach opens a new possibility for stain-free phase and orientation-independent retardation characterization of samples using only one polarizer without needing other additional optical elements traditionally used in polarimetric measurements. Microsc. Res. Tech. 2012. © 2012 Wiley Periodicals, Inc.

Beschreibung: In this work, we proposed and built a multimodal optical setup that extends a commercially available confocal microscope (Olympus VF300) to include nonlinear second harmonic generation (SHG) and third harmonic generation (THG) optical (NLO) microscopy and fluorescence lifetime imaging microscopy (FLIM). We explored all the flexibility offered by this commercial confocal microscope to include the nonlinear microscopy capabilities. The setup allows image acquisition with confocal, brightfield, NLO/multiphoton and FLIM imaging. Simultaneously, two-photon excited fluorescence (TPEF) and SHG are well established in the biomedical imaging area, because one can use the same ultrafast laser and detectors set to acquire both signals simultaneously. Because the integration with FLIM requires a separated modulus, there are fewer reports of TPEF+SHG+FLIM in the literature. The lack of reports of a TPEF+SHG+THG+FLIM system is mainly due to difficulties with THG because the present NLO laser sources generate THG in an UV wavelength range incompatible with microscope optics. In this article, we report the development of an easy-to-operate platform capable to perform two-photon fluorescence (TPFE), SHG, THG, and FLIM using a single 80 MHz femtosecond Ti:sapphire laser source. We described the modifications over the confocal system necessary to implement this integration and verified the presence of SHG and THG signals by several physical evidences. Finally, we demonstrated the use of this integrated system by acquiring images of vegetables and epithelial cancer biological samples. Microsc. Res. Tech. 2012. © 2012 Wiley Periodicals, Inc.

Beschreibung: Microscopic vision measurement precision has been largely limited by inaccurately calibrated model parameters, because image plane is near parallel to reference plane in the narrow depth of field. This article proposes a method of precise microscopic vision measurement based on the adaptive positioning of the camera coordinate frame. The microscopic vision measurement movably attaches the origin of the camera coordinate frame along the optical axis. By finding the optimal position, the nonlinearity of the objective function in calibration optimization is decreased and the optimization sensitivity to initial values is reduced. Therefore, we obtain a high calibration precision and eventually ensure a high measurement precision. Mathematical simulations illustrate that the calibration precision of the proposed microscopic vision measurement model is higher than that of the conventional vision measurement model. The experiment shows that with magnification of 3.024×, the presented system achieves a precision of 0.12% based on the proposed microscopic vision measurement model, which is two times higher than the one based on the conventional vision measurement model. Microsc. Res. Tech. 2012. © 2012 Wiley Periodicals, Inc.

Beschreibung: We discuss here the principles of a novel optical method in which the scanning of a laser spot around a fluorescent object is used to determine its shape, orientation, and fluorophore distribution. The scanning pattern is adapted to the shape of the object according to a feedback principle based on intensity modulation measurements. The modulation of the intensity with respect to the angular coordinate is used to keep the orbit centered on the object. The modulation induced by rapid oscillations of the orbit radius is used to measure the local distance from the surface with nanometer precision. We provide a model to describe the fundamental relationship between modulation and distance and discuss the range of validity of several approximate expressions. According to this model, the distance can be measured with a precision dependent on the steepness of the point spread function and the total number of detected photons. To test our findings, we performed experiments with one or two channels on fluorescent spheres of known size and characterized the modulation function of our microscope setup. We conclude that the method can be used to measure distances in the range 10–200 nm between two surfaces labeled with two different probes. Microsc. Res. Tech. 2012. © 2012 Wiley Periodicals, Inc.

Beschreibung: The aim of this study was to evaluate the penetration of endodontic sealer into the dentin tubules, the integrity of the sealer layer perimeter, and the sealer area at the apical third after different filling techniques by confocal laser scanning microscopy (CLSM). Forty-five mandibular premolars were mechanically prepared with ProTaper files, until F5 file. Thereafter, they were filled with an epoxy-resin sealer (AH Plus) mixed with Rhodamine B dye (0.1% proportion) and allocated in three groups: Group 1, single master cone; Group 2, cold lateral compaction; and Group 3, Thermafil. For confocal laser scanning microscopy analysis, the specimens were transversely sectioned at 4 mm from the apex. The images at ×10 and ×40 were analyzed by Imagetool 3.0 software. Significant differences were not found among the three experimental groups according the dentin-impregnate area by the sealer ( P = 0.68) and between the sealer and root canal perimeter ( P = 0.18). However, root canal filling techniques were significantly different when apical sealer areas were compared ( P = 0.001). Thermafil group showed smaller sealer areas (8.09%) while cold lateral compaction and gutta-percha master cone showed similar areas (17.37 and 21.18%, respectively). The dentin-impregnated area was not dependent on the root canal filling technique. Single master cone, cold lateral condensation and Thermafil techniques presented integrity of the sealer perimeter close to 100% and Thermafil resulted in a significantly thinner sealer layer. Microsc. Res. Tech. 2012. © 2012 Wiley Periodicals, Inc.

Beschreibung: We present three novel reproducible methodologies for the quantification of changes in actin organization from microscope images. Striation and integrative analysis were devised for the investigation of trans-cellular filaments and F-actin localization, respectively, in response to physiological or mechanical actin-modulatory conditions. Additionally, the Parker-Qusous (PQ) formula was developed as a measure of total quantity of F-actin, independent of cell volume changes, whereby fluorescence intensity was divided by the cube root of cell volume, squared. Values obtained were quantified in Mauricean Units (Mu; pixel/μm 3 ). Upon isolation, there was a 49% decrease in total F-actin fluorescence from 1.91 ± 0.16 pixel/μm 3 (Mu) to 0.95 ± 0.55 Mu, whereas upon culture, an apparent increase in total fluorescence was deemed insignificant due to an increase in average cell volume, with a rise, however, in striation units (StU) from 1 ± 1 to 5 ± 1 StU/cell, and a decrease in percentage cortical fluorescence to 30.45% ± 1.52% ( P = 7.8 × 10 −5 ). Freshly isolated chondrocytes exhibited a decrease in total F-actin fluorescence to 0.61 ± 0.05 Mu and 0.32 ± 0.02 Mu, 10 min posthypertonic and hypotonic challenges, respectively. Regulatory volume decrease was inhibited in the presence of REV5901 with maintenance of actin levels at 1.15 Mu. Following mechanical impact in situ, there was a reduction in total F-actin fluorescence to 0.95 ± 0.08 Mu and 0.74 ± 0.06 Mu under isotonic and hypotonic conditions, respectively, but not under hypertonic conditions. We report simple methodologies for quantification of changes in actin organization, which will further our understanding of the role of actin in various cellular stress responses. These techniques can be applied to better quantify changes in localization of various proteins using fluorescent labeling. Microsc. Res. Tech. 2012. © 2012 Wiley Periodicals, Inc.

Beschreibung: OBJECTIVE: To evaluate the oral tissue response to an experimental particle ovine biomaterial by scanning electron microscopy (SEM) and light microscopy (LM). MATERIAL AND METHODS: Forty-eight rats had surgical periodontal defects treated with either blood clotting (control), bovine biomaterial™ (B), or an experimental ovine biomaterial (O). Data from SEM analysis (defect exposure, root surface exposure, diameter of matrix fibers and bundles, and globuli areas; n = 5) were applied to Shapiro–Wilk, Kruskal–Wallis, and Dunn's test, whereas LM analysis (tissue cicatrization characteristics and diameter defect; n = 3) had data applied to two-way analysis of variance. Animals were monitored for 1 and 3 weeks. RESULTS: By SEM, the O samples showed significant differences from B and C in the area of defect exposure ( H 2,15 = 8.66; P 〈 0.05). In both periods, O and B samples showed similar results for matrix fiber diameters, differently than C samples ( H 2,15 = 14.0; P 〈 0.05). All other SEM variables were considered equivalent among the groups ( P 〉 0.05). Under LM, an acute and chronic granulomatous inflammation was seen in the presence of both biomaterials (B and O, 1 week); both the control and the ovine grafting samples showed mature bone in the repair site (3 weeks); the defect diameter showed similar values among groups, at both monitoring periods ( F 2,12 = 1.0401; P 〉 0.05). CONCLUSION: The ovine particles of this study showed a favorable response to oral tissue repair, demonstrating to be a potential source for the development of bone grafting biomaterials. Microsc. Res. Tech. 2012. © 2012 Wiley Periodicals, Inc.

Beschreibung: The aboriginal use of reed and bone as raw materials for knives and daggers, respectively, has been well-documented ethnographically in some geographical areas of Melanesia. Because of the significant role that these weapons played in inter- and intra-ethnic aggression, they can potentially have retained smears from the contact with human blood. To carry out a guiding low-vacuum scanning electron microscopy (SEM) study of specific interest to ethnography, the outsides of a fragment of stalk of giant cane ( Arundo donax ) and tibial diaphysis of domestic sheep ( Ovis aries ) were smeared with peripheral human blood. No biological specimen preparation was applied to the samples. After just over 1 month, bloodstain boundaries and their neighboring inner areas were examined via secondary electrons by a variable-pressure SEM (VP-SEM) working in low-vacuum mode. On both substrates, bloodstains exhibited micro-scales. No janocyte (erythrocyte negative replica) was observed in the examined areas. However, erythrocytes were seen crowded together as grain-shaped corpuscles in the smear on reed, and several hecatocytes (moon-like shaped erythrocytes) were evidenced in the smear on bone. The results of this study suggest that a VP-SEM working in low-vacuum mode can be used fruitfully to detect blood remains in medium-sized reed and bone antique aboriginal artifacts. This procedure can prospectively help to ethnographic museum curators and aboriginal-art surveyors as an easy guiding test in the valuation of antique traditional weapons prior to acquisition, when the real use of a piece has been claimed by the supplier. Microsc. Res. Tech. 2012. © 2012 Wiley Periodicals, Inc.

Beschreibung: OBJECTIVES: The aim of this study was to evaluate the reactivity of steroid hormone receptors (SHRs), dystroglycans (DGs), matrix metalloproteinases (MMPs), insulin-like growth factor receptor (IGFR-1), and laminin (Lam) in both prostatic stromal and epithelial compartments showing different diseases in elderly men. METHODS: Sixty prostatic samples were obtained from 60- to 90-year-old patients (mean 63 years) with and without prostatic lesions from Hospital of the School of Medicine, State University of Campinas (UNICAMP). The Samples were divided into standard (no lesions); high grade prostatic intraepithelial neoplasia (HGPIN); prostatic cancer (PC); and benign prostatic hyperplasia (BPH) groups. The samples were submitted to immunohistochemistry and Western blotting analyses. Research Ethics Committee of the School of Medicine, University of Campinas/UNICAMP (number 0094.0.146.000-08). RESULTS: The results showed increased IGFR-1 and MMPs protein levels in the PC and HGPIN groups. Decreased αDG and βDG protein levels were verified in the PC and HGPIN groups. Androgen receptor (AR) reactivity was similar among all groups. Estrogen receptor α (Erα) immunoreactivity was more intense in the epithelium in the PC and HGPIN groups. Estrogen receptor β (ERβ) immunoreactivity was weak in the epithelium of the HGPIN and PC groups. CONCLUSIONS: To conclude, there was an association among IGFR-1, MMPs, and SHRs, indicating IGFR-1 as a target molecule in prostate therapy, considering the IGF proliferative properties. Also, the distinct SHRs reactivities in the lesions in both prostatic compartments indicated different paracrine signals and pointed out the importance of estrogenic pathways in the activation of these disorders. Microsc. Res. Tech. 2012. © 2012 Wiley Periodicals, Inc.

Beschreibung: The ultrastructure of oogenesis in Macrobrachium rosenbergii, with reference to vitellogenesis, has not been reported. We used light and electron microscopy, as well as vitellin (Vn) purification and antibody production, to study the temporal and spatial production of Vn in the ovary by immunofluorescence. Histologically, the ovary is subdivided into cone-shaped ovarian pouches with a central core containing layers of oogonia. These divide to produce oocytes that migrate outwardly and differentiate into mature oocytes. During the course of differentiation, oocytes undergo modifications, including the rearrangement of nuclear chromatin, the accumulation of ribosomes, rough endoplasmic reticulum (RER), and lipid, and the formation of secretory and yolk granules, resulting in four stages. Ultrastructurally, early previtellogenic oocytes (Oc 1 ) are characterized by the accumulation of new ribosomal aggregates, translocated from the nucleus. Late previtellogenic oocytes (Oc 2 ) show nuclear heterochromatin with a “clock face” pattern, the presence of RER, and three types of secretory granules. Follicular cells occupy the intercellular spaces and surround the Oc 2 . Early vitellogenic oocytes (Oc 3 ) are larger, with nuclei containing predominantly decondensed euchromatin, and cytoplasm with yolk and secretory granules, and few lipid droplets. Late vitellogenic oocytes (Oc 4 ) are characterized by completely euchromatic nuclei, an indistinct plasma membrane, yolk platelets and secretory granules, and abundant lipid. Vitellogenin (Vg) in ovaries of M. rosenbergii consist of two main bands at MW 90 and 102 kDa. Our data indicates that Vn is present, and probably synthesized in Oc 3 and Oc 4 , but there may be some undetected exogenous Vg production. Microsc. Res. Tech. 2012. © 2012 Wiley Periodicals, Inc.

Beschreibung: Absorptive cells have notable importance for proper function of the colon, absorbing water and nutrients. In type I diabetes, hyperglycemia leads to remarkable alterations in cell structure. In absorptive cells, such changes may impair the function of the organ as a whole. Also, the effects of physical training, which plays crucial role in the treatment of diabetes, are not yet known in these cells. For this reason, to analyze the changes in colon epithelial absorptive cells of diabetic rats and the effects of physical training, Wistar rats were divided into four groups: sedentary control (SC), trained control (TC), sedentary diabetic (SD), and trained diabetic (TD). The training protocol consisted of swimming for 60 min a day, 5 days per week, during 8 weeks. Colon samples were collected, processed, and evaluated by histochemical and ultrastructural techniques. Although histochemical analysis did not reveal major differences, significant morphological differences were ultrastructurally observed among groups, especially related to the structure of tight junctions, interdigitations, and microvilli, which became longer in diabetics, and whose length was reduced after physical training, as proved by statistical analysis. There were no relevant changes in organelles. Thus, the development of type I diabetes can lead to changes at ultrastructural level that, even subtle, may cause important alterations in cell function. The practice of physical training, in turn, proved to be an important ally in the treatment of such changes. However, it cannot be used singly for treating this disease, requiring the combined practice of other methods. Microsc. Res. Tech. 2012. © 2012 Wiley Periodicals, Inc.

Beschreibung: The myotendinous junction (MTJ) is a major area for transmitting force from the skeletal muscle system and acts in joint position and stabilization. This study aimed to use transmission electron microscopy to describe the ultrastructural features of the MTJ of the sternomastoid muscle in Wistar rats from newborn to formation during adulthood and possible changes with aging. Ultrastructural features of the MTJ from the newborn group revealed pattern during development with interactions between muscle cells and extracellular matrix elements with thin folds in the sarcolemma and high cellular activity evidenced through numerous oval mitochondria groupings. The adult group had classical morphological features of the MTJ, with folds in the sarcolemma forming long projections called “finger-like processes” and sarcoplasmic invaginations. Sarcomeres were aligned in series, showing mitochondria near the Z line in groupings between collagen fiber bundles. The old group had altered “finger-like processes,” thickened in both levels of sarcoplasmic invaginations and in central connections with the lateral junctions. We conclude that the MTJ undergoes intense activity from newborn to its formation during adulthood. With increasing age, changes to the MTJ were observed in the shapes of the invaginations and “finger-like processes” due to hypoactivity, potentially compromising force transmission and joint stability. Microsc. Res. Tech. 2012. © 2012 Wiley Periodicals, Inc.

Beschreibung: In addition to causing myiasis in humans, Megaselia spiracularis Schmitz has also been reported as a forensically important fly. In this study, we presented the morphology of all larval instars and puparium of M. spiracularis using scanning electron microscopy. The first instar larva was composed of 12 segments. Antennae and maxillary palp complex were visible. Two spiracular slits could be seen at the posterior spiracle. The branch of posterior spiracular hairs was approximately equal to the palm-formed base in length. Second and third larval instars were very similar to first instar, except for the presence of anterior spiracle. The labium of the second instar larva was triangular and ventrally curved, whereas it was a bilobed structure and the tip forked in the first instar. The bubble membrance comprised of ≈40 globules presented at the third instar larvae. Puparia showed a retracted cephalic region and a pair of pupal respiratory horns on the dorsum. A comparison of the morphological features between immature stages of M . spiracularis and M . scalaris , a forensically important fly indoors in Germany, Malaysia, and China, was discussed. Microsc. Res. Tech. 2012. © 2012 Wiley Periodicals, Inc.

Beschreibung: Experiments were conducted to determine the effects that different methods for the preparation of modern plant samples have on the resulting phytoliths using rice husks ( Oryza sp.). The methods that are commonly used in phytolith extraction include (1) dry ashing, (2) acid extraction (3) a combination of both techniques. The results showed that processing methods have an impact on the morphology of two subspecies of rice phytoliths, dry ashing producing more conjoined cell phytoliths or multicells phytoliths than acid extraction. Using a combination of both methods resulted in the presence of fewer conjoined cells than dry ashing alone, but more conjoined cells than acid extraction. Alternative explanations are proposed to explain the formation of conjoined phytolith cells. Microsc. Res. Tech., 2012. © 2012 Wiley Periodicals, Inc.

Beschreibung: This study evaluated the effect of the systemic use of sodium alendronate in rats in vivo. Forty-five Wistar rats aged 36 to 42 days and weighing 200 to 230 g were randomly assigned to a control group ( n = 20), which received distilled water, and an experimental group ( n = 25), which received 2 weekly doses of 1 mg/kg of chemically pure sodium alendronate. The animals were killed after 60 days of treatment. The tibias were removed for analysis of bone mineral density by dual-energy X-ray absorptiometry (DXA). Then, the maxillary incisors were extracted for analysis of the mineralized dental tissues using fluorescence spectroscopy (FS), scanning electron microscopy (SEM), bright field microscopy (BFM), and cross-sectional microhardness (CSMH) testing. DXA and CSMH data were subjected to statistical analysis by Kruskal-Wallis test (5% significance level). The experimental group presented higher bone mineral density than the control group by DXA. FS analysis revealed presence of alendronate in the mineralized dental tissues of the specimens of the experimental group. Significant morphological differences were not found by SEM and BFM. Enamel and dentin (100 and 300 μm from the dentinoenamel junction) CSMH data did not show significant difference between the control and experimental groups. Based on the obtained results, we conclude that while alendronate increased the bone mineral density and was incorporated into the mineralized dental tissues it did not cause significant alterations in the morphology and microhardness of rat incisor enamel and dentin. Microsc. Res. Tech., 2012. © 2012 Wiley Periodicals, Inc.

Beschreibung: Growth rings were used to determine the root age of medicinal Paeonia lactiflora from four producing areas, and their corresponding paeoniflorin content were measured based on the identification of ages. Different P. lactiflora root samples of different ages were collected from the four major growing areas in China: Bozhou, Anhui Province; Pan'an, Zhejiang Province; Zhongjiang, Sichuan Province; and Heze, Shandong Province. The relationship between the number of growth rings and age was analyzed using hand sections and paraffin sections. The paeoniflorin content in the roots of different P. lactiflora cultivars from different growing areas was measured using high-performance liquid chromatography (HPLC). The growth rings in the P. lactiflora roots were consistent with the age of the plant from Heze, Zhongjiang, Pan'an, whereas that for the P. lactiflora from Bozhou was one less than the age of the plant. The HPLC results show that the paeoniflorin content was highest in P. lactiflora ‘Baihuachuanshaoyao,’ followed by ‘Baihuahangshaoyao,’ ‘Honghuachuanshaoyao,’ and ‘Honghuahangshaoyao,’ ‘Bozhoushaoyao’ had the lowest levels of paeoniflorin. With increasing age, the paeoniflorin in the roots of the different P.lactiflora cultivars slowly declined or remained the same. In summary, the age of the roots of P. lactiflora from different growing areas can be determined using growth rings. The paeoniflorin content in the roots of P. lactiflora is correlated with cultivar and it was slowly declined with increasing age. Microsc. Res. Tech., 2012. © 2012 Wiley Periodicals, Inc.

Beschreibung: Background: The early detection of neoplasia is the prime aim of a diagnostician. Altered chromatin distribution is the earliest microscopic change observed in malignant transformation making it a valuable morphometric parameter. This study was aimed to assess and correlate the progressive changes in chromatin texture from normal to varying grades of premalignancies to malignancy of the oral cavity. Methods: Thirty-four archival tissue specimens categorized as normal buccal mucosa (5), low-grade epithelial dysplasia (11), high-grade epithelial dysplasia (7), and squamous cell carcinoma (11) were stained with Feulgen reaction. Pixel optical densitometry histograms were obtained from analysis of an average of 300 cells/case using ImageJ software. Nine histogram curve characteristics (including area under the curve, area integer percentage, center, centroid) were then analyzed statistically for differences between the four groups. Results: Area integer percentage ( P = 0.002), center ( P = 0.038), and centroid ( P = 0.021) were statistically significant within the four groups The parameters showed a dip in their value from normal to low-grade dysplasia but showed a steady increase in high-grade dysplasia and carcinoma. Conclusion: The optical density characteristics had the ability to differentiate the progression of neoplastic changes by extracting information of the chromatin distribution not quantifiable by routine microscopy. The early clumping of chromatin in the periphery in low-grade dysplasia followed by progressively increasing chromatin aggregates in high-grade dysplasia and carcinoma correlated well with the shift observed in the histogram center and centroid. The novel pixel optical densitometry technique efficiently predicted malignant transformation. Microsc. Res. Tech. 2012. © 2012 Wiley Periodicals, Inc.

Beschreibung: There is an increasing variety of endocrine disrupting chemicals (EDCs) either with (anti)estrogenic or (anti)androgenic potential widely present in the environment. These xenosteroids may mimic endogenous steroid hormones disrupting the homeostasis of physiological pathways and leading to several disturbances, especially in tissues highly dependent on steroid hormones such as the prostate. Taking this into account, this comparative study aimed to verify the potential of ethinylestradiol (EE) and testosterone acting as ECDs on the prostate of both male and female adult gerbils exposed to these agents during the embryonic phase. Consequently, pregnant gerbils were treated either with 10 μg/kg/day of EE or with a single dose of 1 mg of testosterone cypionate. The pups that were born 6–8 days after testosterone exposure and the pups that were born after 3 days of EE exposure were allowed to grow but were sacrificed within 4 months. Serological, morphological, stereological, and immunohistochemical analyses were used. Overall, the results showed that both sexes exposed to testosterone and EE during gestation had a prostatic gland with an increased stromal and epithelial and a reduced luminal compartment. Moreover, we observed that glands affected with prostatic intraepithelial neoplasia showed intense stromal reshuffling. In conclusion, although these alterations were observed in both sexes, more relevant to this study was the differential responsiveness of males and females exposed to these different drugs. Whereas the EE affected males more, the testosterone was more harmful to the females. Microsc. Res. Tech. 2012. © 2012 Wiley Periodicals, Inc.

Beschreibung: Development of cell-based therapy entails the use of different types of materials as support for cultured cells. Some of these materials are opaque. For a general microscope study of cell cultures prepared on transparent supports, Giemsa stain with bright field microscopy is useful. With opaque supports or scaffolds, epifluorescence microscopy is necessary. The method the authors describe uses eosin Y to stain cytoplasm and DAPI to stain nuclei under fluorescence microscopy. This method provides easy and fast fluorescent staining for a general morphological study of cultured cells on transparent or opaque supports. Microsc. Res. Tech. 2012. © 2012 Wiley Periodicals, Inc.

Beschreibung: Recent advances in microwave chemical fixation (MCF) and/or high pressure freezing (HPF) combined with transmission electron microscopy have resulted in superior ultrastructural detail in a variety of tissue types. To date, selachian tissue has been fixed and processed using only standard chemical fixation (CF) methods, and the resulting ultrastructure has been less than ideal. In this study, we compared the ultrastructure of the fragile retinal tissue from the brown banded bamboo shark, Chiloscyllium punctatum , obtained using CF, MCF, and HPF methods. For all fixation protocols, ultrastructural preservation was improved by keeping the tissue in oxygenated Ringer solution until the time of fixation. Both MCF and HPF produced superior retinal ultrastructure compared to conventional CF. Although HPF occasionally resulted in very high quality ultrastructure, microwave fixation was almost comparable, quicker and far more consistent. Microsc. Res. Tech., 2012. © 2012 Wiley Periodicals, Inc.

Beschreibung: The Fanniidae is one of four families in the superfamily Muscoidea (Diptera), including some important medical and hygienic flies. There is a paucity of reports on the ultrastructure of olfactory sensilla for the fanniid species. To provide more information on the morphology of the antennal and palpal sensilla of fanniid fly, Fannia hirticeps (Stein, 1892) has been studied using scanning electron microscopy. The first two antennal segments, scape and pedicel, are covered by microtrichiae and several chaetic sensilla. Six distinct morphological types of sensilla are recorded on the antennal funiculus, including one trichoid, two basiconic, two coeloconic sensilla, and one clavate sensilla. The measurement and density of each sensilla type are also provided. The trichoid sensilla tend to be longer and denser toward the apex of antennal funiculus. Basiconic sensilla spread all over the funicular surface. F. hirticeps bears two types of coeloconic sensilla, type 2 coeloconic sensilla distributed on the distal part of the anterior surface, whereas type 1 distributed on the rest of the funiculus. Clavate sensilla are found on the base of antennal funiculus. Only one large sensory pit is located on the posterior surface. Maxillary palps bear one type of basiconic sensilla. These results are compared with eight other muscid flies. Our findings provide a morphological basis for future investigations on olfactory-mediated behavior of this group. Microsc. Res. Tech. 2012. © 2012 Wiley Periodicals, Inc.

Beschreibung: We performed a macroscopic and microscopic study of the tongues of common opossums, Didelphis marsupialis, from South America. We studied two males and two females. We collected morphometric data on the tongue with precision calipers. For the light microscopy and scanning electron microscopy analyses, we fixed tissue fragments in 10% formaldehyde and 2.5% glutaraldehyde, respectively. The opossum tongues averaged 5.87 ± 0.20 cm in length, 3.27 ± 0.15 cm in width at the lingual body, and 3.82 ± 0.15 cm in width at the root. The mean thickness of the lingual body was 1.8 ± 0.1 cm, and the thickness of the root was 3.82 ± 0.15 cm. Sharp filiform papillae were scattered across the entire tongue; conical filiform papillae occurred on the lingual body and tongue tip; fungiform papillae were scattered among the filiform papillae on the lingual body and tongue tip; and there were three vallate papillae at the root of the tongue. We found two strands of papillary projections in the tongue root. Despite the low variability observed in the lingual papillae, the morphological data obtained in this study may be related to the opossum's diverse food habits and the extensive geographic distribution of the species throughout America. Microsc. Res. Tech. 2012. © 2012 Wiley Periodicals, Inc.

Beschreibung: White button mushroom extract was examined in this study on (1) its potential effect on angiogenesis in chorioallantoic culture and (2) its recovering effect on the skin after injury in the ICR mice. Methods used included TUNEL assay on apoptosis, immunohistochemistry for vascular endothelial growth factor (VEGF), proliferative cell nuclear antigen (PCNA), epidermal growth factor (EGF), transforming growth factor β (TGF-β), and immune factor CD4 and western blotting. The results of chorioallantoic culture showed that the mushroom treatment led to significant increase in densities of VEGF sites. In the skin injury, ICR mice model increased EGF, PCNA, and collagen fibers, along with decrease of TUNEL positive apoptotic cells and limited reaction of TGF-β and CD4 indicated that white button mushroom extract appeared to have beneficial effects on skin in regeneration and after injury. Microsc. Res. Tech. 2012. © 2012 Wiley Periodicals, Inc.

Beschreibung: Using fluorescence in situ hybridization technique, expression of three oncogenes, C-myc, RARa, and cyclin-D was tested on a uterine leiomyoma. C-myc and RARa were amplified in approximately 30% and 90% of the cells, respectively. Numerous small signals of C-myc were indicative of the presence of double minutes. Amplification of RARa is being reported for the first time in a leiomyoma. Cyclin-D was normal in diploid cells while it was highly amplified in polyploid cells. Low levels of amplified C- myc and cyclin-D cells seem to be the reason for this tumor to be benign, while RARa could not be effective without the association of some other gene such as PML. Information presented here are significant toward developing new curative strategies such as gene-specific drugs and molecular manipulation to stop the activity of cancer gene. Further study may elucidate that how fibroids grow and maintain their rare benign nature. © 2012 Wiley Periodicals, Inc.

Beschreibung: Titanium surface texture and chemistry modification successfully improves the host response and consequently the bone-to-implant contact surrounding dental implants. The aim of the present study was to investigate, using histomorphometrical-analysis, the effects of titanium surface modification by laser-ablation (Nd:YAG) followed by thin chemical deposition of HA. Forty-eight rabbits received one implant by tibiae of AS-machined (MS), laser-modified (LMS), or biomimetic hydroxyapatite-coated (HA) surface. Bone-to-implant contact (BIC) and bone area (BBT) were evaluated after 4, 8, and 12 weeks, at cortical and cancellous regions. Average BIC in the cortical region was higher ( P 〈 0.001) on the LMS and HA implants for all periods, with no differences between LMS and HA. For the cancellous area, the LMS and HA implants showed higher ( P 〈 0.01) BIC than MS at the initial periods. The LMS and HA showed similar values in the cortical region, but a tendency of higher values for HA in the cancellous region was observed in all periods. For the BBT, the differences were found only between HA and MS after 4 weeks in the cortical region ( P 〈 0.05), and after 12 weeks in the cancellous area ( P 〈 0.05). Our results showed that HA biomimetic coating preceded by laser treatment induced the contact osteogenesis and allowed the formation of a more stable bone–implant interface, even in earlier periods. Microsc. Res. Tech., 2012. © 2012 Wiley Periodicals, Inc.

Beschreibung: Egg morphology of nine species of the cluster fly genus Pollenia Robineau-Desvoidy was studied with scanning electron microscopy. Funnel-shaped micropylar area, chorion covered with centrally depressed hexagons, well developed hatching pleats on the dorsal surface encompassing the median area were found in eggs of all species: P. amentaria (Scopoli), P. angustigena Wainwright, P. atramentaria (Meigen), P. labialis Robineau-Desvoidy, P. mayeri Jacentkovsky, P. pediculata Macquart, P. rudis (Fabricius), P. similis (Jacentkovsky), and P. vagabunda (Meigen). Untypical for Calliphoridae, a dual morphological structure of plastron-bearing area is described here for the first time. On the basis of our observations, all species studied share the presence of a plastron respiratory function on the entire surface between the hatching lines. Differences between species were found in the shape of the longitudinal hatching pleats and the proportion between ‘island pattern’ and ‘hexagonal pattern’ of their chorionic surface, and in the intensity of perforation of hexagons of the median area. Comparisons of Pollenia egg morphology with that of other representatives of Calliphoridae revealed its unique structure, allowing easy differentiation from other representatives of the family. Microsc. Res. Tech. 2012. © 2012 Wiley Periodicals, Inc.

Beschreibung: Apatura ilia (Denis and Schiffermüller, 1775) and A. iris (Linnaeus, 1758) are fascinating butterflies found in the Palaearctic ecozone (excepting the north of Africa). The wings of these insects are covered with a great number of two types of scales positioned like roof tiles. Type I scales are on the surface, while type II scales are situated below them. The structural color of the type I scales is recognized only on the dorsal side of both the fore and hind wings of the males of the aforementioned species. Both types of scales are responsible for pigment color of the wings, but iridescence is observed only in the type I scales. The brilliant structural color is due to a multilayer structure. The features of the scales, their dimensions and fine structure were obtained using scanning electron microscopy. Cross sections of the scales were then analyzed by transmission electron microscopy. The scales of the “normal” and clytie forms of A. ilia have a different nanostructure, but are of the same type. A similar type of structure, but with a different morphology, was also noticed in A. iris . The scales of the analyzed species resemble the scales of tropical Morpho butterflies. Microsc. Res. Tech., 2012. © 2012 Wiley Periodicals, Inc.

Beschreibung: OBJECTIVES: To compare microleakage of three self-etch adhesives and to analyze enamel surface morphology and interfacial morphology of resin–enamel and resin–dentin interface under scanning electron microscope (SEM). EXPERIMENTAL DESIGN: Study was conducted in 65 extracted human premolars. Class V cavities were prepared in 45 teeth and assigned to three groups ( n = 15) according to three self-etch adhesives (OptiBond All-in-One, iBond, and Adper Prompt L-Pop). After restoration, 10 samples from each group were used to assess microleakage at enamel and dentin margin. Five samples from each group were used for analysis of interfacial morphology at resin–enamel and resin–dentin interface under SEM. Remaining 20 teeth were used to prepare flat enamel buccal surfaces to analyze the difference in surface morphology after treatment with three adhesives ( n = 5 each) and 36% phosphoric acid treatment ( n = 5). PRINCIPAL OBSERVATIONS: At enamel margin, Prompt L-Pop depicted least leakage of all the three adhesives and also showed best interfacial adaptation under SEM. At dentin margin, OptiBond All-in-One showed significant less leakage than iBond and Prompt L-Pop. On flat enamel surface, phosphoric acid produced the most retentive etching pattern when compared with the three adhesives. CONCLUSION: Prompt L-Pop showed the best bonding effectiveness in enamel, whereas OptiBond All-in-One performed significantly better in dentin. Microsc. Res. Tech. 2012. © 2012 Wiley Periodicals, Inc.

Beschreibung: Bone marrow is a source of stem cells for greater and easier access, which is widely studied as a provider of hematopoietic and mesenchymal cells for various purposes, mainly therapeutic by the advances in research involving cell therapy. The swine is an animal species commonly used in the pursuit of development of experimental models. Thus, this study aimed to standardize protocol for collection and separation of bone marrow in swines, since this species is widely used as experimental models for various diseases. Twelve animals were used, which underwent bone marrow puncture with access from the iliac crest and cell separation by density gradient followed by a viability test with an average of 98% of viable cells. Given our results, we can ensure the swine as an excellent model for obtaining and isolation of mononuclear cells from bone marrow, stimulating several studies addressing the field of cell therapy. Microsc. Res. Tech., 2012. © 2012 Wiley Periodicals, Inc.

Beschreibung: We have explored the effects of atmospheric environment on Kelvin force microscopy (KFM) measurements of potential difference between different regions of test polycrystalline diamond surfaces. The diamond films were deposited by microwave plasma-assisted chemical vapor deposition, which naturally produces hydrogen terminations on the surface of the films formed. Selected regions were patterned by electron-beam lithography and chemical terminations of oxygen or fluorine were created by exposure to an oxygen or fluorine plasma source. For KFM imaging, the samples were mounted in a hood with a constant flow of helium gas. Successive images were taken over a 5-h period showing the effect of the environment on KFM imaging. We conclude that the helium flow removes water molecules adsorbed on the surface of the samples, resulting in differences in surface potential between adjacent regions. The degree of water removal is different for surfaces with different terminations. The results highlight the importance of taking into account the atmospheric environment when carrying out KFM analysis. © 2012 Wiley Periodicals, Inc.

Beschreibung: The liver has a remarkable ability to regenerate after partial hepatectomy (PH), although the factors governing such ability are still poorly understood. During the prereplicative phase of the regeneration, ultrastructural alterations of periportal hepatocytes were seen, including mitochondrial swelling, abnormal accumulation of lipids, and myelin figures which could lead to the formation of lipid droplets. As it has been hypothesized that caveolin-1 is involved in lipidogenesis and in mitochondrial homeostasis, we aimed to study the subcellular distribution of caveolin-1 in hepatocytes at an early stage following PH. Liver samples were processed for light and electron microscopy at 0 h, 24 h, and 96 h after PH. The expression and subcellular distribution of caveolin-1 was assessed by immunohistochemical and immunocytochemical techniques. Following PH, at 24 h, membranes of altered mitochondria of periportal hepatocytes exhibited significant decrease of caveolin-1 expression compared with control. Myelin figures showing high expression of caveolin-1 were also seen. At 96 h, hepatocytes became ultrastructurally similar to the control liver, and the expression of caveolin-1 on mitochondria showed a moderate increase compared with 24 h after PH. Decrease of expression of caveolin-1 in the altered liver mitochondrial membranes at 24 h following PH, and the high expression of caveolin-1 observed on myelin figures, suggests involvement of caveolin-1 is in both mitochondrial homeostasis and lipidogenesis. Addressing the role played by caveolin-1 during liver regeneration might disclose additional features of mitochondrial homeostasis and lipidogenesis during frequent metabolic liver diseases. Microsc. Res. Tech., 2012. © 2012 Wiley Periodicals, Inc.

Beschreibung: In this study, we aimed to increase the sensitivity of protein labeling using 1.4 nm gold nanoparticles and glutamate δ2 receptor (GluD2) from the postsynaptic membrane of the Purkinje cells. The very small marker size of the particles reduces the steric hindrance between antibodies leading to a higher labeling efficiency of more than one subunit per single receptor molecule. The nanoparticles are visible in 200 kV dark-field scanning transmission electron microscope on freeze-fractured carbon replica of nervous tissue after plasma cleaning treatment. The different elemental composition of nanoparticles as Au nanogold or CdS quantum dot can be distinguished by energy dispersive X-ray spectroscopy. This method ensures detection of an average of three subunits per GluD2 and often labels all four of them with 1.4 nm Au nanoparticles. It is concluded that this high-resolution microscopic method is useful for exploring the quaternary structure of membrane proteins. Microsc. Res. Tech., 2012. © 2012 Wiley Periodicals, Inc.

Beschreibung: Electron microscopy has been used to visualize chromosome since it has high resolution and magnification. However, biological samples need to be dehydrated and coated with metal or carbon before observation. Ionic liquid is a class of ionic solvent that possesses advantageous properties of current interest in a variety of interdisciplinary areas of science. By using ionic liquid, biological samples need not be dehydrated or metal-coated, because ionic liquid behaves as the electronically conducting material for electron microscopy. The authors have investigated chromosome using ionic liquid in conjunction with electron microscopy and evaluated the factors that affect chromosome visualization. Experimental conditions used in the previous studies were further optimized. As a result, prewarmed, well-mixed, and low concentration (0.5∼1.0%) ionic liquid provides well-contrasted images, especially when the more hydrophilic and the higher purity ionic liquid is used. Image contrast and resolution are enhanced by the combination of ionic liquid and platinum blue staining, the use of an indium tin oxide membrane, osmium tetroxide-coated coverslip, or aluminum foil as substrate, and the adjustment of electron acceleration voltage. The authors conclude that the ionic-liquid method is useful for the visualization of chromosome by scanning electron microscopy without dehydration or metal coating. Microsc. Res. Tech., 2012. © 2012 Wiley Periodicals, Inc.

Beschreibung: The mammalian skin has developed sensory structures (mechanoreceptors) that are responsible for different modalities of mechanosensitivity like touch, vibration, and pressure sensation. These specialized sensory organs are anatomically and functionally connected to a special subset of sensory neurons called mechanosensory neurons, which electrophysiologically correspond with Aβ fibers. Although mechanosensory neurons and cutaneous mechanoreceptors are rather well known, the biology of the sense of touch still remains poorly understood. Basically, the process of mechanosensitivity requires the conversion of a mechanical stimulus into an electrical signal through the activation of ion channels that gate in response to mechanical stimuli. These ion channels belong primarily to the family of the degenerin/epithelium sodium channels, especially the subfamily acid-sensing ion channels, and to the family of transient receptor potential channels. This review compiles the current knowledge on the occurrence of putative mechanoproteins in mechanosensory neurons and mechanoreceptors, as well as the involvement of these proteins on the biology of touch. Furthermore, we include a section about what the knock-out mice for mechanoproteins are teaching us. Finally, the possibilities for mechanotransduction in mechanoreceptors, and the common involvement of the ion channels, extracellular membrane, and cytoskeleton, are revisited. Microsc. Res. Tech., 2012. © 2012 Wiley Periodicals, Inc.

Beschreibung: Changes in physicochemical properties of the human hair shaft induced by UVB during the summer climate were investigated. Using atomic force microscopy and thermogravimetric analyzer measurements, we investigated the changes in hair shaft physical properties including morphology, roughness, and water content. The changes in chemical properties were investigated by using FTIR spectroscopy. Hair samples from 20 females without any chemical or physical stresses were investigated. The samples were exposed to various UVB doses of 400, 2,000, and 4,000 mJ/cm 2 , which correspond to the radiation times of 0.44, 2.22, and 4.44 h, respectively. The morphology and surface of the hair shaft was not significantly affected by UVB irradiation. The components of keratin were affected by the UVB irradiation where the amide A bands at 3,263 and 3,246 cm −1 of the proximal and distal roots, respectively, were shifted to higher band region because α-helix converted to β-sheet, and the stretching modes of C H lipid alkyl chains at 2,965 and 2,850 cm −1 in the proximal and distal roots, respectively, were developed. As the UVB intensity increased, the water content of the proximal root decreased. Microsc. Res. Tech., 2012. © 2012 Wiley Periodicals, Inc.

Beschreibung: The shallow-water bivalve Codakia orbiculata which harbors gill-endosymbiotic sulfur-oxidizing γ-proteobacteria can lose and acquire its endosymbionts throughout its life. Long-term starvation and recolonization experiments led to changes in the organization of cells in the lateral zone of gill filaments. This plasticity is linked to the presence or absence of gill-endosymbionts. Herein, we propose that this reorganization can be explained by three hypotheses: (a) a variation in the number of bacteriocytes and granule cells due to proliferation or apoptosis processes, (b) a variation of the volume of these two cell types without modification in the number, and (c) a combination of both number and cell volume variation. To test these hypotheses, we analyzed cell reorganization in terms of proliferation and apoptosis in adults submitted to starvation and returned to the field using catalyzed reporter deposition fluorescence in situ hybridization, immunohistochemistry, and structural analyses. We observed that cell and tissue reorganization in gills filaments is due to a variation in cell relative abundance that maybe associated with a variation in cell apparent volume and depends on the environment. In fact, bacteriocytes mostly multiply in freshly collected and newly recolonized individuals, and excess bacteriocytes are eliminated in later recolonization stages. We highlight that host tissue regeneration in gill filaments of this symbiotic bivalve can occur by both replication of existing cells and division of undifferentiated cells localized in tissular bridges, which might be a tissue-specific multipotent stem cell zone. © Microsc. Res. Tech. 2012. © 2012 Wiley Periodicals, Inc.

Beschreibung: The glycoconjugate pattern of developing ovarian follicles in wild and cultured Senegalese sole Solea senegalensis was investigated by means of lectin histochemistry. Ovaries from cultured fish contained oocytes up to the late vitellogenic stage, whereas they reached the hydration stage in wild specimens. The follicular cells bound MAL II, SBA, HPA, DBA, Con A, KOH-sialidase (K-s)-WGA, GSA I-B 4 in the late vitellogenic stage, and in wild fish also SNA and K-s-PNA, whereas in the hydration stage SBA, HPA, DBA, and GSA I-B 4 only. The zona radiata reacted with SBA, HPA, DBA, Con A, and GSA I B 4 in the late vitellogenic stage and in cultured fish also with UEA I, whereas in the hydration stage it stained with SBA only. The cortical alveoli bound SBA, HPA, RCA 120 during the late vitellogenic stage, also SNA, PNA, K-s-PNA, GSA I-B 4 in cultured fish, DBA, and K-s-WGA in wild ones which stained with SBA, HPA, and GSA I-B 4 in the hydrated stage. The yolk reacted with Con A in the late vitellogenic oocytes, and also with MAL II, SNA, K-s-PNA, SBA, HPA, K-s-WGA, GSA I-B 4 , UEA I in the hydrated ones. From perinucleolus to late vitellogenic stages, the oocyte nucleoplasm bound Con A, GSA I-B 4 , GSA II, UEA I, and in wild fish also MAL II, SNA, LTA but only GSA I-B 4 reactivity in the early maturation stage. These findings demonstrate that the glycan pattern of fish ovarian follicles changes during the maturative stages and that it is affected by culture-rearing conditions. Microsc. Res. Tech. 2012. © 2012 Wiley Periodicals, Inc.

Beschreibung: Mammalian choroid plexuses (CPs) are vascularized structures involved in numerous exchange processes that supply nutrients and hormones to the brain, and that remove deleterious compounds and metabolites from the brain. Studies in the adult Mediterranean buffalo have investigated the morphology of CPs using histochemical and immunohistochemical techniques. To date, however, there have been no studies conducted on ruminants regarding this removal process which serves to repair functional vascular damage in the CPs. Each of these vascular repair processes is a very complex and none of these has not yet been completely understood. Then, the aim of the present study is to investigate the morphological processes during angiogenesis in the CPs of healthy adult buffaloes, utilizing transmission electron microscopy (TEM), scanning electron microscopy (SEM), and immunogold-labeling SEM analysis (biomarkers: angiopoietin-2 [Ang-2], vascular endothelial growth factor receptor-3 [VEGFR-3], and CD133). At TEM, the inner surface of the blood capillaries sometimes showed pillar-like cells, which in contact with endothelial cells formed prominences, which in turn formed neo-blood capillaries. With immunogold-labeling SEM analysis, the CP blood capillaries showed Ang-2 and VEGF-3, respectively, in positive particles and spheroid formations. In addition, the external surface of the blood capillaries showed spheroid formations that originated from the neo-vascular capillaries whose terminals formed a capillary network, positive to CD133. On the basis of these results, the following hypothesis can be made, namely, that these CPs are vascular structures which play a fundamental role in maintaining brain homeostasis and self-repairing of functional vascular damage, independently of the presence of rete mirabile in this species. Microsc. Res. Tech. 2012. © 2012 Wiley Periodicals, Inc.

Beschreibung: The stretch zone width (SZW) data for 15-5PH steel CTOD specimens fractured at −150°C to + 23°C temperature were measured based on focused images and 3D maps obtained by extended depth-of-field reconstruction from light microscopy (LM) image stacks. This LM-based method, with a larger lateral resolution, seems to be as effective for quantitative analysis of SZW as scanning electron microscopy (SEM) or confocal scanning laser microscopy (CSLM), permitting to clearly identify stretch zone boundaries. Despite the worst sharpness of focused images, a robust linear correlation was established to fracture toughness ( K C ) and SZW data for the 15-5PH steel tested specimens, measured at their center region. The method is an alternative to evaluate the boundaries of stretched zones, at a lower cost of implementation and training, since topographic data from elevation maps can be associated with reconstructed image, which summarizes the original contrast and brightness information. Finally, the extended depth-of-field method is presented here as a valuable tool for failure analysis, as a cheaper alternative to investigate rough surfaces or fracture, compared to scanning electron or confocal light microscopes. Microsc. Res. Tech. 2012. © 2012 Wiley Periodicals, Inc.

Beschreibung: Here we report a new sample preparation method for three-dimensional electron tomography. The method uses the standard film deposition and focused ion beam (FIB) methods to significantly reduce the problems arising from the projected sample thickness at high tilt angles. The method can be used to prepare tomography samples that can be imaged up to a ±75° tilt range which is sufficient for many practical applications. The method can minimize the problem of Ga + contamination, as compared to the case of FIB preparation of rod-shaped samples, and provides extended thin regions for standard 2D projection analyses. Microsc. Res. Tech., 2012. © 2012 Wiley Periodicals, Inc.

Beschreibung: Chrysomya megacephala is a forensic important fly, and its immature forms also cause myiasis. The adults are the first insects to reach a carcass and can oviposit just a few hours after arrival. Therefore, the knowledge of immature stages of flies is essential for correct identification of the species found on corpses. Scanning electron microscopy (SEM) gives detailed information on the morphological characters that can help identify the immature forms of flies. The aim of this study is to identify larvae and puparia of C. megacephala using SEM. The larval instar body of C. megacephala is similar at all instars. The integument is smooth with small spines located at the limit of all segments. The cephalic region has a group of robust spines with one or two tips. The puparia are very similar to third instar larvae, except for the cephalic structures that are retracted. The integument shows the wrinkles from the third instar larvae and posterior spiracle disc with three spiracular openings localized on the top of an elevation. In conclusion, SEM provides some characteristics to distinguish among Chrysomya species that could help entomologists to identify immature found on corpses. Microsc. Res. Tech. 2012. © 2012 Wiley Periodicals, Inc.

Beschreibung: Potentilla fulgens , a member of the family Rosaceae, has been known since ancient times in Northeast India for its antiparasitic, antitumour and antidiabetic properties. The crude ethanolic extract of the plant was assayed against Raillietina echinobothrida , the intestinal cestode parasite of domestic fowl to authenticate the putative anthelmintic efficacy and cestocidal potential in particular of the plant. Treatment of the worm in-vitro with 25, 50 and 100 mg of crude extract per ml of phosphate buffered saline caused paralysis leading to death taking 4.02–4.20, 3.05–3.75 and 2.05–2.83 h respectively. Scanning electron microscopic observations on treated worms revealed deformation of surface architecture, particularly in scolex and surface microtriches. Severe distortion and disorganization of the tegumental and subtegumental layer along with cytoplasmic organelle was also observed under transmission electron microscope. The possible use of the plant as a potential anthelmintic against cestode parasite is discussed. Microsc. Res. Tech., 2012. © 2012 Wiley Periodicals, Inc.

Beschreibung: Despite the large number of species, their wide distribution, and unique reproductive characteristics, Neotropical bats have been poorly studied, and important aspects of the reproduction of these animals have not been elucidated. We made an ultrastructural analysis of spermatogenesis in Molossus molossus (Molossidae). The process of spermatogonial differentiation is similar to that found in other bats and is also relatively similar to that of Primates, with three main spermatogonia types: A d , A p , and B. Meiotic divisions proceed similarly to those of most mammals, and spermiogenesis is clearly divided into 12 steps, in the middle of the range known for bats (9–16 steps). Formation of the acrosome is similar to that known from other mammals; however, the ultrastructure of spermatozoa was found to have unique characteristics, including many wavy acrosomal projections on its surface, which seems to be specific for the family Molossidae. Comparing the ultrastructure of the spermatozoon of M. molossus with other bats already study, we observed that three characters vary: morphology of the outer dense fibers, of the perforatorium, and of the spermatozoon head. The great similarity of morphological characters between M. molossus and Platyrrhinus lineatus suggests that M. molossus is more closely related to the Phyllostomidae than to the Rhinolophidae and the Vespertilionidae. Microsc. Res. Tech. 2012. © 2012 Wiley Periodicals, Inc.

Beschreibung: In this study, healing of cutaneous wounds in Labeo rohita using scanning electron microscope is reported. Wound area could be divided into three regions. Immediately after infliction of wound, edges retract exposing underlying tissues in wound gap (Region I). Simultaneously, at region close to wound edge (Region II), mucous goblet cell openings are observed with copious mucous secretions. Within 1 h, Region I gets covered by mucous secretions, and epidermis at edges starts migrating. Opposing fronts gradually advance and by 4–6 h come in contact to epithelialize wound gap. Zone of contact of fronts is demarcated by epidermal ridge, which is relatively prominent at 8 h. It gradually diminishes and is not distinguished at 24 h and afterward. At 1–4 h, microridges on epithelial cell surfaces appear irregularly arranged, widely spaced, short with abrupt ends at Region I; relatively extensive at Region II; and similar to those in controls at region surrounding Region II (Region III). At 12 h and afterward, microridges appear similar to those in controls at Regions I and II. At 1–2 h, isolated swollen epithelial cells, often in process of detachment and exfoliation at surface, are observed at Regions I and II. Such cells are infrequent at 8 h and afterward. Region I covered by migrated epidermis appears trough like at 4 h to 2 days, level of which gradually rises and at Day 4, surface of epidermis appears at a level similar to that at Regions II and III. Changes have been associated with the imbalance of osmotic homeostasis due to disruption of barrier between internal and external environment of skin. Microsc. Res. Tech. 2012. © 2012 Wiley Periodicals, Inc.

Beschreibung: This study evaluated the presence of residues after removal of calcium hydroxide [Ca(OH) 2 ] associated with different vehicles. Thirty single-rooted teeth were instrumented to a master apical file #25 using 2.5% NaOCl as main irrigant and 17% trisodium EDTA (ethylenediaminetetraacetic acid) as final agent irrigant. Then, the root canals were dressed with Ca(OH) 2 associated with silicone oil (Group 1), 2% chlorhexidine gluconate (Group 2), or propylene glycol (Group 3). After coronal sealing, all teeth were kept in a moist environment at room temperature. After 7 days, the teeth were reopened and medicaments were removed using 5 mL of saline solution and instrumentation with master apical file followed by new irrigation with 5 mL of 2.5% NaOCl. Subsequently, teeth were split longitudinally and assessed by scanning electron microscopy. The wall cleanliness of the cervical and apical thirds of the roots were evaluated and scored by three blinded examiners. Statistical analysis was performed using Kruskal–Wallis and Wilcoxon tests at 5% level of significance. All roots had residues of Ca(OH) 2 on the canal walls. All experimental groups had similar results ( P 〉 0.05) regardless of the third evaluated. There was significant difference between the apical and cervical thirds only in Group 3 ( P 〈 0.05). Association of different vehicles to Ca(OH) 2 does not influence the persistence of residues on the root canal walls. Microsc. Res. Tech. 2012. © 2012 Wiley Periodicals, Inc.

Beschreibung: Diplopods are agile saprophagous invertebrates with the ability to colonize several soil layers. They feed on waste and decaying organic matter, making the soil in which they are present better aerated and mineral-enriched. Because of their habits, diplopods have been suggested to be used in studies to analyze possible toxic effects of impacted soils in edafic fauna. In that respect, this study aimed at checking the feasibility of using morphological analysis from millipede's midgut as a biomarker for soils exposed to sewage sludge from sewage treatment plants. Histological and histochemical techniques, applied to the species Rhinocricus padbergi 's midgut, were used. Two sewage sludge samples were used; they were collected in two small towns in the countryside of São Paulo State. Both cities are part of the Piracicaba–Capivari–Jundiaí basin (PCJ-1 and PCJ-3). For 7 and 15-day periods, the animals were exposed to 10% concentrations and crude sludge for the PCJ-1sample and to 1, 10, and 50% concentrations and sludge for the PCJ-3 sample. The material exposed to the PCJ-1 sample showed an increase in the presence of cytoplasmic granules in both concentrations in all periods and a slight increase in the rate of epithelial renewal. The material of PCJ-3 sample presented the same alterations observed in PCJ-1 sample in higher frequence and over that high quantity of neutral polysaccharides in the hepatic cells layer. We can conclude that with the increase of sewage sludge concentrations tested, there is an increase in morphophysiologicals alterations frequencies found in the diplopods' midgut. Microsc. Res. Tech. 2012. © 2012 Wiley Periodicals, Inc.