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.

In most anatomical studies developed with mammals, the tongue is described as highly differentiated among different species. However, studies on the tongue of aquatic mammals are still limited as compared to those on terrestrial mammals. The aim of this study was to describe the tongue morphology of the Franciscana dolphin (Pontoporia blainvillei) using macroscopic observations, light, and scanning electron microscopy. Microscopically, the dorsal surface was covered by a keratinized stratified epithelium. Salivary gland acini were found on the middle and caudal third of the tongue. The dorsal surface was totally covered by filiform papillae with a connective tissue core and a connective tissue structure round in shape in the middle and caudal regions. Microsc. Res. Tech., 2012. © 2012 Wiley Periodicals, Inc.

The transient receptor potential (TRP) channels are involved in sensing mechanical/physical stimuli such as temperature, light, pressure, as well as chemical stimuli. Some TRP channels are present in the vertebrate retina, and the occurrence of the multifunctional channel TRP vanilloid 4 (TRPV4) has been reported in adult zebrafish. Here, we investigate the expression and distribution of TRPV4 in the retina of zebrafish during development using polymerase chain reaction (PCR), Western blot, and immunohistochemistry from 3 days post fertilization (dpf) until 100 dpf. TRPV4 was detected at the mRNA and protein levels in the eye of zebrafish at all ages sampled. Immunohistochemistry revealed the presence of TRPV4 in a population of the retinal cells identified as amacrine cells on the basis of their morphology and localization within the retina, as well as the co-localization of TRPV4 with calretinin. TRPV4 was first (3 dpf) found in the soma of cells localized in the inner nuclear and ganglion cell layers, and thereafter (10 dpf) also in the inner plexiform layer. The adult pattern of TRPV4 expression was achieved by 40 dpf the expression being restricted to the soma of some cells in the inner nuclear layer and ganglion cell layers. These data demonstrate the occurrence and developmental changes in the expression and localization of TRPV4 in the retina of zebrafish, and suggest a role of TRPV4 in the visual processing. Microsc. Res. Tech., 2012. © 2012 Wiley Periodicals, Inc.

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.

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, the 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.

Background: Diabetes mellitus results in many complications, also compromising the salivary glands. The current treatment for this condition should be a substituting method to exogenous insulin. In this aspect, the immunotherapy has been tested, but, it can be inefficient as an agent for the control of damage caused by diabetes. Thus, the aim of this study was to evaluate the anti-CD3 monoclonal antibody as alternative immunotherapy in the recovery of salivary glands of spontaneously diabetic NOD (nonobese diabetic) mice. Methods: NOD mice were divided into two groups of 10 animals: group I (untreated diabetic mice) and group II (anti-CD3-treated diabetic mice). After treatment, the samples of salivary glands were collected for histological examination under both transmitted and polarized light microscopy. Results: Alterations in tissue architecture; increase in extracellular matrix and presence of inflammatory process were observed in untreated animals. Recovery of the salivary acinar cells occurred in treated animals. The parotid glands demonstrated a smaller amount of collagen fibers and were not observed severe inflammatory processes. Conclusion: These results indicate that immunotherapy contributed to reestablishment of tissue damaged by the hyperglycemic condition, demonstrating that the immunomodulation plays an important role in the recovery of salivary glands. Microsc. Res. Tech., 2012. © 2012 Wiley Periodicals, Inc.

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.

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.

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.

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.

Although the Geographical Information System (GIS), which integrates computerized drawing computer assisted design (CAD) and relational databases (data base management system (DBMS)), is best known for applications in geographical and planning cartography, it can also use many kinds of information concerning the territory. A multidisciplinary project was initiated since 5 years a multidisciplinary study was initiated to use GIS to integrate environmental and ecological data with findings on animal health, ethology, and anatomy. This study is chiefly aimed at comparing two different methods for measuring the absorptive surface of rumen papillae. To this scope, 21 female sheep (Ovis aries) on different alimentary regimes (e.g., milk and forage mixed diet, early herbaceous diet, dry hay diet, and fresh hay diet at the maximum of pasture flowering and at the maximum of pasture dryness) were used; after slaughtering, 20 papillae were randomly removed from each sample collected from four indicator regions of rumen wall, placed near a metric reference and digitally photographed. The images were developed with the ArcGIS™ software to calculate the area of rumen papillae by means of GIS and to measure their mid-level width and length to calculate the papillae area as previously performed with a different method. Spatial measurements were analyzed using univariate and multivariate methods. This work demonstrates that the GIS methodology can be efficiently used for measuring the absorptive surface of rumen papillae. In addition, GIS demonstrated to be a rapid, precise, and objective tool when compared with previously used method. Microsc. Res. Tech. 2012. © 2012 Wiley Periodicals, Inc.

To help understand the mechanism of host discrimination during oviposition of a parasitoid wasp, Venturia canescens (Gravenhorst) (Hymenoptera: Ichneumonidae), the ultrastructure and morphology of the sense organs on the dorsal and ventral stylets of the ovipositor were examined using scanning and transmission electron microscopy. Ovipositor stylets were scanned for sense organs, because only unsheathed ovipositors are used for probing, stabbing, and host discrimination. Five types of sensilla and secretary pores were identified. Based on their pattern of innervation, the surface-dome and pitted-dome sensilla were thought to have a gustatory function and, thus, are the best candidates for host-discrimination. The arrangements of dendrites of type I and type II campaniform sensilla suggested that these acted as pressure- and mechano-receptors, respectively. Coeloconic sensilla might act as thermo-hygroreceptors, whilst secretary pores might provide lubrication during the process of stabbing for oviposition. Surface-dome sensilla were the most numerous type found followed by secretary pores, pitted-dome sensilla, and coeloconic sensilla. Almost all the sensilla and secretary pores were more numerous on the dorsal than on the ventral stylets, and the maximum number of these sensilla and secretary pores occurred on the distal end of the ovipositor stylets and decreased toward the proximal end. The surface-dome sensilla and secretary pores occurred in alternate rows while the pitted-dome sensilla were scattered all over the surface of the ovipositor stylets, especially on the sides of the stylets. Microsc. Res. Tech. 2012. © 2012 Wiley Periodicals, Inc.

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.

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.

The speed and accuracy of Förster resonance energy transfer (FRET) measurements can be improved by rapidly alternating excitation wavelengths between the donor and acceptor fluorophore. We demonstrate FRET efficiency measurements based on a fiber laser and photonic crystal fiber as the source for two-photon excitation (TPE). This system offers the potential for rapid wavelength switching with the benefits of axial optical sectioning and improved penetration depth provided by TPE. Correction of FRET signals for cross excitation and cross emission was achieved by switching the excitation wavelength with an electrically controlled modulator. Measurement speed was primarily limited by integration times required to measure fluorescence. Using this system, we measured the FRET efficiency of calmodulin labeled with Alexa Fluor 488 and Texas Red dyes. In addition, we measured two-photon induced FRET in an E⁰GFP-mCherry protein construct. Results from one-photon and two-photon excitation are compared to validate the rapid wavelength switched two-photon measurements. Microsc. Res. Tech., 2011. © 2011 Wiley Periodicals, Inc.

We present a modern, light portable device specifically designed for environmental samples for cryogenic transmission-electron microscopy (cryo-TEM) by on-site cryo-plunging. The power of cryo-TEM comes from preparation of artifact-free samples. However, in many studies, the samples must be collected at remote field locations, and the time involved in transporting samples back to the laboratory for cryogenic preservation can lead to severe degradation artifacts. Thus, going back to the basics, we developed a simple mechanical device that is light and easy to transport on foot yet effective. With the system design presented here we are able to obtain cryo-samples of microbes and microbial communities not possible to culture, in their near-intact environmental conditions as well as in routine laboratory work, and in real time. This methodology thus enables us to bring the power of cryo-TEM to microbial ecology. Microsc. Res. Tech., 2011. © 2011 Wiley Periodicals, Inc.

Colon layers, especially the submucosa, as well as the secretion of goblet cells are extremely important for the functioning and transit of substances in this organ. However, the damages arising from type I diabetes and the effects of physical training, which plays crucial role in the treatment of this disease, are not yet known in these regions. To analyze the changes in colon submucosa and goblet cells of diabetic rats, as well as the effects of physical training, Wistar rats were divided into four groups: sedentary control, trained control, 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. Goblet and submucosa cells did not show alterations in shape, size, protein and carbohydrate content, in all treatment groups. Decreased amount of collagen fibers, however, was observed in the submucosa and lamina propria of SD rats, but this alteration was recovered in TDs. The ultrastructural analysis, in turn, revealed greater quantity of Golgi apparatus cisterns in SDs, distinctly than TDs, which showed improvement in this diabetic condition. Thus, physical training was responsible for the recovery of some important diabetic alterations, possibly improving the motility of substances in the large intestine. Nevertheless, it cannot be considered alone in the treatment of this disease, requiring the combined practice of other methods. Microsc. Res. Tech., 2011. © 2011 Wiley Periodicals, Inc.

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.

The purpose of this study was to compare the inorganic content and morphology of one nanofilled and one nanohybrid composite with one universal microhybrid composite. The Vickers hardness, degree of conversion and scanning electron microscope of the materials light-cured using LED unit were also investigated. One nanofilled (Filtek™ Supreme XT), one nanohybrid (TPH®₃) and one universal microhybrid (Filtek™ Z-250) composite resins at color A₂ were used in this study. The samples were made in a metallic mould (4 mm in diameter and 2 mm in thickness). Their filler weight content was measured by thermogravimetric analysis (TG). The morphology of the filler particles was determined using scanning electron microscope equipped with a field emission gun (SEM-FEG). Vickers hardness and degree of conversion using FT-IR spectroscopy were measured. Filtek™ Z-250 (microhybrid) composite resin shows higher degree of conversion and hardness than those of Filtek™ Supreme XT (nanofilled) and TPH®₃ (nanohybrid) composites, respectively. The TPH₃® (nanohybrid) composite exhibits by far the lowest mechanical property. Nanofilled composite resins show mechanical properties at least as good as those of universal hybrids and could thus be used for the same clinical indications as well as for anterior restorations due to their high aesthetic properties. Microsc. Res. Tech., 2011. © 2011 Wiley Periodicals, Inc

Brazilian endemic species Diplodon expansus (Küster, 1856) is found in freshwater bodies in the country's southeast, in large anthropogenic influence regions especially with an extensive agriculture emphasis. One of the main pesticides used in the species occurrence region is the atrazine herbicide, which has a great contamination potential in the aquatic environment. Therefore, several studies into its toxicity in aquatic systems have been developed. However, the tested concentrations are usually very high and rarely found in the environment and the short-term exposure responses in other aquatic organisms such as native bivalves are still scarce. Thus, this study sought to consider the potential effects of environmentally realistic concentrations of atrazine herbicide on the surface morphology of gill filaments of the bivalve D. expansus under laboratory-controlled conditions after short-term exposure. None of the animals died before the end of the experiment. The main alterations were observed on the frontal surface of filaments, which include mucus accumulation, cilia loss, and disruption. Mucus increased secretion and accumulation in the frontal filaments region preceded as a protective mechanism. Cilia loss and disruption on the frontal surface of the gill filament indicated that ciliated frontal cells were more sensitive to atrazine exposure and these alterations may cause gills functional damages, compromising the uptake of food particles and respiration. Therefore, higher sublethal concentrations of atrazine may compromise the survival and consequently the population of D. expansus in agriculture areas after a longer period of continuous exposure. Microsc. Res. Tech. 2011. © 2011 Wiley Periodicals, Inc.

For the study of cellular ultrastructure, the sample needs to be stabilized by fixation, with the ultimate aim to preserve the native tissue organization and to protect the tissue against later stages of preparation. Chemical and freezing fixation are most used, and chemical fixation employs agents that permeate tissues and cells by diffusion and covalently bind with their major biochemical constituents to fix them. Most widely used chemical fixatives are aldehydes, e.g., formaldehyde and glutaraldehyde, which are noncoagulating, crosslinking agents. Cryofixation methods for ultrastructural studies are also popular, and high-pressure freezing immobilizes all cell constituents and arrests biological activity by removing the thermal energy from the system. In the current research, we used platelet-rich plasma (PRP) to study expansive fibrin fibers and platelet ultrastructure to compare the two fixation techniques. We also used thrombin and calcium chloride as a clotting agent to determine the technique most suitable for the formation of extensive fibrin networks. Chemically fixated fibrin fibers were more compact and condensed and also showed a banding pattern on longitudinal sections. High-pressure frozen samples were more dispersed while platelets fixated showed better preserved cellular membranes and organelle structure. PRP coagulated by addition of CaCl₂ showed blood platelets that are noticeably more activated compared with PRP; however, with thrombin, a sharp ultrastructure was seen. We conclude that PRP mixed with thrombin, and freeze substituted, is the most suitable method for the study of extensive fibrin fibers as well as platelets. Microsc. Res. Tech. 2011. © 2011 Wiley Periodicals, Inc.

OBJECTIVE: Signal pattern enumeration of Urovysion Fluorescence in Situ Hybridization test is tedious and requires great experience. Our aim was to eliminate human interaction by automating the process, using an adoptable, automated image acquisition, and analysis system. METHODS: For extensive analytical analysis control, cell populations were used, while preliminary clinical study was performed on 21 patients with clinical suspicion for bladder cancer. All investigations were carried out using an automated user-trainable workstation (Metafer4-Metacyte). RESULTS: The system identified nuclei with a specificity and sensitivity of 92.7 and 96.6%, respectively, while signal detection accuracy was 81.1% on average. Both analytical and diagnostic accuracy of automated analysis was comparable to manual approach (94.8 and 71% vs. 97.9 and 76%, respectively), but classification accuracy increased with degree of polysomy, thus diagnostic sensitivity in low grade, low stage cases was poor. CONCLUSION: It is possible to automate signal enumeration of Urovysion using a user-trainable system, and achieve efficiency comparable to manual analysis. Previously introduced automated immunophenotypic targeting should further increase diagnostic sensitivity, while resulting in a comprehensively automated method. However, the problem of reduced detection accuracy in cases featured with low polysomy is likely to remain a great challenge of automated signal enumeration. Microsc. Res. Tech. 2011. © 2011 Wiley Periodicals, Inc.

This study presents the morpho-histological and histochemical characterization of the testes, integument, Malpighian tubules, and midgut of engorged Rhipicephalus sanguineus nymphs on the detachment day, showing the morphological and physiological characteristics to this phase in the life cycle of these individuals. The testis is constituted by germinative cells (only spermatogonia) with large, round-shaped and strongly stained nuclei which are organized into cysts by a thin layer of somatic cells. The integument consists of a cuticle subdivided into epicuticle (lipoprotein) and procuticle (glycoproteic), and a layer of epithelial cells which present glycolipoprotein elements. The procuticle presents two distinct regions: the exocuticle (next to the epicuticle) and the endocuticle (next to the epithelial layer). The Malpighian tubules present a simple epithelium with small flat and/or cubic cells, which form its wall and delimitates a lumen full of lipoprotein material. The midgut consists of an epithelial wall formed by two types of digestive cells, spent cells and empty digest cells, and by generative cells supported by a basal lamina and a thin layer of muscular tissue. This study described the main organs of engorged nymphs of R. sanguineus, to generate information that can help researchers to better understand the biology of these ectoparasites; which is fundamental for the development of compounds that are less aggressive to the environment. In addition, if the immature stages of the ticks are controlled, the number of adult ticks able to cause damages to the animals—and to the man as well—is also under control. Microsc. Res. Tech. 2011. © 2011 Wiley Periodicals, Inc.

Purpose: The aim of this study was to evaluate the nanoleakage patterns between dental enamel and reinforced leucite ceramic, bonded with resin luting systems and a flowable composite resin. Materials and Methods: Twelve crowns of bovine incisors were randomly divided into four groups (n = 3) according to the luting procedure: Excite/Variolink II, Clearfil SE Bond/Panavia F, Scotchbond Multi-Purpose Plus/RelyX ARC, and Single Bond 2/Filtek Z350 Flow. To evaluate the nanoleakage patterns, IPS Empress Esthetic disks (5 mm Ø and 1.2-mm thick) were bonded to enamel, and, after 24 h, the specimens were immersed in a 50% (w/v) solution of silver nitrate (24 h), fixed, dehydrated, and processed scanning electron microscopy (SEM). Results: None nanoleakage on interface of the groups that Single Bond 2 followed by the flowable composite were used. The highest percentage of nanoleakage was shown by the Excite/Variolink II protocol. Also, in all conditions tested, none silver nitrate uptake was observed between the leucite-reinforced ceramic and the resin luting cement. Conclusions: The use of a two-step etch-and-rinse adhesive with flowable composite was able to promote an adequate seal of the bond interface at the enamel. Moreover, the conventional dual-cured resin cements associated with simplified and dual-cured adhesives tested are also indicated to bond thin ceramics to enamel, since all presented low silver nitrate uptake. Microsc. Res. Tech., 2011. © 2011 Wiley Periodicals, Inc.

This study aimed to evaluate, ex vivo, the nanoleakage in dentinal tubules, the linear infiltration of silver nitrate in the dentin wall/root-end filling material interface, and the presence of gaps in this interface in root-end cavities filled with 4 filling materials. Forty-eight disto-buccal root canals of maxillary molars were instrumented and filled. Retrograde cavities were prepared with ultrasonic points (apical 2 mm). The samples were divided into 2 control groups (n = 4) and 4 experimental groups (n = 10): Group I – white mineral trioxide aggregate (MTA); Group II – Super EBA; Group III – Portland cement; and Group IV – Sealer 26. After 1 week, the specimens were subjected to silver nitrate and prepared for SEM (backscattered electrons). In the apical-apical segment, an area with significantly higher leakage was observed for Super EBA, followed by Portland cement, MTA, and Sealer 26 (P = 0.0054). In the medium and cervical segments, all materials showed the same leakage behavior (P = 0.1815 and P = 0.1723, respectively). The linear infiltration at the dentin wall/root-end filling material interface was higher with Super EBA than the other groups. No differences in the percentage of gaps along the 3 mm of dentin wall/root-end filling material interface between the 4 materials were evident (P > 0.05). Nanoleakage occurred mainly in the apical segment of the samples, and Super EBA showed the highest values. The area and linear leakage were lower in the middle and coronal segments, regardless of the root-end filling material. No material perfectly sealed the root-end cavities, which allowed for the leakage occurrence. Microsc. Res. Tech., 2011. © 2011 Wiley Periodicals, Inc.

Nanofillers are added to dental adhesives to improve mechanical properties of the hybrid layer. Ethanol or water added to the demineralized dentin to improve adhesive infiltration may produce filler aggregation. OBJECTIVE: To assess the effect of 5 vol% water or ethanol addition on nanoparticles distribution in dental adhesives. METHODS: Six available commercial adhesives systems were selected: Clearfil SE Bond (CSE), Clearfil Protect Bond (CPB), FL-Bond (FLB), Clearfil S3 (CS3), Bond Force (BF), One Up Bond F plus (OUB), and an experimental adhesive system without filler (EXP). Polymer films were obtained by adding 0 (control) or 5 vol% water or ethanol into the bonding resins. Preparations were light-cured (40 s). Three specimens were analyzed for each mixture. Three phases and 3D images were taken from each specimen by means of an atomic force microscope in taping mode (TM/AFM). Cluster sizes and surface nanoroughness were assessed. RESULTS: Control specimens from CSE, FLB, OUB, and BF presented clusters. The addition of solvents lead to particles aggregation in tested bonding resins. Ethanol addition produced more aggregates, particularly in adhesives containing fluoraluminosilicate as fillers. CONCLUSIONS: Nanofillers aggregation occurred in all adhesive systems in presence of additional solvents. In general, aggregate sizes were higher after the addition of ethanol. Formed clusters size values are always above the dimensions of the spaces existing between the demineralized collagen fibers. Microsc. Res. Tech. , 2011. © 2011 Wiley Periodicals, Inc.

Scanning electron microscopy (SEM) can be used to analyze the presence of debris and smear layer on the internal walls of root canal. This study evaluated the debris and smear removal in flattened root canals using SEM after use of different irrigant agitation protocols. Fifty mandibular incisors were distributed into five groups (n = 10) according to the irrigant agitation protocol used during chemomechanical preparation: conventional syringe irrigation with NaviTip needle (no activation), active scrubbing of irrigant with brush-covered NaviTip FX needle, manual dynamic irrigation, continuous passive ultrasonic irrigation, and apical negative pressure irrigation (EndoVac system). Canals were irrigated with 5 mL of 2.5% NaOCl at each change of instrument and received a final flush with 17% EDTA for 1 min. After instrumentation, the roots were split longitudinally and SEM micrographs at ×100 and ×1,000 were taken to evaluate the amount of debris and smear layer, respectively, in each third. Data were analyzed by Kruskal–Wallis and Dunn's post-hoc tests (α = 5%). Manual dynamic activation left significantly (p < 0.05) more debris inside the canals than the other protocols, while ultrasonic irrigation and EndoVac were the most effective (p < 0.05) for debris removal. Regarding the removal of smear layer, there was no statistically significant difference (p > 0.05) either among the irrigant agitation protocols or between the protocol–canal third interactions. Although none of the irrigant agitation protocols completely removed debris and smear layer from flattened root canals, the machine-assisted agitation systems (ultrasound and EndoVac) removed more debris than the manual techniques. Microsc. Res. Tech., 2011. © 2011 Wiley Periodicals, Inc.

The umbilical cord blood (UCB) is an important source of hematopoietic stem cells with great deal of interest in regenerative medicine. The UCB cells have been extensively studied as an alternative to the bone marrow transplants. The challenge is to define specific methods to purify and characterize these cells in different animal species. This study is aimed at morphological characterization of progenitor cells derived from UCB highlighting relevant differences with peripheral blood of adult in dog and cats. Therefore, blood was collected from 18 dogs and 5 cats' umbilical cords from fetus in various developmental stages. The mononuclear cells were separated using the gradient of density Histopaque-1077. Characterization of CD34+ cells was performed by flow cytometric analysis and transmission electron microscopy. Granulocytes (ancestry of the basophiles, eosinophiles, and neutrophiles) and agranulocytes (represented by immature lymphocytes) were identified. We showed for the first time the ultrastructural features of cat UCB cells. Microsc. Res. Tech. , 2011. © 2011 Wiley Periodicals, Inc.

In this article, the reproductive system's morphology of three young animals of the species Saguinus midas, from the bauxite mine in Paragominas, is described. The specimens were fixed and preserved in a solution of 10% aqueous formaldehyde, followed by dissection, measurement of the genital organs (uterus, vagina, ovaries, and uterine tubes), and histological processing. The vulva is delimited by the labia, with a clitoris. It is lined by keratinized stratified squamous epithelium with sebaceous glands of holocrine secretion. The vagina is an elongated tube with an average length of 26 mm and diameter of 1 mm, presenting a non-keratinized squamous epithelium, disposed between the vestibule of the vagina and cervix, the latter being relatively short. The uterus is simple, has globular shape and is located in the caudal portion of the abdominal cavity, with an average length of 14 mm and average width of 7 mm. It is formed by vascular and serous layers of muscles, and undergoes a bifurcation to form two structures on the bottom of blind sac. The uterine tubes are long and convoluted with an average length of 35 mm (right) and 36 mm (left), consisting of loose connective tissue and muscle layer lined by simple ciliated columnar epithelium. The ovaries are large and ellipsoid with smooth surface. Histologically, one animal showed ovulation fosse. Microsc. Res. Tech., 2011. © 2011 Wiley Periodicals, Inc.

This article explores the achievable transmission electron microscopy specimen thickness and quality by using three different preparation methods in the case of a high-strength nanocrystalline Cu–Nb powder alloy. Low specimen thickness is essential for spatially resolved analyses of the grains in nanocrystalline materials. We have found that single-sided as well as double-sided low-angle Ar ion milling of the Cu–Nb powders embedded into epoxy resin produced wedge-shaped particles of very low thickness (<10 nm) near the edge. By means of a modified focused ion beam lift-out technique generating holes in the lamella interior large micrometer-sized electron-transparent regions were obtained. However, this lamella displayed a higher thickness at the rim of ≥30 nm. Limiting factors for the observed thicknesses are discussed including ion damage depths, backscattering, and surface roughness, which depend on ion type, energy, current density, and specimen motion. Finally, sections cut by ultramicrotomy at low stroke rate and low set thickness offered vast, several tens of square micrometers uniformly thin regions of ∼10-nm minimum thickness. As major drawbacks, we have detected a thin coating on the sections consisting of epoxy deployed as the embedding material and considerable nanoscale thickness variations. Microsc. Res. Tech., 2011. © 2011 Wiley Periodicals, Inc.

Purpose: This work aims to study the erosion on restorative materials and on surrounding dentin. Fifty root dentin samples were obtained from bovine incisors. Methods: Twenty samples were not restored and thirty received cavity preparations. Samples were assigned to five groups: G1, G2: sound dentin (D); G3: composite resin (CR); G4: resin-modified glass-ionomer cement (RMGIC); G5: glass-ionomer cement (GIC). The samples of groups 2–5 were submitted to six cycles (demineralization–remineralization). Samples were analyzed by micro energy-dispersive X-ray fluorescence spectrometry (μ-EDXRF) and by scanning electron microscopy (SEM). Results: Mineral loss was greater in G2 samples than in RMGI > CR > GIC > D (control). SEM images showed pronounced dentin demineralization in groups 2 and 4. The acid erosion has a significant effect on mineral loss (Ca and P) of root dentin without restoration. Conclusions: Composite resin had the best chemical resistance to erosion among all the materials. Fluoride contained in GIC seemed to cause some protection, however, with material degradation. Chemical interaction of tooth-colored dental materials with root dentin could be assessed by μ-EDXRF. Microsc. Res. Tech., 2011. © 2011 Wiley Periodicals, Inc.

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., 2011. © 2011 Wiley Periodicals, Inc.

This article reports a novel color tuning method by local sputtering nanolayers on microstructured porous alumina (PA) templates with different pore depths. With the aid of scanning electron microscopy observation, physical models of the original and sputtered PA templates are set up, and the details of the color tuning method are further proposed. Two series of colors covering the whole visible range are first obtained by respectively sputtering Cr and Ag nanolayers on two groups of PA templates with pore-depths ranging from 230 to 490 nm. A vivid colorful pattern of “Butterfly wings” is then prepared by local sputtering such Cr and Ag nanolayers on the surface of a PA with 310 nm pore-depth. The scanning electron microscopy images of Cr and Ag sputtered PA surfaces show different microstructures, which is in agreement with different color exhibiting. This method is expected to have a potential of being widely applied in the fields of micro-optics, microstructures, advanced materials, and micro/nanotechnology. Microsc. Res. Tech., 2011. © 2011 Wiley Periodicals, Inc.

Using live-cell confocal microscopy and particle tracking technology, the simultaneous transport of intracellular vesicles of the endo-lysosomal pathway and nonviral polyethylenimine (PEI)/DNA nanocomplexes was investigated. Due to potential problems associated with the use of acid-sensitive probes in combination with a gene vector that is hypothesized to buffer the pH of intracellular vesicles, the biological location of PEI/DNA gene vectors was revealed by probing their trafficking in cells expressing fluorescent versions of either early endosome antigen 1, a protein that localizes to early endosomes, or Niemann Pick C1, a protein that localizes to late endosomes and lysosomes. Studies directly show that PEI/DNA nanoparticles are actively transported within both early and late endosomes, and display similar overall transport rates in each. Additionally, gene vector transfer between endosomes is observed. Over time post-transfection, gene vectors accumulate in late endosomes/lysosomes; however, real-time escape of vectors from membrane-bound vesicles is not observed. Microsc. Res. Tech., 2011. © 2011 Wiley Periodicals, Inc.

The highly complex techniques of electron microscopy made it bound to the sensitive and critical micrograph analysis. The accurately interpreted micrographs are of paramount values in basic investigations. Interpretation skills and quality of the micrographs are the two fundamental keys in accomplishment of these goals but there are many mistakes and errors that can happen during the sample preparation, sectioning, EM operation, and photo publishing. The mentioned mistakes and errors effect directly in the final result which is a micrograph and can lead the analyzer who can be a pathologist to an interpretation followed by serious danger for patient. Artifacts caused by any given stimuli expected to be bothersome for investigators. Even best qualified equipments can be regarded as source of artifact generation. In this article, seven serious errors in electron micrographs which usually occur in transmission electron microscopy are addressed. Microsc. Res. Tech., 2011. © 2011 Wiley Periodicals, Inc.

Full-field hard X-ray tomography could be used to obtain three-dimensional (3D) nanoscale structures of biological samples. The image of the fission yeast, Schizosaccharomyces pombe, was clearly visualized based on Zernike phase contrast imaging technique and heavy metal staining method at a spatial resolution better than 50 nm at the energy of 8 keV. The distributions and shapes of the organelles during the cell cycle were clearly visualized and two types of organelle were distinguished. The results for cells during various phases were compared and the ratios of organelle volume to cell volume can be analyzed quantitatively. It showed that the ratios remained constant between growth and division phase and increased strongly in stationary phase, following the shape and size of two types of organelles changes. Our results demonstrated that hard X-ray microscopy was a complementary method for imaging and revealing structural information for biological samples. Microsc. Res. Tech., 2011. © 2011 Wiley Periodicals, Inc.

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., 2011. © 2011 Wiley Periodicals, Inc.

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., 2011. © 2011 Wiley Periodicals, Inc.

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., 2011. © 2011 Wiley Periodicals, Inc.

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 μm² 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., 2011. © 2011 Wiley Periodicals, Inc.

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., 2011. © 2011 Wiley Periodicals, Inc.

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., 2011. © 2011 Wiley Periodicals, Inc.

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., 2011. © 2011 Wiley Periodicals, Inc.

Root canal preparation is the most important phase of endodontic procedure and it consists of adequate canal space cleaning and shaping. In recent years, rotary instruments and techniques have gained importance because of the great efficacy, speed, and safety of the preparation procedure. AIM: The aim of this research was to investigate measurement of maximal and minimal residual dentine thickness (RDT) and canal diameter after the canal preparation with different NiTi rotary files. METHODS: The research has been conducted on extracted human teeth in vitro conditions. The teeth have been divided in seven groups (20 teeth per group) depending on the kind of instruments used for root canal preparation: ProTaper, GT, ProFile, K-3, FlexMaster, hand ProTaper, and hand GT. The canals have been shaped in a crown-down manner and irrigated with 5.25% sodium hypochlorite. The roots of the teeth have been cut 1 and 3 mm from the apex. Apical preparation quality has been assessed under the polarized light microscope. RESULTS: The maximal residual dentine thickness at distance 1 mm from apex, ranged from 1.16 to 1.45 mm, and at distance 3 mm from apex, from 1.44 to 1.84 mm. The minimal dentine thickness at distance 1 mm from apex ranged from 0.52 to 0.73 mm, and at distance 3 mm from apex, from 0.66 to 0.83 mm. The canal diameters after preparation at distance 1 mm from apex ranged from 0.42 to 0.49 mm, and at distance 3 mm from apex, from 0.53 to 0.63 mm. CONCLUSIONS: There was no significant difference neither in maximal and minimal RDT, nor in canal diameters shaped with different NiTi instruments tested. All tested NiTi files have accomplished good quality preparation of apical root canal parts. Microsc. Res. Tech., 2011. © 2011 Wiley Periodicals, Inc.

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., 2011. © 2011 Wiley Periodicals, Inc.

The S100 protein in nervous tissue appears to play important roles in regulating neuronal differentiation, glial proliferation, plasticity, development, axonal growth, and in neurogenetic processes. In fish, the adult neurogenic activity is much higher than in mammals. In this study, the localization of S100 protein was investigated in the brain of annual teleost fish, Nothobranchius furzeri, which is an emerging model organism for aging research. By immunohistochemical techniques, S100 immunoreactivity (IR) was detected in glial cells, small neurons, and fibers throughout all regions of central nervous system (CNS) with different pattern of distribution. In the telencephalon, S100 IR was seen in the olphactory bulbs and in different areas of the telencephalic hemispheres. In the diencephalon, S100 positivity was observed in the habenular nuclei of the epithalamus, in the cortical thalamic nucleus, in the dorsal, ventral and caudal portions, the latter with the posterior recessus nucleus, and in the diffuse inferior lobe of the hypothalamus, along the diencephalic ventricle and in the dorsal optic tract. In the mesencephalon, S100 IR was observed in the longitudinal tori, in the optic tectum, and along the mesencephalic ventricle. In the rhombencephalon, S100 IR was shown in valvula and body of the cerebellum, and in some nuclei of the medulla oblongata. The results suggest that S100 may play a key role in the maintenance of the CNS and in neurogenesis processes in the adulthood. © 2011 Wiley-Liss, Inc.

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., 2011. © 2011 Wiley Periodicals, Inc.

Heat shock proteins 70 (Hsp70) are the most extensively studied heat shock proteins for the cellular abundance and cytoprotective effects. Hsp70 induction and subsequent quantification has been used as a sensitive system for aquatic toxicity risk assessment. In this study, the confocal microscopy was used to localize Hsp70 in Carassius auratus (C. auratus) with immunohistochemical technology. There are different zooms to select to analyze the object at the same field of vision with one objective lens with confocal microscopy. It need not change objective lens to observe the details of tissues. In this study, the tissue slices of C. auratus were observed with the 20-fold objective lens. Furthermore, the zooms of 1, 2, and 3 were used to acquire the distribution of Hsp70 in the tissue slices of C. auratus, and the clearer images of Hsp70 in the tissues were acquired. The results indicated that Hsp70 were present in the gill, liver, and cardiac muscle of C. auratus, and a method was established to detect Hsp70 in the tissues of C. auratus with confocal microscopy. Microsc. Res. Tech., 2012. © 2012 Wiley Periodicals, Inc.

The frequent use of some rare earths in the medical and industrial domains make us worry about their intracellular behavior into the body. Reason for which we have investigated the subcellular localization of one of these elements, the samarium, in the mammary gland of lactating female wistar rats using two very sensitive methods of observation and microanalysis, the transmission electron microscopy and the secondary ion mass spectrometry. The ultrastructural study showed the presence of electron dense deposits in the lactating mammary glandular epithelial cell lysosomes of the samarium-treated rats, but no loaded lysosomes were observed in those of control rats. The microanalytical study allowed both the identification of the chemical species present in those deposits as samarium isotopes (¹⁵²Sm⁺) and the cartography of its distribution. Our results confirm the previous ones showing that lysosomes of the glandular epithelial cells are the site of the intracellular concentration of foreign elements such as gallium. The intralysosomal deposits observed in the mammary glandular cells of the samarium-treated rats are similar in their form and density to those observed with the same element in other varieties of cells, such as liver, bone marrow, and spleen cells. Our ultrastructural and microanalytical results and those obtained in previous studies allow deducing that the intralysosomal deposits are very probably composed of an insoluble samarium phosphate salt. © 2011 Wiley-Liss, Inc.

This article presents a new approach for extracting high level semantic concepts from digital histopathological images. This strategy provides not only annotation of several biological concepts, but also a coarse location of these concepts. The proposed approach is composed of five main steps: (1) a stain decomposition stage, which separates the contribution of hematoxylin and eosin dyes, (2) a color standardization that corrects color image differences, (3) a part-based representation, which describes the image in terms of the conditional probability of relevant local patches, selected by their stain contributions, (4) a discriminative classification model, which bridges out the found patterns and the biological concepts, (5) a block-based annotation strategy that identifies the multiple biological concepts within an image. A set of 655 skin images, containing 10 biological concepts of skin tissues were used for assessing the proposed approach, obtaining a sensitivity of 84% and a specificity of 67% when annotating images with multiple concepts. Microsc. Res. Tech., 2011. © 2011 Wiley-Liss, Inc.

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., 2011. © 2011 Wiley-Liss, Inc.

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., 2011. © 2011 Wiley-Liss, Inc.

Functional restoration for orthopedic, systemic, or dental purposes uses a diverse range of biomaterials. Monitoring for any subsequent failures associated with pathology is vital. To this end, an alternative methodology beyond that of cutting sections by conventional microtome knife-based histomethodologies was pioneered. The method was to saw a block of tissue followed by grinding the unsupported slice to the required thickness. The sawing and grinding of unsupported bones or teeth exposed not only the specimen fragility, but also the delicacy of the associated soft tissues. Although incomplete, the significance of the technique was recognized and improvements were incorporated which persist to the present day addressing the histopathology of challenging specimens. One major improvement for preserving cellular structure as well as reducing fracture incidence was achieved by impregnating tissue samples with cross-linked resins prior to the cutting and grinding processes. Methyl and glycol methacrylate resins superseded all other choices because of the advantages of photo-initiated curing. Further pioneering work led to the formulation of increasingly appropriate resin systems and the subsequent processing protocols evolved, as clinical needs for histology-specific techniques became apparent. Over the decades, several key pioneers entered this scientific arena to develop the now established resin formulation, processing protocols, and allied applications. This has led to the commercialization of the various resin systems. The aim here is to discuss the commercially available glycol and methyl methacrylate resin systems and their practical application to a variety of important techniques used in the histological assessment of complex pathology specimens. Microsc. Res. Tech., 2011. © 2011 Wiley-Liss, Inc.

Starch is the most important carbohydrate in the human diet and contained in many staple foods such as rice, wheat, corn, potatoes and cassava, etc. Currently, microscopic imaging technology is one of the most effective ways to investigate and analyze the structure of starches. Segmentation of starch granules is a necessary step for starch granule structure analysis such as starch granules detection, shape recognition, and size computation. This article investigates a new method based on GVF (gradient vector flow) snake deformable model for starch granules segmentation. The proposed method focuses on full automatic segmentation on granules, especially on separation of adjacent and contacted starch granules, which occur widely in microscopic images. A novel energy function based on position and intensity is introduced into the directional gradient computation, thus the directional gradient is used to obtain the directional GVF snake, which drives the deforming contours to the real contours of multiple granules. To demonstrate the good ability of the proposed method, we segment 30 starch granule images and compare it with the level set method, experimental results show that the new method can separate multiple starch granules successfully; especially it works much better on overlapping objects segmentation than the level set method. Microsc. Res. Tech. 2011. © 2011 Wiley-Liss, Inc.

Variable bright-darkfield contrast (VBDC) is a new technique in light microscopy which promises significant improvements in imaging of transparent colorless specimens especially when characterized by a high regional thickness and a complex three-dimensional architecture. By a particular light pathway, two brightfield- and darkfield-like partial images are simultaneously superimposed so that the brightfield-like absorption image based on the principal zeroth order maximum interferes with the darkfield-like reflection image which is based on the secondary maxima. The background brightness and character of the resulting image can be continuously modulated from a brightfield-dominated to a darkfield-dominated appearance. When the weighting of the dark- and brightfield components is balanced, medium background brightness will result showing the specimen in a phase- or interference contrast-like manner. Specimens can either be illuminated axially/concentrically or obliquely/eccentrically. In oblique illumination, the angle of incidence and grade of eccentricity can be continuously changed. The condenser aperture diaphragm can be used for improvements of the image quality in the same manner as usual in standard brightfield illumination. By this means, the illumination can be optimally adjusted to the specific properties of the specimen. In VBDC, the image contrast is higher than in normal brightfield illumination, blooming and scattering are lower than in standard darkfield examinations, and any haloing is significantly reduced or absent. Although axial resolution and depth of field are higher than in concurrent standard techniques, the lateral resolution is not visibly reduced. Three dimensional structures, reliefs and fine textures can be perceived in superior clarity. Microsc. Res. Tech. 2011. © 2011 Wiley-Liss, Inc.

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. 2011. © 2011 Wiley-Liss, Inc.

The aim of this study is to show histological and immunofluorescence analysis of renal parenchyma of agoutis affected by gentamicin-induced renal disease after the infusion of bone marrow mononuclear cells (BMMC) stained with Hoechst®. Nine agouti's males were divided into three groups: Test group (TG): renal disease by gentamicin induced (n = 3), cell therapy group (CTG): renal disease by gentamicin induced and BMMC infusion (n = 3), and control group (CG): nonrenal disease and BMMC infusion (n = 3). TG and CTG were submitted to the protocol of renal disease induction using weekly application of gentamicin sulfate for 4 months. CG and CTG received a 1 × 108 BMMC stained with Hoechst and were euthanized for kidney examination 21 days after BMMC injection and samples were collected for histology and immunofluorescence analysis. Histological analysis demonstrated typical interstitial lesions in kidney similarly to human disease, as tubular necrosis, glomerular destruction, atrophy tubular, fibrotic areas, and collagen deposition. We conclude that histological analysis suggest a positive application of agouti's as a model for a gentamicin inducing of kidney disease, beyond the immunofluorescence analysis suggest a significant migration of BMMC to sites of renal injury in CTG. Microsc. Res. Tech., 2011. © 2011 Wiley-Liss, Inc.

An experimental approach was performed on 20 giant rabbits to establish the possibilities and limitations of μ-CT for routine processing of nondemineralized bone tissue. Hydroxyapatite (HA) or β-tricalciumphosphate (β-TCP) bead implants or a melange of both, microchambered and solid, were implanted into a standardized and precise defect in the patellar groove. The bone-healing phase was chosen for the histology considering 1 or 2 days, and 2, 3, and 6 weeks. Normal X-ray and μ-CT were applied on all specimens; five specimens in the 6-week stage were additionally processed according to the full range of conventional nondemineralized bone processing methods. μ-CT increased the possibilities of nondemineralized histology with respect to bone morphometry and a complete sequence of sections, thus providing a complete analysis of the bone response. μ-CT was limited in differentiating bone quality, cell analyses, and mineralization stages. The investigation based on normal X-rays is limited to defining integration and excluding the fibrous and bony encapsulation of loose implants. μ-CT allows a 3D evaluation of newly formed bone which is clearly marked against the ceramic implant. It does not allow, however, for the differentiation between woven and lamellar bone, the presentation of the canalicular lacunar system, or on the cell level, revealing canaliculi or details of the mineralization process which can be documented by high-resolution microradiography. Titer dynamics of bone formation remains the domain of polychromatic sequential labeling.

The complete sequence of μ-CT slices enhances the possibilities for routine histology, tremendously allowing to the focus on detail histology to topographically well-defined cuts, thus providing more precise conclusions which take into consideration the whole implant. Microsc. Res. Tech., 2011. © 2011 Wiley-Liss, Inc.

A better understanding of the remodeling process of postextraction sockets is essential in dental treatment planning. The aim of this study was to evaluate whether confocal laser scanning microscopy (CLSM) can be applied to imaging contour changes of postextraction sites, as well as to its quantification with image analysis of obtained three-dimensional images. This work describes a new application of the CLSM technique. The system used was the OLS3100-USS, LEXT model (Olympus®). CLSM was used for the surface analysis of the extraction site. The measurements taken with CLSM were: (1) mesio-distal distance, (2) alveolar ridge thickness, and (3) vestibular and lingual alveolar ridge height. Results of study cast scanning at baseline, 1 and 3 months after tooth extraction, with CLSM are well-detailed images of postextraction areas. The CLSM technique used in study casts is a valid method to measure the dimensional changes that happen in the edentulous area after tooth extraction. This technique allows the evaluation of changes in mesio-distal distance, thickness of the alveolar ridge and alveolar ridge height based on the measurements on the alveolar contours. Microsc. Res. Tech., 2011. © 2011 Wiley-Liss, Inc.

This study was designed to evaluate the effect of various buffers on the fluorescence signal intensity of two fluorochromes (IP and CFDA) when used to assess the membrane integrity of ram sperm. Second ejaculates (18) from nine adult males were collected using an artificial vagina and diluted in either MOPS, TRIS, TES, HEPES, citrate, or phosphate-based extenders. Semen samples were stored at 15°C and the membrane integrity was assessed within the first 24 h of storage. Mean fluorescence intensity (FI) of PI- and CDFA-labeled sperm heads and fluorescence background noise (FBN) were determined quantitatively using Image J software. Fluorescence contrast (FC) was expressed as the difference between FI and FBN. Significantly, higher FI and FC were recorded when TRIS diluent was used, rather than the other diluents, both in the propidium- and fluorescein-labeled cells. The citrate and phosphate-based extenders showed intermediate results of FC between those of TRIS and zwitterionic (MOPS, TES and HEPES) groups for the PI-labeled sperm. However, in the CFDA-labeled sperm, the lower values of FC were obtained in the citrate and phosphate groups due to increased levels of FBN. For the membrane-damaged sperm, fluorescent labeling was limited to the sperm heads when TRIS-buffer was used, whereas in the other groups, the sperm tail was also frequently observed. It was concluded that TRIS buffer solution markedly increases the fluorescence yield of IP/CFDA-labeled sperm cells in the ram and that this should be considered when evaluating their membrane integrity. Microsc. Res. Tech., 2011. © 2011 Wiley-Liss, Inc.

We present combined epi-coherent anti-Stokes Raman scattering (CARS) and multiphoton imaging with both chemical discrimination and subcellular resolution on human skin invivo. The combination of both image modalities enables label-free imaging of the autofluorescence of endogenous fluorophores by two-photon excited fluorescence, as well as imaging of the distribution of intercellular lipids, topically applied substances and water by CARS. As an example for medical imaging, we investigated healthy and psoriasis-affected human skin with both image modalities in vivo and found indications for different lipid distributions on the cellular level. Microsc. Res. Tech., 2011. © 2011 Wiley-Liss, Inc.

Effective medical image analysis is possible by the use of technique known as segmentation. Segmentation is a very challenging task because there is not any standard segmentation method is available for any medical application. In this article, we have proposed an automatic brain MR image segmentation method. Fast discrete curvelet transform and spatial fuzzy C-mean algorithm is used for noise removal and segmentation of brain MR image. Fuzzy entropy has been used for calculating adaptive and optimal threshold to separate out the image segments. Our proposed system is exclusively based on the information contained by the image itself. No extra information and no human intervention are required in our proposed system. We have tested our proposed system on different T1, T2 and PD brain MR images. Microsc. Res. Tech., 2011. © 2011 Wiley-Liss, Inc.

Myoepithelial cells (MECs) mainly surround acini and intercalated ducts in the human salivary glands. The contraction of MECs provides the expulsive force to promote salivation. We previously found functional transient receptor potential vanilloid subtype 1 (TRPV1) was expressed in rabbit and human submandibular glands and increased saliva secretion. However, it was unknown whether TRPV1 was expressed in MECs of submandibular glands. In this study, we observed the immunoflourescence of TRPV1 was not only located in serous acini and ducts but also surround the basal layer of the acinus and intercalated ducts of human submandibular glands. Double immunofluorescence staining revealed colocalization of TRPV1 with calponin, vimentin, and α-smooth muscle actin, which indicated the myoepithelial expression of TRPV1. Treating submandibular gland tissues with capsaicin, an agonist of TRPV1, substantially increased the phosphorylation of the 20-kDa regulatory light-chain subunit of myosin (MLC₂₀), a crucial molecule for contraction of smooth muscle cells, in MECs. Pretreatment with capsazepine, a specific TRPV1 inhibitor, blocked capsaicin-induced MLC₂₀ phosphorylation. These results suggest that TRPV1 is expressed in MECs of the human submandibular gland and mediates myoepithelial contraction via a mechanism involving MLC₂₀ phosphorylation. Microsc. Res. Tech., 2011. © 2011 Wiley-Liss, Inc.

Atom probe tomography (APT) is a mass spectrometry method with atomic-scale spatial resolution that can be used for the investigation of a wide range of materials. The main limiting factor with respect to the type of problems that can be addressed is the small volume investigated and the randomness of common sample preparation methods. With existing site-specific specimen preparation methods it is still challenging to rapidly and reproducibly produce large numbers of successful samples from specifically selected grain boundaries or interfaces for systematic studies. A new method utilizing both focused ion beam (FIB) and transmission electron microscopy (TEM) is presented that can be used to reproducibly produce damage-free atom probe samples with features of interest at any desired orientation with an accuracy of better than 50 nm from samples that require very little prior preparation. Microsc. Res. Tech. 2011. © 2011 Wiley-Liss, Inc.

Scaphoideus titanus Ball (Hemiptera: Cicadomorpha) is a leafhopper vector of a phytoplasma disease, the Flavescence dorée (FD), belonging to the vine yellows group. A scanning and transmission electron microscope study has been carried out to investigate the antennal sensory structures. The first two segments, the scape and the pedicel, are short, covered by cuticular scales and devoid of sensilla, with exception of some scattered hairs on the pedicel. The flagellum consists of a unique, elongated segment in which numerous subunits can be recognized, being separated by a sort of cuticular crown. The proximal five subunits bear most of the sensilla. We discovered the presence of single- and double-walled coeloconic sensilla, campaniform sensilla, basiconic sensilla, and trichoid sensilla. A scolopidium is located within the proximal region of the flagellum. Ultrastructural investigations suggest that the antennal sensilla could be involved in the perception of air-borne vibrations, temperature, and humidity variations. The most relevant feature is the extreme reduction of the olfactory sensilla, both in terms of number of sensory structures and sensory neurons per sensillum. The strong reduction in antennal olfactory sensilla to which this specie has undergone is discussed as possible consequence of the specificity toward the host plant. Microsc. Res. Tech. 2011. © 2011 Wiley-Liss, Inc.

Surface ultrastructures of gill filaments and secondary lamellae of Rita rita and Cirrhinus mrigala, inhabiting different ecological habitat, were investigated to unravel adaptive modifications. R. rita is a sluggish, bottom dwelling carnivorous catfish, which inhabits regions of river with accumulations of dirty water. It retains its viability for long time if taken out of water. C. mrigala is an active bottom dwelling Indian major carp, which lives in relatively clean water and dies shortly after taken out of water. In R. rita, gill septa between gill filaments are reduced. Microridges on epithelial cells covering gill filaments are often continuous and arranged concentrically. Secondary lamellae are extensive. The epithelium appears corrugated, show irregular elevations and shallow depressions, and microridges on epithelial cells appear fragmented. In C. mrigala, in contrast, the gill septa are extensive. Microridges on epithelial cells covering gill filaments are fragmented. Secondary lamellae are less extensive. The epithelium appears smooth and microridges on epithelial cells are relatively inconspicuous. These differences have been considered adaptive modification in relation to habit and ecological niches inhabited by two fish species. Presence of mucous goblet cells on gill filaments is discussed in relation to their functions including precipitation of the sediments and preventing clogging of gill filaments. Infrequent mucous goblet cells in the epithelium of secondary lamellae in two fish species are considered an adaptation, minimizing thickness of the epithelium to reduce barrier between blood and water for favoring gasses exchange with increased efficiency. Microsc. Res. Tech., 2011. © 2011 Wiley-Liss, Inc.

Mucociliary clearance is essential to maintain the defense function of the maxillary sinus; however, no literatures described the age changes in its lining epithelium. Therefore, the current work sought to describe the morphological postnatal age-related changes of maxillary sinus lining epithelium in rats using light, transmission, and scanning electron microscopes. Eighteen albino rats were divided into six groups according to their ages: 2-week-old, 1-month-old, 2-month-old, 3-month-old, adults, and senile rats. One-month-old-rats' group was the first to have recognizable maxillary sinus cavities that were lined by either single flat cellular layer or two distinct epithelial layers. These cells were devoid of microvilli and cilia, none of them showed evidence of differentiation into identifiable cell types. In 2- and 3-month-old rats, the mucosa of maxillary sinus started to be lined with pseudostratified epithelium with apparent increase in both microvilli and cilia. The first indication of goblet cell differentiation was observed in 3-month-old-rats. In the adult rats, the sinuses became completely lined by mature respiratory epithelium. However, in senile rats the epithelium exhibited polyps with clumped cilia and some areas of stratification and desquamation. Goblet cells were scanty and degenerating. The impaired mucociliary components (epithelium, cilia, and goblet cells' mucus) found in young and old ages of the current work might be correlated to human to explain predisposition of rhino-sinusitis in these age groups. Microsc. Res. Tech., 2011. © 2011 Wiley-Liss, Inc.

Morphological properties of atmospheric particles are directly related to their residence time and transport behaviors, and their deposition patterns in human respiratory systems. The projected properties of particles measured by transmission electron microscopy (TEM) were combined with the particle height measured by atomic force microscopy (AFM) to determine volume equivalent diameter of submicrometer particles. For nonvolatile (refractory) laboratory-generated spherical polystyrene latex and cubic NaCl particles, the measured volume equivalent diameters agreed well with the true values (within 4%). However, for nonrefractory (NH₄)₂SO₄ particles, the measured volume equivalent diameter was much smaller than the true value due to evaporation of volatile species at low vacuum pressure and high electron-beam intensity conditions in TEM, and deformation of particles in AFM. We observed that the volume equivalent diameter of 100 nm mobility-classified atmospheric particles was 35 ± 5 nm, suggesting that these particles contain nonrefractory species, whereas that of 20 nm mobility-classified atmospheric particles was found to be 19 ± 6 nm, suggesting that these particles were refractory and spherical. Microsc. Res. Tech., 2011. © 2011 Wiley-Liss, Inc.

AIMS: To evaluate measurements of collagen fibril spacing using different shaped regions of interest (ROI) on transmission electron micrograph (TEM) images of rabbit corneal stroma. METHODS: Following glutaraldehyde fixation and phosphotungstic acid staining, TEM images of collagen fibrils in cross section were projected at a final magnification close to 250,000 × to obtain overlays. Interfibril distances (IFDs; center-to-center spacing) were measured within different ROIs of the same nominal area (0.25 μm²) but different shape (with the length to width, L:W, ratio from 1:1 to 6:1). The IFD distribution was analyzed, and the 2D organization assessed using a radial distribution analysis. RESULTS: The fibrils had an average diameter of 35.3 ± 3.8 (SD) nm, packing density of 393 ± 4 fibrils / μm² and a fibril volume fraction of 0.39 ± 0.02. IFDs ranged from 29 to 1400 nm depending on the shape of the ROI, with average values ranging from 263 to 443 nm. By artificially selecting IFD data only to a radial distance of 250 nm, the average IFDs were just 145–157 nm. The radial distributions, to 250 nm, all showed a nearest neighbors first peak which shifted slightly from predominantly at 45–54 nm with more rectangular ROIs. The radial distribution profiles could be shown to be statistically different if the ROI L:W ratio was 2:1 or greater. CONCLUSION: Selection of an ROI for assessment of packing density and interfibril distances should be standardized for comparative assessments of TEMs of collagen fibrils. Microsc. Res. Tech., 2011. © 2011 Wiley-Liss, Inc.

Virtually every study that has used transmission electron microscopy (TEM) to estimate viral diversity has acknowledged that loss of phage tails during sample preparation may have biased the results. However, the magnitude of this potential bias has yet to be constrained. To characterize biases in virus morphological diversity due to tail loss, six phage strains representing the order Caudovirales were inoculated into sterile sediments and soils. Phage particles were then extracted using standard methods. Morphologies of extracted phage particles were compared to those of unmanipulated control samples to determine the extent of tail breakage incurred by extraction procedures. Podoviruses exhibited the smallest frequency of tail loss during extraction (1.2–14%), myoviruses were moderately susceptible to tail breakage (15–40%), and siphoviruses were highly susceptible (32–76%). Thus, TEM assessments of viral diversity in soils or sediments by distribution of tail morphologies may be biased toward podoviruses and virions lacking tails, while simultaneously underestimating the abundance of siphoviruses. However, since the majority of viral capsids observed under TEM were intact, estimates of viral diversity based on the distribution of capsid diameters may provide a more reliable basis for morphological comparisons within and across ecosystems. Microsc. Res. Tech., 2011. © 2011 Wiley-Liss, Inc.

Digestive tubules of Strombidae are composed by three cell types: digestive cells, vacuolated cells, and crypt cells. The last one is characterized by the presence of intracellular granules identified as spherocrystals. Such structures are known to occur in basophilic cells of gastropod digestive gland, where they are supposed to be involved in the regulation of some minerals and in detoxification. In this study, energy-dispersive X-ray analysis (EDX) and energy filtered transmission electron microscopy (EFTEM) were used to determine the elemental content of spherocrystals in two Strombidae, Strombus gigas and Strombus pugilis. In freshly collected individuals of both species, the following elements were detected: Ca, Fe, Mg, P, and Zn. Aluminum and Mn were also detected in S. gigas. Their presence in spherocrystals indicates that, in Strombidae, spherocrystals are involved in the regulation of minerals and essential trace metals. In order to answer the question “are spherocrystals involved in nonessential trace metals scavenging?,” artificial cadmium and lead exposure by both waterborne and dietary pathways was applied to S. pugilis. No evidence of cadmium (Cd(NO₃)₂) or lead (Pb(NO₃)₂) provided by food was found in spherocrystals. Cadmium provided in water (Cd(NO₃)₂ and CdCl₂) causes structural modifications of the digestive gland; however, this element was not trapped in spherocrystals. These results suggest that spherocrystals are not involved in detoxification of such nonessential trace metals. Microsc. Res. Tech., 2011. © 2011 Wiley-Liss, Inc.

Quantification of immunohistochemical results constitutes an important tool in the analysis of cells and tissue that is not readily replaced by other techniques. For reliable quantification, it is essential to consider factors such as tissue fixation and tissue sampling. We report a study on the model of the intestine of Isospora suis-infected piglets, in which we addressed (1) whether the quantity of detectable T cells in the intestinal mucosa is the same in formalin-, HOPE®-, and cryo-conserved material or whether the amounts of T cells at least correlate with one another; and (2) whether single jejunal segments differ in regard to the quantity of mucosal T cells and variability of lymphocyte infiltration. Quantification of T cells in histological sections of different parts of the jejunum of 15-22 day old piglets infected with I. suis was performed using an anti-CD3-antibody and stereological point counting. Area fractions of T-cell profiles per intestinal mucosa profile were higher in cryo-conserved samples than in HOPE®- and formalin-conserved material but no correlation between different fixations could be found. The proximal part of the jejunum contained fewer T cells compared with mid- and end-jejunum. Coefficients of variation did not differ between the intestinal segments. For quantification of T cells in the gut mucosa of piglets infected with I. suis, the cryo-conserved mid jejunum seems most suitable in cases when unbiased sampling of the complete intestine is not feasible. It is generally not possible to compare quantitative results of immunostaining in samples conserved by different methods. Microsc. Res. Tech., 2011. © 2011 Wiley-Liss, Inc.

This study presents an application of analytical electron microscopy in biology to investigate the chemical composition of the spherites and to elucidate the importance of these methods in the life sciences. The structure of the spherites in the midgut cells of first, second, and third instar larvae Euroleon nostras was investigated by a combination of transmission electron microscopy (TEM), energy dispersive X-ray spectroscopy (EDXS), electron energy-loss spectroscopy (EELS), and energy filtering TEM (EFTEM). The structure and chemical composition of the spherites changed during the metamorphosis. In first larvae, the spherites are composed of amorphous, flocculent material, containing C, N, and O. In second larvae and third ones, the spherites have concentric layers of alternating electron-dense and electron-lucent material. In second larvae, Si, P, Ca, and Fe are accumulated in the spherite organic matrix, composed of C, N, and O. In the spherites of third larvae, additionally Al was found. Therefore, the spherites are thought to store organic compounds in all three larval stages of E. nostras and additionally inorganic compounds in second and third ones. In first larvae, spherites are present in the midgut cells; in second and third larvae, they are present in the cells of the midgut and in its lumen. It could be suggested that the spherites might be involved in the regulation of the appropriate mineral composition of the internal environment and could serve as the accumulation site of nontoxic waste materials that cannot be metabolized. Microsc. Res. Tech., 2011. © 2011 Wiley-Liss, Inc.

There is no descriptive information about morphology of the oropharyngeal cavity including tongue, palate, and laryngeal region in Corvidae family. This study not only presents the first definitive anatomical description of the structures in the oropharyngeal cavity of magpie and raven but also reviews and compares the scattered information on the morphology of the other avian species available in the literature. In this study, the organs of four birds (two magpies and two ravens) were used. The tongue in magpie and raven was considerably elongated and terminated with an oval-shaped apex. Although the lingual apex in the magpie was divided by fissure, no such bifurcation existed on that of raven. Lingual apex was quite distinctive in both species, and multiple acicular processes covered the apex. These occurrences on the tongue of magpie were replaced by thread-like processes as approaching the lingual body. In raven, these processes were mostly foliated, becoming longer toward the back of the lingua, and denser compared with raven. Moreover, we observed conical papillary crest, which was pointed backward and located between the lingual body and the radix of the tongue and pharyngeal conical papillae around the glottis and palate clefts in both species. There was a transversal fold separating the choanal cleft from the infundibular cleft in raven. Anatomy of the oropharyngeal cavity in the raven and magpie, which are the member of the same family, Corvidae, showed little differences and overall their oral morphological features were excessively similar. Microsc. Res. Tech., 2011. © 2011 Wiley-Liss, Inc.

All microscopical images contain noise, increasing when (e.g., transmission electron microscope or light microscope) approaching the resolution limit. Many methods are available to reduce noise. One of the most commonly used is image averaging. We propose here to use the mode of pixel values. Simple Python programs process a given number of images, recorded consecutively from the same subject. The programs calculate the mode of the pixel values in a given position (a, b). The result is a new image containing in (a, b) the mode of the values. Therefore, the final pixel value corresponds to that read in at least two of the pixels in position (a, b). The application of the program on a set of images obtained by applying salt and pepper noise and GIMP hurl noise with 10–90% standard deviation showed that the mode performs better than averaging with three-eight images. The data suggest that the mode would be more efficient (in the sense of a lower number of recorded images to process to reduce noise below a given limit) for lower number of total noisy pixels and high standard deviation (as impulse noise and salt and pepper noise), while averaging would be more efficient when the number of varying pixels is high, and the standard deviation is low, as in many cases of Gaussian noise affected images. The two methods may be used serially. Microsc. Res. Tech., 2011. © 2011 Wiley-Liss, Inc.

Anoplistes halodendri halodendri (Pallas, 1776) and Anoplistes halodendri ephippium (Stevens and Dalman, 1817) are two subspecies of the longicorn beetle A. halodendri (Coleoptera, Cerambycidae). In the recent years, these subspecies have been spreading rapidly in Hippophae rhamnoides and Hedysarum scoparium shrubberies in the Chinese provinces of Shanxi and Kingie, causing mass mortality of these shrubberies species and consequently leading to great damage to local ecological environment construction as well as high economic, ecological, and societal losses. To control their hazards effectively, here, we study and compare the types, densities, and distribution of antennal sensilla of A. h. halodendri and A. h. ephippium using scanning electron microscopy. Eight sensilla types were observed on the antennae of these two subspecies, including placoid sensilla (Ps), chaetica sensilla (ch) (types I–III), sensilla basiconica (b) (types I and II), sensilla gemmiformium (G), and auricillica sensilla (au). Ps predominated on the antennae of both A. h. halodendri and A. h. ephippium, followed by b and ch, and G and au. There were differences between the subspecies in the distribution and density of the different sensilla types, with ch I being found exclusively on the antennae of A. h. ephippium and b I and au on the antennae of A. h. halodendri only. With the exception of Ps, the densities of ch II, ch III, b II, and G on the antennae of A. h. halodendri were much higher than on the antennae of A. h. ephippium. These results provide sufficient evidence to clarify the receptive mechanisms used by these two subspecies of longicorn beetles and their responses to volatile semiochemicals released by their host plants as well as discuss their differences with respect to host and habitat selection. Microsc. Res. Tech. 2011. © 2011 Wiley-Liss, Inc.

Thirty adult agoutis (Dasyprocta primnolopha) from the Nucleus of Study and Preservation of Wild Animals at the Federal University of Piauí were used. Blood scrubs of these animals were colored by the Leishman method and analyzed in light microscopy. The cells had been measured using programs that analyze images (Leica QWin – Image Processing and Analysis Software). Mature erythrocytes, basophil reticulocytes, lymphocytes, eosinophils, neutrophils, monocytes, and thrombocytes were identified. Agoutis' erythrocytes presented elliptical form, without nucleus with an average diameter of 5.64 micromeres ± 0.38. The lymphocytes are spherical cells with scarce cytoplasm, dense and with a very centralized rounded nucleus measuring an average diameter of 13.20 micromeres ± 0.35. The monocytes are slightly basophilic, with a spherical nucleus, central constriction, and an average diameter of 20.59 micromeres ± 0.32. The neutrophils are spherical, with a polymorphic lobulated nucleus, with an average diameter of 11.2 micromeres ± 0.20. The eosinophils are spherical with lobulated nucleus and with an average diameter of 14.25 micromeres ± 0.36. Only five basophils were observed, with abundance of cytoplasmic granules with 9.8 micrometers of diameter ± 0.30. Thrombocytopenic pleomorphism was frequent. There were similarities in the cellular constituents in peripheral blood of agoutis and of other rodents and humans. The cellular types from the peripheral blood, the morphology, and morphometry of the blood's cells did not vary according to sex. © Microsc. Res. Tech. 00:000?000, 2011. © 2011 Wiley-Liss, Inc.

Callithrix penicillata belongs to the family Callitrichidae, Callithrix genus. They are basically insectivorous, but they consume fruits. The mucosa of the tongue is composed of some papillary types, revealing different levels of expertise. The present study attempted to describe the morphological and ultrastructural aspects of the dorsal surface of the C. penicillata, describing the characteristics and distribution of papillae found. Five tongues of C. penicillata (two females and three males), obtained from breeding colonies of CENP-Ananindeua-PA, died from natural causes. The material was fixed partly in a buffer solution paraformaldehyde 10% and partly in modified Karnovsky solution, divided into apex, body, and root, and then the fragments were used in light microscopy and scanning electron microscopy. The average length of the tongue of the females was 22 mm and for males 20.5 mm. Three types of papillae were described: filiform (along all tissue extension with 154 μm of diameter), fungiform (along all tissue extension with 275 μm of diameter), and vallate (just three units in caudal (dorsal) portion with 672 μm of diameter). Data analysis indicates that the distribution and ultrastructural morphology of the C. penicillata lingual papillae are some similar to other primates. Microsc. Res. Tech. 2011. © 2011 Wiley-Liss, Inc.

Morphological observation and analysis of cerebral microvascular network is an essential way to study cerebral function. Automated labeling of cerebral microvascular in microscopy images is one of the key steps for quantitative analysis of microvascular network in the specimens of brain mantle. It is presented in this work that an automated image processing approach based on curvilinear structure detector is applied to label and analyze the microvascular in the image. Steerable filter is also introduced to address the detecting confusion in branching regions. And then the vascular morphology analysis, such as average microvascular density, is also performed after image processing. Validation has demonstrated that the results from proposed approach are satisfied. The proposed method is finally applied in the study of cerebral microvascular dysfunction induced by γ-ray irradiation. Microsc. Res. Tech., 2011. © 2011 Wiley-Liss, Inc.

We present here the phasor approach to biosensor Förster resonance energy transfer (FRET) detection by fluorescence lifetime imaging microscopy (FLIM) and show that this method of data representation is robust towards biosensor design as well as the fluorescence artifacts inherent to the cellular environment. We demonstrate this property on a series of dual and single chain biosensors, which report the localization of Rac1 and RhoA activity, whilst performing concomitant ratiometric FRET analysis on the acquired FLIM data by the generalized polarization (GP) approach. We then evaluate and compare the ability of these two methods to quantitatively image biosensor FRET signal as a function of time and space. We find that with lifetime analysis in the phasor plot each molecular species is transformed into a two-dimensional coordinate system where independent mixtures of fluorophores can be distinguished from changes in lifetime due to FRET. This enables the fractional contribution of the free and bound state of a dual chain biosensor or the low and high FRET species of a single chain biosensor to be quantified in each pixel of an image. The physical properties intrinsic to each biosensor design are also accurately characterized by the phasor analysis; thus, this method could be used to inform biosensor optimization at the developmental stage. We believe that as biosensors become more sophisticated and are multiplexed with other fluorescent molecular tools, biosensor FRET detection by the phasor approach to FLIM will not only become imperative to their use but also their advancement. Microsc. Res. Tech., 2011. © 2011 Wiley-Liss, Inc.

The zebrafish pineal gland plays a fundamental role in the regulation of the circadian rhythm through the melatonin secretion. The pinealocytes, also called photoreceptive cells, are considered the morphofunctional unit of pineal gland. In literature, the anatomical features, the cellular characteristics, and the pinealocytes morphology of zebrafish pineal gland have not been previously described in detail. Therefore, this study was undertaken to analyze the structure and ultrastructure, as well as the immunohistochemical profile of the zebrafish pineal gland with particular reference to the pinealocytes. Here, we demonstrated, using RT-PCR, immunohistochemistry and transmission electron microscopy, the expression of the mRNA for rhodopsin in the pineal gland of zebrafish, as well as its cellular localization exclusively in the pinealocytes of adult zebrafish. Moreover, the ultrastructural observations demonstrated that the pinealocytes were constituted by an outer segment with numerous lamellar membranes, an inner segment with many mitochondria, and a basal pole with the synapses. Our results taken together demonstrated a central role of zebrafish pinealocytes in the control of pineal gland functions. Microsc. Res. Tech., 2011. © 2011 Wiley-Liss, Inc.

In vitro interaction of Entamoeba histolytica trophozoites with fibronectin (FN) induces redistribution of the amoebic fibronectin receptor (β1EhFNR). Trafficking of beta1 integrins is important for cell adhesion and migration in higher eukaryotes and requires the participation of Rab proteins. In E. histolytica, the machinery involved in integrin trafficking is not completely known. EhRab7 is a 24.5-kDa protein whose alignment with other Rab7 proteins demonstrated that it shared significant homology with Rab7 proteins from other organisms, including humans. Using different types of microscopy (fluorescence and confocal microscopy), it was established that Rab7 and the actin cytoskeleton participated in the mobilization of β1EhFNR in FN-stimulated trophozoites. β1EhFNR and Rab7 co-localized only in vesicular structures at 5 min, and at longer time (1 h), both co-localized in both plasma membrane and in vesicular structures; at the same time, Rab7 co-localized with specific actin structures (phagocytic vacuoles). At 5 h the β1EhFNR, Rab7, and actin co-localized at the plasma membrane, and only β1EhFNR and Rab7 decorated vesicles of different sizes. Actin and Rab7 co-localized in a cap-like structure at one end of the cell. Fluorescence resonance energy transfer and electron microscopy confirmed the close interaction between β1EhFNR and Rab7. Moreover, the use of a lysosome-specific marker (LysoTracker) and a Golgi-specific marker (NBD C₆-ceramide) allowed us to establish that, at some point within the endocytic route, β1EhFNR and Rab7 co-localized within a lysosome-type organelle, but not a Golgi-like organelle, which indicated that this integrin-like molecule was returned to the plasma membrane via exocytic or secretory vesicles.Microsc. Res. Tech., 2011. © 2011 Wiley-Liss, Inc.

Fipronil, active ingredient of Frontline® acaricide, is a phenyl-pyrazolic derivative, and its efficacy in the elimination of several plagues, even in lower concentrations, has already been demonstrated; however, its effect on nontarget organisms has not been thoroughly explained. In this sense, the present study had the objective to evaluate the effects of different doses of fipronil in mice and determine its cytotoxic potential and possible compromises on nontarget organisms in artificial conditions. The results showed that animals exposed to fipronil presented significant alterations in the thyroidean tissue with evident follicular disorganization, decreased in the size of most follicles culminating with the rupture of smaller follicles and still with posterior fusion in a big and irregular “follicle” filled with colloid. The present study also revealed that the action of fipronil not only causes disorganization in the thyroidean tissue but alsoalters the chemical composition of the colloid itself (prehormone), modifying its proteic basis, which was confirmed by histochemical test for protein detection, making it clear that, in thyroids exposed to fipronil, the colloid was weakly positive or even negative for the presence of proteins in its constitution. Microsc. Res. Tech. 2011. © 2011 Wiley-Liss, Inc.

A major challenge of cancer biology is to visualize the dynamics of the metastatic process in secondary organs at high optical resolution in vivo real-time. Here, we presented intravital, dual-colored imaging of liver metastasis formation from a single cancer cell to metastatic colonies in the living liver of living mice using two photon laser scanning microscopy (TPLSM). Red fluorescent protein expressing murine (SL4) or human (HT29) colorectal cancer cell lines were inoculated to the spleen of green fluorescent protein expressing mice. Intravital TPLSM was performed by exteriorizing and fixing the liver lobe of living mice. This was repeated several times for the long-term imaging of the same mouse. Viable cancer cells in the living liver of living mice were visualized intravitally at a magnification of over 600×. Single cancer cells were arrested within hepatic sinusoids 2 h after injection. Platelet aggregation surrounding a cancer cell was observed, indicating a phenomenon of tumor-cell induced platelet aggregation. Cancer cells were extravasated from hepatic sinusoids to the space of Disse. Protrusions of Kupffer cells surrounding a cancer cell were observed, indicating that Kupffer cells appear to phagocytose cancer cells. SL4 cells formed liver metastatic colonies with extensive stromal reaction. Liver metastases by HT29 cells were observed as a cluster of micrometastatic nodules. High-resolution, dual-colored, real-time visualization of cancer metastasis using intravital TLPSM can help to understand spatiotemporal tumor-host interactions during metastatic processes in the living organs of living animals. Microsc. Res. Tech. 2011. © 2011 Wiley-Liss, Inc.

Cellular composition of the adult zebrafish (Danio rerio) optic tectal cortex was examined in this study. Morphological techniques such as 1μm thick serial plastic sections stained with osmium tetroxide and toluidine blue, modified rapid Golgi silver impregnation, GFAP immunohistochemistry, confocal microscopy, as well as scanning and transmission electron microscopy were used. Neuronal and glial components are described and the layers of the cortex are revisited. Specific neuronal arrangements as well as unique glial/ependymal cells are described. A three dimensional rendering of the astrocytic fiber arrangement in the marginal zone is presented and a composite drawing summarizes the cellular composition of the optic tectum. Microsc. Res. Tech. 2011. © 2011 Wiley-Liss, Inc.

To investigate the structural anisotropy in bovine septal cartilage, quantitative procedures in microscopic magnetic resonance imaging (μMRI), polarized light microscopy (PLM), and mechanical indentation were used to measure the tissue in three orthogonal planes: vertical, medial, and caudocephalic. The quantitative T2 imaging experiments in μMRI found strong anisotropy in the images of both vertical and caudocephalic planes but little anisotropy in the images from the medial plane. The PLM birefringent experiments found that the retardation values in the medial section were only about 10% of these in the vertical and caudocephalic sections and that the angle values in all three sections followed the rotation of the tissue section in the microscope stage. The stress relaxation experiments in mechanical indentation showed reduced stiffness in the medial plane compared to stiffness in either the vertical or caudocephalic planes. Collectively, the results in this project coherently indicate a marked structural anisotropy in cartilage from the nasal septum, where the long axis of the collagen fibrils is oriented in parallel with the medial axis. Microsc. Res. Tech., 2011. © 2011 Wiley-Liss, Inc.

To study the deleterious effects of ketamine and the potential interaction effects between ethanol and ketamine on the cerebellum, functional magnetic resonance imaging (fMRI) tests were performed on the habitual ketamine users (n = 3) when they flexed and extended their upper limbs. Another fMRI test was performed on the same participants in which they consumed alcohol (12%, 200 mL) 1 h before the test. Downregulation on the activity of cerebellum was observed and the level of activation in the cerebellum decreased dramatically in habitual ketamine users with alcohol consumption before the test. Further studies were performed by using male ICR mice receiving treatment of ketamine only [30 mg kg⁻¹ intraperitoneally (i.p.)] or ethanol only everyday (0.5 mL 12% orally) and those with coadministration of the above dosages of ketamine and ethanol for 3 months. Fewer Purkinje cells were observed in the cerebellar sections of ketamine treated mice under silver staining. For TUNEL test, a significant increase in the apoptotic cells were observed in the cerebella of the ketamine treated mice (P = 0.016) and of those with co-administration of ketamine and ethanol (P < 0.001), when compared with the control. A statistical significance (P < 0.001) in two-way ANOVA test indicated that there might be an interactive mechanism between ethanol and ketamine acting on the cerebellum. Microsc. Res. Tech. 2011. © 2011 Wiley-Liss, Inc.

By investigating the identities, amounts, and distributions of the major components of human gallstones, the formation mechanism of calculi may be elucidated. Using FT-IR specular reflection spectroscopy, the compositions of five common types of gallstones, including cholesterol calculus, bile pigment calculus, cholesterol-bilirubin calculus, mixed calculus, and black stones, were positionally, qualitatively, and quantitatively analyzed. Several organic and inorganic substances were found in human gallstones, including cholesterol, calcium bilirubinate, calcium carbonate, calcium phosphate, and calcium stearate. Among them, cholesterol and calcium bilirubinate were the main components. The amounts of these substances varied in different types of gallstone. The distributions of cholesterol and calcium salts (with the exception of calcium bilirubinate) were also closely associated with location. In the various calculi, the amounts of cholesterol and calcium bilirubinate in the cores, the layered structures outside the cores, and the outer shells were higher than those of other substances. The identities of the major components of calculi were similar in different types of gallstones, but their amounts and distributions were significantly different. The analytical method used in this study, which was able to accurately determine the location, quality, and quantity of the major components of gallstones, would be an effective tool to study the formation mechanism of calculi. Microsc. Res. Tech., 2011. © 2011 Wiley-Liss, Inc.

Fluorescence microscopy and microspectrofluorometry are important tools in the characterization and identification of proteins, offering a great range of applications in conservation science. Because of their high selectivity and sensitivity, the combination of these techniques can be exploited for improved recognition and quantification of proteinaceous binders in paintings and polychromed works of art. The present article explores an analytical protocol integrating fluorescence microscopy and fluorometry for both identification and mapping of proteinaceous binders (in particular egg and glues) in paint samples. The study has been carried out on historically accurate reconstructions simulating the structure and composition of tempera and oil paints containing these binders. To assess the spatial distribution of specific proteins within the paint layers, cross-sections from the reconstructions were analyzed by fluorescence imaging after staining with an exogenous fluorophore. Reference fluorescence spectra for each layer were acquired by a multichannel spectral analyzer and compared after Gaussian deconvolution. The results obtained demonstrated the effectiveness of the integrated protocol, highlighting the potential for the use of fluorescent staining coupled with microspectrofluorometry as a routine diagnostic tool in conservation science. The current work creates a set of fully characterized reference samples for further comparison with those from actual works of art. Microsc. Res. Tech., 2011. © 2011 Wiley-Liss, Inc.

A pre-cryogenic holder (cryo-holder) facilitating cryo-specimen observation under a conventional scanning electron microscope (SEM) is described. This cryo-holder includes a specimen-holding unit (the stub) and a cryogenic energy-storing unit (a composite of three cylinders assembled with a screw). After cooling, the cryo-holder can continue supplying cryogenic energy to extend the observation time for the specimen in a conventional SEM. Moreover, the cryogenic energy-storing unit could retain appropriate liquid nitrogen that can evaporate to prevent frost deposition on the surface of the specimen. This device is proved feasible for various tissues and cells, and can be applied to the fields of both biology and material science. We have employed this novel cryo-holder for observation of yeast cells, trichome, and epidermal cells in the leaf of Arabidopsis thaliana, compound eyes of insects, red blood cells, filiform papillae on the surface of rat tongue, agar medium, water molecules, penicillium, etc. All results suggested that the newly designed cryo-holder is applicable for cryo-specimen observation under a conventional SEM without cooling system. Most importantly, the design of this cryo-holder is simple and easy to operate and could adapt a conventional SEM to a plain type cryo-SEM affordable for most laboratories. Microsc. Res. Tech., 2011. © 2011 Wiley Periodicals, Inc.

The most practical approach to reduce morbidity and mortality of cancer is to delay the process of carcinogenesis by usage of anticancer agents. This necessitates that safer compounds are to be critically examined for anticancer activity especially, those derived from natural sources. A spice commonly found in India and the surrounding regions, is turmeric, derived from the rhizome of Curcuma longa and the major active component is a phytochemical termed curcumin. Green tea is one of the most popular beverages used worldwide, produced from the leaves of evergreen plant Camellia sinensis and the major active ingredients are polyphenolic compounds known as catechins. In this study, synergistic anticancer activity of curcumin and catechin was evaluated in human colon adenocarcinoma HCT 15, HCT 116, and human larynx carcinoma Hep G-2 cell lines. Although, both curcumin or catechin inhibited the growth of above cell lines, interestingly, in combination of both these compounds highest level of growth control was observed. The anticancer activity shown is due to cytotoxicity, nuclear fragmentation as well as condensation, and DNA fragmentation associated with the appearance of apoptosis. These results suggest that curcumin and catechin in combination can inhibit the proliferation of HCT 15, HCT 116, as well as Hep G-2 cells efficiently through induction of apoptosis. Microsc. Res. Tech., 2011. © 2011 Wiley Periodicals, Inc.

This study analyzed the newly formed bone tissue after application of recombinant human BMP-2 (rhBMP-2) and P-1 (extracted from Hevea brasiliensis) proteins, 2 weeks after the creation of a critical bone defect in male Wistar rats treated or not with a low-intensity laser (GaAlAs 780 nm, 60 mW of power, and energy density dose of 30 J/cm²). The animals were divided into two major groups: (1) bone defect plus low-intensity laser treatment and (2) bone defect without laser irradiation. The following subgroups were also analyzed: (a) 5 μg of pure rhBMP-2; (b) 5 μg of pure P-1 fraction; (c) 5 μg of rhBMP-2/monoolein gel; (d) 5 μg of P-1 fraction/monoolein gel; (e) pure monoolein gel. Comparisons of the groups receiving laser treatment with those that did not receive laser irradiation show differences in the areas of new bone tissue. The group treated with 5 μg of rhBMP-2 and laser irradiation was not significantly different (P >0.05) than the nonirradiated group that received the same treatment. The irradiated, rhBMP-2/monoolein gel treatment group showed a lower area of bone formation than the nonirradiated, rhBMP-2/gel monoolein treatment group (P < 0.001). The area of new bone tissue in the other nonirradiated and irradiated groups was not significantly different (P > 0.05). Furthermore, the group that received the 5 μg of rhBMP-2 application showed the greatest bone formation. We conclude that the laser treatment did not interfere with the area of new bone tissue growth and that the greatest stimulus for bone formation involved application of the rhBMP-2 protein. Microsc. Res. Tech. 2011. © 2011 Wiley Periodicals, Inc.

The purpose of this study was to assess the influence of Er:YAG laser pulse repetition rate on the thermal alterations occurring during laser ablation of sound and demineralized primary dentin. The morphological changes at the lased areas were examined by scanning electronic microscopy (SEM). To this end, 60 fragments of 30 sound primary molars were selected and randomly assigned to two groups (n = 30); namely A sound dentin (control) and B demineralized dentin. Each group was divided into three subgroups (n = 10) according to the employed laser frequencies: I–4 Hz; II–6 Hz, and III–10 Hz. Specimens in group B were submitted to a pH-cycling regimen for 21 consecutive days. The irradiation was performed with a 250 mJ pulse energy in the noncontact and focused mode, in the presence of a fine water mist at 1.5 mL/min, for 15 s. The measured temperature was recorded by type K thermocouples adapted to the dentin wall relative to the pulp chamber. Three samples of each group were analyzed by SEM. The data were submitted to the nonparametric Kruskal-Wallis test and to qualitative SEM analysis. The results revealed that the temperature increase did not promote any damage to the dental structure. Data analysis demonstrated that in group A, there was a statistically significant difference among all the subgroups and the temperature rise was directly proportional to the increase in frequency. In group B, there was no difference between subgroup I and II in terms of temperature. The superficial dentin observed by SEM displayed irregularities that augmented with rising frequency, both in sound and demineralized tissues. In conclusion, temperature rise and morphological alterations are directly related to frequency increment in both demineralized and sound dentin. Microsc. Res. Tech., 2011. © 2011 Wiley Periodicals, Inc.

Osteoporosis is a global public health that affects postmenopausal women due to the deficiency of estrogen, a hormone that plays an important role in the microarchitecture of bone tissue. Osteoporosis predisposes to pathological bone fracture that can be repaired by conventional methods. However, depending on the severity and quantity of bone loss, the use of autogenous grafts or biomaterials such as hydroxyapatite might be necessary. The latter has received increasing attention in the medical field because of its good biological properties such as osteoconductivity and biocompatibility with bone tissue. The objective of this study was to evaluate using histologic and radiographic analyses, the osteogenic capacity of hydroxyapatite implanted into the femur of rats with ovariectomy-induced osteoporosis. Eighteen rats were divided into three groups with six animals in each: group nonovariectomized, bilaterally ovariectomized not receiving estrogen replacement therapy, and bilaterally ovariectomized submitted to estrogen replacement therapy. Defects were created experimentally in the distal epiphysis of the femur with a surgical drill and filled with porous hydroxyapatite granules. The animals were sacrificed 8 weeks after surgery. The volume of newly formed bone in the implant area was quantified by morphometrical methods. The results were analyzed by ANOVA followed by the Tukey test (P < 0.05). The hydroxyapatite granules showed good radiopacity. Histological analysis revealed less quantity of newly formed bone in the ovariectomized group not submitted to hormone replacement therapy. In conclusion, bone neoformation can be expected even in bones compromised by estrogen deficiency, but the quantity and velocity of bone formation are lower. Microsc. Res. Tech., 2011. © 2011 Wiley Periodicals, Inc.

Taking poly(lactic acid) microbubbles and purple membranes as examples, a general in situ observation and relocation method of nanomaterial samples based on microscope systems was reported. First, a four-grade coordinate with different precisions was marked around a substrate by UV lithography. Second, using optical microscope and scanning probe microscope, special positions of poly(lactic acid) microbubbles and purple membranes were observed, respectively. Third, the four-grade coordinate value corresponded to the special sample position, and the distance between the special position and coordinate edge were recorded, respectively. Finally, the special position can be easily found again, or the sample in the special position can be manipulated and secondary processed based on the recorded coordinate value and distance, after the sample was removed and then was reset on the sample stage of microscope. The in situ observation and relocation method can be applied in different microscope systems and different sample substrates, and will have potential applications in the manipulation and the secondary process of micro- and nano-devices. Microsc. Res. Tech., 2011. © 2011 Wiley Periodicals, Inc.

Background: The aim of this work was to study the new bone tissue formation after bone morphogenetic protein type 2 (rhBMP-2) and P-1 application, using 5 and 10 μg of each, combined to a material carrier, in critical bone defects. Methods: It was used 70 Wistar rats (male, ∼250 g) that were divided in 10 groups with seven animals on each. Groups are the following: critical bone defect only, pure monoolein gel, 5 μg of pure P-1, 5 μg of pure rhBMP-2, 5 μg of P-1/monoolein gel, 5 μg of rhBMP-2/monoolein gel, 10 μg of pure P-1, 10 μg of pure rhBMP-2, 10 μg of P-1/monoolein gel, 10 μg of rhBMP-2/monoolein gel. Animals were sacrificed after 4 weeks of the surgical procedure and the bone samples were submitted to histological, histomorphometrical, and immunohistochemical evaluations. Results: Animals treated with pure P-1 protein, in both situations with 5 μg and 10 μg, had no significant difference (P > 0.05) for new bone formation; other groups treated with 10 μg were statistically significant (P < 0.05) among themselves and when compared with groups in which it was inserted the monoolein gel or critical bone defect only (P < 0.05). In the group involving the 10 μg rhBMP-2/monoolein gel association, it was observed an extensive bone formation, even when compared with the same treatment without the gel carrier. Conclusion: Using this experimental animal model, more new bone tissue was found when it was inserted the rhBMP-2, especially when this protein was combined to the vehicle, and this process seems to be dose dependent. Microsc. Res. Tech., 2011.© 2011 Wiley Periodicals, Inc.

A software package for computing radial distribution functions and other pair correlation functions from electron diffraction patterns of disordered solids is presented. The package, called RDFTools, is freely available via the internet and allows rapid in situ measurements of such quantities as interatomic nearest neighbor distances, average bond angles and coordination numbers. The software runs under DigitalMicrograph™ (Pleasanton, California, Gatan), a very widely used program in transmission electron microscopy. All implemented algorithms have been designed to compute diffraction integrals and data-processing averages in a fast and efficient manner to enable quick processing of publication ready, quantitative pair distribution function information. In the development of RDFTools, significant attention was paid to provide a robust and intuitive user-interface for deriving reliable semiquantitative information. For example, RDFTools enables accurate pair separation distances to be revealed upon immediate interrogation at the microscope; even for potentially thick specimens and/or regions of unknown elemental composition. Microsc. Res. Tech., 2011. © 2011 Wiley Periodicals, Inc.

Authentication of Chinese Materia Medica (CMM) decoction dregs is important for ensuring the efficacy and safety of CMM when they are used in decoction. If someone got worse or poisoned after taking a decoction while the formula is appropriate, the authentication of CMM dregs is the effective method to explore the reasons. Therefore, a systematic study on the authentication of CMM dregs was carried out. In this study, two pairs of easily confused CMM dregs, Fenge (Puerariae Thomsonii Radix) and Shanyao (Dioscoreae Rhizoma), Dihuang (Rehmanniae Radix) and Huangjing (Polygonati Rhizoma), which mostly comprise storage tissues, were investigated by comparing the morphological and microscopic characteristics. Fenge and Shanyao contain abundant starch granules. After decoction their dregs were hard, nonstarchy and horn-like. Fully gelatinized starch granules were found in the powder of Fenge dregs while incompletely gelatinized starch granules were occasionally found in the Shanyao dregs. In contrast, Dihuang and Huangjing contain water-soluble sugars. After decoction, their dregs were hard and brittle. Their parenchymatous cells were shrunken and unknown crystals were found under microscope. Hence, the morphological and microscopic characteristics of CMM before and after decoction were different, and different changes in the CMM dregs can be illustrated by the different nature of their ergastic substance. These differences could be used to authenticate CMM dregs. Microsc. Res.Tech., 2011. © 2011 Wiley Periodicals, Inc.

Changes in fish macrophages (Macs) are useful indicators of environmental pressures, but responses due to chemical and nonchemical stresses may be confounded by natural sources of variability. These may include sex and gonadal stage. In this study, we start addressing the following question: is the seasonally dependent ovary stage a factor to consider when using kidney Macs as biomarkers? To tackle this problem, the relative amount of pigmented Macs in kidney (head, trunk, and tail portions) was stereologically estimated in Ohrid trout, and related with the breeding status. The amount of Macs significantly increased from pre vitellogenesis to late vitellogenesis and showed a decreasing trend then after, with lower values noted after spawning in the head (1.9% versus 7.5% versus 2.0%), trunk (1.8% versus 7.5% versus 2.5%), and tail (2.5% versus 6.7% versus 2.9%) kidney. The decrease seen at spawning was significant in head and trunk kidney, and at post spawning it was significant for all kidney portions. The amounts of Macs were positively correlated with the ovary relative weights and plasma estradiol levels. We proved for the first time that fish kidney pigmented Macs can vary in amount during the breeding cycle. Our data, combined with literature, strongly support that the sex-steroid profile and kidney status-seasonal remodeling both influence the Macs pool; likely not only in female trout. So, while increases in Macs may warn of ecosystem problems, we show that using kidney Macs for biomonitoring should also take into account seasonally, particularly that related with ovary maturation. Microsc. Res. Tech. 2011. © 2011 Wiley Periodicals, Inc.

The subcellular behavior of aluminum and indium, used in medical and industrial fields, was studied in the gastric mucosa and the liver after their intragastric administration to rats, using, two of the most sensitive methods of observation and microanalysis, the transmission electron microscopy, and the secondary ion mass spectrometry. The ultrastructural study showed the presence of electron dense deposits, in the lysosomes of parietal and principal gastric mucosa cells but no loaded lysosomes were observed in the different studied hepatic territories. The microanalytical study allowed the identification of the chemical species present in those deposits as aluminum or indium isotopes and the cartography of their distribution. No modification was observed in control rats tissues. In comparison to previous studies describing the mechanism of aluminum concentration in the gastric mucosa and showing that this element was concentrated in the lysosomes of fundic and antral human gastric mucosa, our study provided additional informations about the types of cells involved in the phenomenon of concentration of aluminum and indium, which are the parietal and the principal cells of the gastric mucosa. Our study demonstrated that these cells have the ability to concentrate selectively aluminum and indium in their lysosomes, as a defensive reaction against intoxication by foreign elements. Microsc. Res. Tech., 2011 © 2011 Wiley Periodicals, Inc.

Electron dense granules (EDGs) were identified by transmission electron microscopy in Entamoeba histolytica trophozoites recovered from hamster liver lesions. Abundant granules were present in trophozoites recovered after 15 min of liver inoculation. Variation in the size and morphology of these EDGs was also observed. Numerous granules were present in the plasma membrane when these parasites were incubated for 5 min with MDCK monolayers. Release of these EDGs was suggested by the presence of granules in contact with the surface of the target cell plasma membrane. Parasite phagocytic invaginations were observed after 10 min of parasite-monolayer interaction. In these structures, scarce granules were seen. Granules secretion was corroborated by obtaining of a pellet of these small structures from the incubation of trophozoites with collagen supernatant. Collagenase and gellatinase activity of this pellet was identified in SDS-PAGE gels. EDGs were also present in amebic hamster liver lesions. Our observations corroborate that these granules are secreted and suggest that may participate in the cytopathic effect of E. histolytica both in vitro and in vivo. Microsc. Res. Tech., 2011. © 2011 Wiley Periodicals, Inc.

Fipronil, active ingredient of the acaricide Frontiline®, is a phenyl-pyrazolic derivative, and its efficacy in the elimination of several plagues, even in low concentrations, has already been demonstrated; however, its effect on nontarget organisms has not been thoroughly explained. In this sense, the objective of this study was to evaluate the effects of different dosages of fipronil on the liver of mice in artificial conditions. Results showed that the animals exposed to fipronil present significant ultrastrucutural changes in hepatic cells with evident cellular and cytoplasm disorganization in hepatocytes characterized by an increase in the number of organelles, mainly mitochondria and rough endoplasmic reticulum, organelles that, in the case of the exposed animals, were probably responsible for the enzymes' synthesis that have the function of inactivating the toxic metabolites. A fat accumulation in the hepatocytes' cytoplasm (steatosis) was observed, in addition to extended vacuolated areas, mainly in regions next to the cell nucleus. Alterations observed in the nuclei of the hepatocytes pointed out cell death processes. Moreover, Kupffer cells increased in number (hyperplasia) suggesting an increase in the phagocytic activity of the liver in the exposed animals. Microsc. Res. Tech., 2011. © 2011 Wiley Periodicals, Inc.

Forensic entomology is an area of science that serves as a tool in crime scene investigations. Usually, flies are the first insects to reach a carcass and can oviposit just a few hours after arrival. Therefore, the knowledge of immature stages is essential for correct identification of the species found on corpses. Scanning electron microscopy (SEM) gives detailed information about morphological characters helping to identify the immature forms of flies. Chrysomya putoria (Wiedemann) is a very important fly for forensic entomology, because it has high population densities and is easily found in colonizing carcasses, moreover, it is also a possible causative agent for secondary myiasis. The aim of this study is to identify larvae and puparia of C. putoria using SEM. The first instar larvae were composed of 12 smooth segments separated by spines. Antennae and maxillary palps were visible. Anterior spiracle was absent and only one spiracular opening could be seen at the posterior spiracle. Second and third larval instars were similar to first instar, except for the presence of anterior spiracle that is composed by 11–12 spiracular ramifications. At the anal segment, two spiracular openings were found in second instars and three openings in third instar larvae. Puparia showed a retracted cephalic region and none of the head structures were visible. Microsc. Res. Tech. 2011., © 2011 Wiley Periodicals, Inc.