ALBERT

Description: ABSTRACT We present a technique that records transient changes in the fluorescence lifetime of a sample with spatial resolution along a one-dimensional scan. The technique is based on scanning the sample with a high-frequency pulsed laser beam, detecting single photons of the fluorescence light, and building up a photon distribution over the distance along the scan, the arrival times of the photons after the excitation pulses and the time after a stimulation of the sample. The maximum resolution at which lifetime changes can be recorded is given by the line scan period. Transient lifetime effects can thus be resolved at a resolution of about one millisecond. We demonstrate the technique for recording photochemical and nonphotochemical chlorophyll transients in plants and transient changes in free Ca 2+ in cultured neurons. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT Gonadotropin releasing hormone (GnRH) is a peptide that is conserved in both vertebrate and invertebrate species. In this study, we have demonstrated the distribution pattern of two isoforms of GnRH-like peptides in the neural ganglia and testis of reproductively mature male abalone , H. asinina , by immunohistochemistry and whole mount immunofluorescence. We found octopus (oct) GnRH and tunicate-I (t) GnRH-I immunoreactivities (ir) in type 1 neurosecretory cells (NS1) and they were expressed mostly within the ventral horn of the cerebral ganglion, whereas in pleuropedal ganglia they were localized primarily in the dorsal horn. Furthermore, tGnRH-I-ir were strongly detected in fibers at the caudal part of the cerebral ganglia and both ventral and dorsal horns of the pleuropedal ganglia. In the testis, only octGnRH-ir was found primarily in the granulated cell and central capillaries within the trabeculae. These results suggest that multiple GnRH-like peptides are present in the neural ganglia which could be the principal source of their production, whereas GnRH may also be synthesized locally in the testis and act as the paracrine control of testicular maturation. Microsc. Res. Tech. 77:110–119, 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT Microtubules are important targets when studying potential anticancer agents since disturbance of these microtubule dynamics results in cell cycle arrest and cell death. 2-Methoxyestradiol is a naturally occurring metabolite that exerts antiproliferative activity and induces apoptosis. Due to limited biological accessibly and rapid metabolic degradation, several analogs were synthesized. This study investigated the antiproliferative influence of an 2-methoxyestradiol analog, (8R, 13S, 14S, 17S)-2-Ethyl-13-methyl-7, 8, 9, 11, 12,13, 14, 15, 16, 17-decahydro-6H-cyclopenta[a]phenanthrane-3, 17-diyl bis(sulfamate) (EMBS) on cell proliferation, morphology and apoptosis induction in a estrogen receptor-positive breast adenocarcinoma cells line (MCF-7), estrogen receptor-negative highly metastatic breast cell line (MDA-MB-231) and a non-tumorigenic breast epithelial cell line (MCF-12A). Spectrophotometry results indicated that EMBS exerted differential antiproliferative activity in the three cell lines. Cell growth of the breast adenocarcinoma and highly metastatic breast cell line reached a plateau effect at 0.4 μM after 24 h of exposure. Light microscopy and polarization-optical transmitted light differential interference contrast demonstrated compromised cell density, cells blocked in metaphase and the presence of apoptotic characteristics after EMBS exposure for 24 h in all three cell lines. Transmission electron microscopy and scanning electron microscopy revealed hallmarks of apoptosis namely the presence of apoptotic bodies, shrunken cells and cell debris in EMBS-exposed cells. This investigation demonstrated that EMBS does exert antimitotic activity and induces apoptosis contributing to elucidating the signal transduction of EMBS in tumorigenic and non-tumorigenic breast cell lines. Findings warrant in-depth analysis of specific targets in vitro and subsequent in vivo investigation for anticancer therapy. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT The sperm ultrastructure of the short-faced scorpionfly Panorpodes kuandianensis Zhong et al. was investigated using transmission electron microscopy. The spermatozoon is composed of a short head containing a bilayered acrosome and an elongate nucleus, a neck transition region, and a long flagellum. The acrosome consists of an acrosomal vesicle and a central perforatorium. The nucleus has two deep, U-shaped lateral grooves and its chromatin contains a series of parallel, regularly arrayed nuclear fibers. The barrel-shaped centriolar adjunct occupies the most volume of the neck region. The flagellum is helical for its whole length and is formed by an axoneme, two mitochondrial derivatives, a pair of accessory bodies, and peculiar accessory structures. The axoneme has nine accessory microtubules at the anterior flagellum region, displaying a 9 + 9 + 2 microtubular pattern, but the accessory microtubules are short and disappear quickly. The two mitochondrial derivatives differ in length and diameter. In the more posterior region, the remaining anchor-shaped mitochondrial derivative has a circular crystalline region, differing from other mecopteran species. The filiform materials are peculiar and lie beside the nucleus and in the flagellum region. Sperm ultrastructural comparison of P. kuandianensis with other families supports a close affinity of Panorpodidae with Panorpidae. In addition, the occurrence of the nine accessory microtubules in the sperm axoneme of Panorpodes indicates that the 9 + 9 + 2 axonemal pattern might be a symplesiomorphy of the Mecoptera. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT The wing of a dragonfly is thin and light, but can bear high frequent alternating stress and present excellent antifatigue properties. The surface morphology and microstructure of the wings of dragonfly Pantala flavescens were observed using SEM in this study. Based on the biological analysis method, the configuration, morphology, and structure of the vein were studied, and the antifatigue properties of the wings were investigated. The analytical results indicated that the longitudinal veins, cross veins, and membrane of dragonfly wing form a optimized network morphology and spacially truss-like structure which can restrain the formation and propagation of the fatigue cracks. The veins with multilayer structure present high strength, flexibility, and toughness, which are beneficial to bear alternating load during the flight of dragonfly. Through tensile-tensile fatigue failure tests, the results were verified and indicate that the wings of dragonfly P. flavescens have excellent antifatigue properties which are the results of the biological coupling and synergistic effect of morphological and structural factors. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT Muscle fiber images play an important role in the medical diagnosis and treatment of many muscular diseases. The number of nuclei in skeletal muscle fiber images is a key bio-marker of the diagnosis of muscular dystrophy. In nuclei segmentation one primary challenge is to correctly separate the clustered nuclei. In this article, we developed an image processing pipeline to automatically detect, segment, and analyze nuclei in microscopic image of muscle fibers. The pipeline consists of image pre-processing, identification of isolated nuclei, identification and segmentation of clustered nuclei, and quantitative analysis. Nuclei are initially extracted from background by using local Otsu's threshold. Based on analysis of morphological features of the isolated nuclei, including their areas, compactness, and major axis lengths, a Bayesian network is trained and applied to identify isolated nuclei from clustered nuclei and artifacts in all the images. Then a two-step refined watershed algorithm is applied to segment clustered nuclei. After segmentation, the nuclei can be quantified for statistical analysis. Comparing the segmented results with those of manual analysis and an existing technique, we find that our proposed image processing pipeline achieves good performance with high accuracy and precision. The presented image processing pipeline can therefore help biologists increase their throughput and objectivity in analyzing large numbers of nuclei in muscle fiber images. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT Cystoisospora belli is an opportunistic protozoan that causes human cystoisosporiasis, an infection characterized by diarrhea, steatorrhea, abdominal pain, fever, and weight loss. The lack of animal models susceptible to C. belli , and the difficulty in obtaining clinical samples with fair amounts of oocysts have limited the research pertaining to the basic biology of this parasite. This study aimed to describe the ultrastructure of endogenous stages of C. belli in Monkey Rhesus Kidney Cells (MK2) and Human Ileocecal Adenocarcinoma cells (HCT-8). Zoites of C. belli exhibited typical morphological features of coccidia, which included a trilaminar pellicle, an apical complex formed by a conoid, polar rings, rhoptries, and micronemes, in addition to dense granules and the endoplasmic reticulum. No crystalloid body was observed but various lipid and amylopectin granules were usually present in the cytoplasm of zoites. We observed a tendency of the endoplasmic reticulum of the host cell to be located near the parasitophorous vacuole membrane. Merozoites were formed by endodyogeny and during replication, the apical complex of the mother cell remained intact. The formation of gametes or oocysts was not observed. The ultrastructural findings of C. belli are further evidence of its proximity to Sarcocystidae family members and corroborate their reclassification as Cystoisospora spp. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT Arge pullata Zadd is an important phytophagous pest that damages red birch Betula albo-sinensis in Hubei Province, South China. Massive ecological and economic losses have been caused by this species, which threatens the ecological security of the Shennongjia Nature Reserve. To investigate the mechanoreception, chemoreception, and oviposition processes of A. pullata , scanning electron microscopy and optical confocal microscopy were used to reveal the typology, morphology, and distribution of ovipositor and antennal sensilla. The results show that A. pullata has clavate antennae and eight types of sensilla in total, including sensilla chaetica, sensilla trichodea (types 1–3), sensilla basiconica, sensilla coeloconica (types 1 and 2), and Böhm's bristles. Sensilla trichodea type 1 distributed only on male antennae; the densities of sensilla trichodea type 2 and sensilla basiconica differed between the sexes. The binding pattern of ovipositor valvulae was discovered, and one type of sensilla chaetica, two types of sensory pits, and tooth-like cones as well as two types of microtrichia were found in the ovipositor. Based on morphological evidence and research on Hymenoptera, putative functions are suggested to increase our understanding of the mechanisms by which this species finds hosts and mates, and how oviposition takes place. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT The longicorn beetle Xylotrechus grayii (White, 1855) has been spreading rapidly in China, causing mass mortality of honeysuckle which is economically and medicinally important. In order to elucidate the mechanisms of mate and host location and to advance efficient control methods, antennal sensilla features were investigated in both sexes of X. grayii using scanning electron microscopy (SEM). The filiform antennae of both sexes consist of scape, pedicel, and nine flagellomeres (f1–9). Five types of sensilla were observed: sensilla chaetica (5 subtypes, SC1–5), sensilla basiconica (4 subtypes, SB1–4), Böhm bristles (Bm), grooved peg sensilla (Gp), and sensilla campaniformia (Ca). SC were most common on the antennae, followed by SB and Bm. No significant sexual differences in the type, amounts, and distribution of antennal sensilla were found except for the distribution of SB clusters and Ca. SB clusters and Ca occurred on f1–8 of male antennae but were absent on those segments in females, suggesting a potential function as receptors for female sex pheromones. The putative functions of other sensilla are discussed based on their characteristics in related species. This study provides an important foundation for further research on sensory mechanisms and control measures of X. grayii . Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT In mammals, the cerebral cortex microvasculature (CCM) of the neopallium plays important roles in the physiological and pathological processes of the brain. The aim of the present work is to analyze the CCM by use of the SEM-vascular corrosion cast technique, and to examine the immunocytochemical characteristics of the CCM in adult domestic ruminants (cattle, buffalo, and sheep) by using the SEM-immunogold technique. The CCM originated from the very small, finger-like terminal branches of the macrovasculature of the brain. The superficial cortical arterioles were more numerous than the deep straight arterioles which proceeded toward the white matter. The surface casts of the arterioles and capillaries of the cerebral cortex showed ring-shaped formations in the arterioles and at the origin of the capillaries. All capillaries down-stream from these ring-shaped formations were flaccid. Casts of the capillaries showed wrinkles due to the presence of endothelial folds, which is characteristic of varying blood pressure. Formations having intense anti-GIFAP immunoreactivity were frequently evident along the course of the blood capillaries in the cerebral cortex. These formations were probably astrocytes that might regulate the cerebral microcirculation based on physiological and pathological stimuli, such as neuronal activation. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT The effects of anti-angiogenic therapies in guiding tumor angioarchitecture prompted us to examine the modifications in the vascular network of the oral squamous cell carcinoma (SCC) produced by the multitargeted tyrosine kinase inhibitor sunitinib malate. Twelve Syrian hamsters had their right buccal pouches submitted to tumor induction with dimethylbenzanthracene and carbamide peroxide for 55 days. The animals were then divided into two groups of six animals each; group I was treated with sunitinib malate and group II (control) was remained untreated. After 4 weeks, the hamsters had their vascular networks casted by Mercox ® resin and analyzed by scanning electron microscopy. The qualitative study of the vascular network of the control tumor-bearing pouches showed images of intussusception and sprouting angiogenesis, flattened blood vessels, abrupt variations in their diameter, and a tortuous course. The samples treated with sunitinib exhibited a qualitative reduction of the signs of vascular proliferation. In addition, these casts presented an attenuation of the morphological features observed in the untreated tumor-bearing pouches. Quantitative analysis demonstrated that the pouches treated with sunitinib did not show a decrease ( P 〉 0.05) in the vascular diameter and intervessel distances when compared with the control group. The results of the present study suggest that sunitinib may act on the vascular network of oral SCC, normalizing the blood vessels. However, further experiments should be performed in order to determine a judicious dose of this anti-angiogenic therapy. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT Due to its expansion, agriculture has become increasingly dependent on the use of pesticides. However, the indiscriminate use of insecticides has had additional effects on the environment. These products have a broad spectrum of action, and therefore the insecticide affects not only the pests but also non-target insects such as bees, which are important pollinators of agricultural crops and natural environments. Among the most used pesticides, the neonicotinoids are particularly harmful. One of the neonicotinoids of specific concern is thiamethoxam, which is used on a wide variety of crops and is toxic to bees. Thus, this study aimed to analyze the effects of this insecticide in the midgut and Malpighian tubule cells of Africanized Apis mellifera . Newly emerged workers were exposed until 8 days to a diet containing a sublethal dose of thiamethoxam equal to 1/10 of LC 50 (0.0428 ng a.i./l L of diet). The bees were dissected and the organs were processed for transmission electron microscopy. The results showed that thiamethoxam is cytotoxic to midgut and Malpighian tubules. In the midgut, the damage was more evident in bees exposed to the insecticide on the first day. On the eighth day, the cells were ultrastructurally intact suggesting a recovery of this organ. The Malpighian tubules showed pronounced alterations on the eighth day of exposure of bees to the insecticide. This study demonstrates that the continuous exposure to a sublethal dose of thiamethoxam can impair organs that are used during the metabolism of the insecticide. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT Autofocusing technology is indispensable for routine use of microscopes on a large scale in biological field. The autofocusing method using the angle of Hilbert space is brought forward to measure whether the image is focused or not. The angle of Hillbert space can be used to evaluate accurately the similarity degree of two images. The experiment results show that the autofocusing method can decrease the computational cost and get accuracy for real-time biological and biomedical images with noise robustness. The focus curves are smooth and possess the unimodality, the monotonicity and the symmetry. Compared with other classic and optimum focus method, the Hilbert method demonstrates its robustness to noise and can improve the focus speed. The experiments showed that the proposed method can increase the overall performance of an autofocus system and has strong applicability in various autofocusing algorithms. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT The structure of ovary in a representative of the scale insect family Matsucoccidae, Matsucoccus pini , is described at the ultrastructural level. The paired ovaries of M. pini are composed of about 50 ovarioles of telotrophic type that develop asynchronously. An individual ovariole consists of an anterior tropharium (trophic chamber) and posterior vitellarium. The tropharium encloses trophocytes (nurse cells) and early previtellogenic oocytes termed arrested oocytes. In the vitellarium from 1 to 6, linearly arranged oocytes may develop. Analysis of serial sections has shown that each ovariole contains 32 germ cells (trophocytes, arrested oocytes, and developing oocytes). In the cytoplasm of all these cells, small rod-shaped bacteria are present. In the early vitellogenic oocytes, accessory nuclei arise. As vitellogenesis progresses, these nuclei migrate toward the cortical ooplasm. The obtained results are discussed in a phylogenetic context. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT Many species that belong to Artiodactyls order show an interdigital sinus (IS), as it occurs in sheep, in all four extremities. These are considered to be scent glands responsible for sexual communication having strong attractiveness to mature males at the peak of the breeding season. The aim of this study was to evaluate, in IS in cyclic ewes, the microscopic and ultrastructure (scanning and transmission electron microscopy) anatomy, secretion composition, and mRNA and protein expression of estrogen receptors α and β and progesterone receptors. Glandular sebaceous structures occupy a superficial area of the pouch. The other glands present in the IS show a coiled tubular structure and tall and polyhedral secretory cells with irregular luminal surface resulting from the secretory process. Protein and mRNA gene transcription studies were performed to determine the presence of ER (α and β) and P4r in IS. At the follicular phase, IS cell populations analyzed using flow cytometry expressed higher levels of ERβ compared with ERα ( P  〈 0.05), whereas no difference was observed between them in the luteal phase. The IS amount of secretion was the highest in the follicular phase compared with luteal phase ( P  〈 0.05) or pregnancy ( P  〈 0.001).To the best of our knowledge, for the first time, the presence of ER (α and β) within the IS was demonstrated. As estrogen action is mediated by specific receptors in target cells, the presence of these receptors in IS might be needed to trigger signaling pathways involved in conspecific chemical (sexual) communication attributed to this area. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT Light microscopy has undergone a revolution with the advent of super-resolution microscopy methods that can surpass the diffraction limit. These methods have generated much enthusiasm, in particular with regards to the new possibilities they offer for biological imaging. The recent years have seen a great advancement both in terms of new technological developments and exciting biological applications. Here, we review some of the important milestones in the field and highlight some recent biological applications. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT This study focused on test the null hypothesis that there is no difference between the degree of conversion and biocompatibility of different resin reinforced glass ionomer cements (RRGICs). Forty-eight male Wistar rats were used, distributed into four groups ( n  = 12), as follows: Group C (Control, polyethylene), Group FOB (Fuji Ortho Band), Group UBL (Ultra band Lok), and Group MCG (Multicure Glass), in subcutaneous tissue. The events of edema, necrosis, granulation tissue, multinuclear giant cells, young fibroblasts, and collagen formation were analyzed at 7, 15, and 30 days. The degree of conversion was evaluated by the Fourier method. Biocompatibility and degree of conversion were assessed using the Kruskal–Wallis and Dunn tests, and ANOVA and Tukey's test, respectively ( P  〈 0.05). It was observed that, there was significant difference between Groups FOB and UBL for the presence of young fibroblasts at 15 days ( P  = 0.034) and between the Control and MCG Groups for the presence of multinucleated giant cells at 30 days ( P  = 0.009). Monomer conversion increased progressively until day 30, with significant difference between Group FOB and Groups UBL and MCG ( P  = 0.013) at 15 days. The null hypothesis was partially accepted, Fuji Ortho Band showed a less monomer conversion and a smaller number of young fibroblasts in the time of 15 days. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT Several treatments have been developed aiming the prevention of bone loss. There are discussions about the best prophylactic and therapeutic procedures for osteoporosis. This study evaluated the effects of physical exercise associated with risedronate as a prophylactic and therapeutic procedure in osteopenic bones of rats submitted to ovariectomy. We used 48 Wistar rats divided into: ovariectomized or subjected to sham surgery. Ovariectomized rats were divided into the following sub-groups: OVX, 12 weeks sedentary; OVX-EX, treadmill training for 12 weeks; OVX-RA, 12 weeks with risedronate administration; and OVX-EX-RA, 12 weeks with risedronate administration and treadmill training. Rats subjected to sham surgery were divided into the following sub-groups: SH, 12 weeks sedentary; SH-EX, treadmill training for 12 weeks; SH-RA, 12 weeks with risedronate administration; and SH-EX-RA, 12 weeks with risedronate administration and training on the treadmill. The effectiveness of the treatment was evaluated in tibias using biomechanical, radiological, histomorphometric, and immunohistochemical analyses. Data were analyzed by statistical tests, with significance level of P  〈 0.05. Results of mechanical tests showed that the SH-RA group had lower values compared with OVX-RA group; densitometry showed no significant differences; according to histomorphometric methods, OVX group presented lower results than the SH-EX, OVX-RA, SH-EX-RA, and OVX-EX-RA groups, and SH-EX-RA and OVX-EX-RA groups showed values higher than SH-RA, SH, and OVX-EX groups. The SH-EX-RA and OVX-EX-RA groups had decreased immunostaining for tartrate-resistant acid phosphatase and receptor activator of nuclear factor kappa-B ligand and increased osteoprotegerin immunostaining. In this experimental model, it was concluded that the physical training associated with use of risedronate exerted positive effects on biomechanical and microstructural properties in bones of ovariectomized rats. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: The study presents an old icon painted in egg tempera on lime wood, with a poor conservation condition and clogged dirt deposits. The icon is attributed to an anonymous painter of XIXth century and to the neo-classical style of painting. The painting layer was done with only a hand full of pigments, earth colors that were often used in painting the icons from XVIIth to XIXth century in Eastern Europe, that have Byzantine influences. Taking into consideration the nature and the structure of the materials from the upper layers of the painting, affected by deposits of dirt over time, a series of cleaning recipes were studied, using the so called cleaning tests with compatible mixtures of different juices and infusion from indigenes plants, that were freshly done and odorless. A low alkaline 95% ethyl alcohol solution, combined with a few drops of ammoniac 25%, was used as a reference system, due to its compatibility with the greasy deposits found on the polychrome layer and on the wood. The cleaning capacity of the new systems used, in comparison with the standard solution, was analyzed through modern analytical methods of evaluating the degree of cleaning, more exactly by means of visible and UV reflectography, CIE L*a*b* colorimetry by reflection assisted by SEM-EDX and IR spectroscopy. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT Background : There is a discrepancy between the interest in joint-reconstructions and the current knowledge about the healing-processes involved. Major reconstructions are performed with osteosynthesized allografts and fresh allografts for cartilage. Objectives : The main question to be answered is: what do we know about metaphyseal and epiphyseal cancellous bone healing, contact healing of the subchondral bone and its influence on cartilage healing? Can we achieve healing of all four compartments in contact? Purpose : The purpose is to systematically investigate through animal testing the healing processes of metaphyseal and epiphyseal bone, including the subchondral bone and the healing of cartilage of press-fit-inserted grafts, considering nondemineralized high-resolution histology. Material and Methods : Primary cancellous-bone healing of osteosynthesized hemi-osteotomies was studied in 13 canine tibial heads, the contact healing was investigated in 7 dogs and 18 giant-rabbits comparing contact-healing of press-fit-inserted autologs cylindrical grafts with empty defects applying the wet-grinding diamond-technology. Bench-experiments on the epiphyseal bones of swine including pullout-tests of cylindrical grafts formed the basis for validation of that press-fit diamond technology. Results : Primary metaphyseal and epiphyseal contact healing, including hyaline cartilage, was found in all compartments of the meta-and epiphysis in the precisely performed experiments. The press-fit principle, which employs cylindrical grafts and diamond instrumentation featuring a difference of 15/100 mm between graft and recipient bed, achieved high loads between 73.48 and 178.95 N (mean value 118.16 and standard deviation 32.79) in the pullout tests. Conclusion : Autologous press-fit grafting with alignment of the bony baseplate using wet-grinding precision has attained promising histo-morphological results. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT Due to its very low hardness and atomic number, pure lithium cannot be prepared by conventional methods prior to scanning electron microscopy analysis. Here, we report on the characterization of pure lithium metallic sheets used as base electrodes in the lithium-ion battery technology using electron backscatter diffraction (EBSD) and x-ray microanalysis using energy dispersive spectroscopy (EDS) after the sheet surface was polished by broad argon ion milling (IM). No grinding and polishing were necessary to achieve the sufficiently damage free necessary for surface analysis. Based on EDS results the impurities could be characterized and EBSD revealed the microsctructure and microtexture of this material with accuracy. The beam damage and oxidation/hydration resulting from the intensive use of IM and the transfer of the sample into the microscope chamber was estimated to be 〈50 nm. Despite the fact that the IM process generates an increase of temperature at the specimen surface, it was assumed that the milling parameters were sufficient to minimize the heating effect on the surface temperature. However, a cryo-stage should be used if available during milling to guaranty a heating artefact free surface after the milling process. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT Biological reactions between biomaterials and surrounding tissues, analyzed by histology, may be important predictors of clinical healing pattern and selection of slide preparation techniques requires a careful consideration regarding sample properties. In this study, we compared histology of bone specimens prepared with or without decalcification and performed histological and histomorphometrical assessments. For the histological evaluation, one-wall intrabony defects were created around the mandibular molars of six adult dogs, filled with biphasic calcium phosphate, synthetic bone graft material/recombinant human bone morphogenic protein-2, and healing pattern was histologically evaluated at 4 and 12 weeks. New bone formation in 5 × 4 × 4 mm defects and the length of new cementum, connective tissue attachments around the teeth and number of osteoclasts were measured by histomorphometric analysis. After decalcification, new cementum was easily observed and was significantly increased at week 4. In nondecalcified samples, significantly increased connective tissue attachments were seen at week 12. After 12 weeks, the number of countable multinucleated osteoclasts was significantly increased by 62% in nondecalcified versus calcified tissue sections ( P = 0.030). Histomorphometric results may be significantly affected by histological preparation method and therefore, selecting the most appropriate histological preparation method is essential for reliable diagnosis and evaluation of bony samples in studies analyzing tissue regeneration. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT This article presents the experimental results of a research on six manuscripts (three of the XVIII century and three of XIX century) belonging collection of old religious books to the Moldovan Metropolitan Church of Romania. Non-invasive techniques (optical microscopy [OM], scanning electron microscopy/energy dispersive X-ray system, X-ray fluorescence analysis, shrinkage temperature, and Fourier transform infrared spectroscopy/attentuated total reflectance) provided information on the degree of degradation and identification of the leather bookbinding type. Moreover, visual assessment and OM revealed the extent of the surface degradation (wane, biological attack, change color, etc.). The degradation extent of the skin bindings was determined on the 12 samples. The insight on the mechanism of degradation was accomplished by analyzing the deterioration of collagen fibers in terms of shrinkage temperature and chemical modifications induced by oxidative and hydrolytic processes. Shrinkage temperature values were lower compared with the literature data for collagen, indicating that the leather bookbinding suffered intrinsic damage. Morphological analysis was accomplished by microscopy and allowed the identification of skin type and provided information about its processing technique. Mineral elements were identified for leather composition and contributed to the information regarding the origin and the extent of degradation of the leather bookbinding, of the studied manuscripts. The analyzed results were useful in determining the state of preservation and were able to provide an increased efficiency of further restoration. The correlation of the obtained data brought new contributions to the knowledge of the leather covers for the book technique in the XVIII and XIX centuries in monastic workshops of Eastern Europe. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT Vascular remodeling in the brain occurs as a plastic change following neural over-activity. The auditory midbrain (or inferior colliculus, IC) is an ideal place to study sound-induced vascular changes because it is the brain's most vascularized structure and it is tonotopically organized. However, its micro-vascular pattern remains poorly understood. Since the IC is a sphere-like structure, the histological assessment of vasculature could depend on the angle of sectioning. Here, we studied the effects of cutting the IC at different angles on microvascular assessment, specifically: micro-vascular density and the shape of microvascular lumen. Photomicrographs were taken from 5 µm toluidine blue-stained histological sections obtained at two angles of sectioning: (a) the conventional coronal sectioning, and (b) a novel “tangential” sectioning (tangential to the dorso-medial surface of the IC). Results showed that the tangential sections, in comparison with the coronal sections, yielded (a) a higher count of micro-vascular density and (b) a higher proportion of round-shaped micro-vascular lumens. This discrepancy in results between two cut angles is likely related to the spatial pattern of blood vessels supplying the IC. We propose that the tangential sectioning should be adopted as standard for the accurate study of microvasculature in the IC. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT The effect of different Ca contents on the microstructure and mechanical properties of Mg-5Al-1Bi-0.3Mn (AMB501) magnesium alloys was investigated by conventional melting and casting technique using different Ca contents (1.0, 2.0, and 3.0 wt %). Increasing the Ca content resulted in higher hardness and yield strength, but decreased elongation. The improved tensile properties of the AM50-1Bi- x Ca alloys were due to the changes in AMB501 alloy microstructure when the Ca content increased, as demonstrated by scanning electron microscope, energy dispersive spectrum, and X-ray diffractometer. The alloy microstructure indicated that the amount of β-Mg 17 Al 12 phase on grain boundaries decreased and the morphology of β-Mg 17 Al 12 phase on grain boundaries changed from quasicontinuous-net shape to dispersed particles. The Mg 17 Al 12 phase disappeared and a new secondary phase Al 2 Ca appeared after a 3.0 wt % Ca addition. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT The cysts of nine Chinese populations of parthenogenetic Artemia were studied by scanning electron microscope. In the 270 cysts examined, 15 different morphological patterns were recognized with most of them not recorded in previous studies and the “tubercled shell surface” being the most common pattern. Results also displayed high intrapopulation variability, with the maximum of 11 patterns (in 30 cysts) recorded from the Barkol population. No positive correlation between the diversity of cyst shell patterns and ploidy compositions was found. Principal components analysis suggests higher similarity among coastal populations than among inland populations, which may be attributed to the identity of physicochemical conditions among coastal salterns and dissimilarity among inland saline lakes. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT The results of microstructural characterization of mortars containing fly ash class C (High Calcium Fly Ash) from combustion of lignite are presented. The evaluation of the microstructure was performed using scanning electron microscope, optical, and confocal microscope. The tested beams were bent till the crack and microcracks opening, which were healed during the different curing time. The results showed that the replacement of cement with fly ash class C influenced the process of crack healing. The addition of HCFA, at both 30% and 60%, speeds up the self-healing process in cracks and particularly in micro-cracks. In the research, the completely filling up of the cracks by new phases has not been observed, only the beginning of such process has been noticed. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT We propose two-photon excitation-based light-sheet technique for nano-lithography. The system consists of 2 -configured cylindrical lens system with a common geometrical focus. Upon superposition, the phase-matched counter-propagating light-sheets result in the generation of identical and equi spaced nano-bump pattern. Study shows a feature size of as small as few tens of nanometers with a inter-bump distance of few hundred nanometers. This technique overcomes some of the limitations of existing nano-lithography techniques, thereby, may pave the way for mass-production of nano-structures. Potential applications can also be found in optical microscopy, plasmonics, and nano-electronics. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT This study evaluated the bond strength (BS) and the adhesive interface of four endodontic sealers to root canal dentine, before, and after immersion in phosphate buffered saline (PBS) to simulate an in vivo environment. Eighty roots were instrumented using ProTaper rotatory files, under irrigation with 17% EDTA and 1% NaOCl. Posteriorly were divided into four groups ( n = 20) according to the sealer used: Endofill, AH Plus, Sealapex, and MTA Fillapex. Each group was divided into two subgroups ( n = 10) and stored at 37°C immersed in water for 7 days and in PBS for 60 days. From each subgroup, 1 mm thick sections were obtained. One section of each region (coronal, middle, and apical) was submitted to the push-out test and failures were observed. Twelve sections of each subgroup (four from each region) were evaluated under SEM. Three-way ANOVA evaluation for BS showed significant differences between groups and regions ( P 〈 0.0001), but not between subgroups ( P 〉 0.05). AH Plus had significantly higher BS than the others sealers, regardless of the analyzed subgroup (Tukey's test, P 〈 0.5). The most common failures were adhesive to dentine and cohesive of the sealer. The SEM evaluation (Kruskal–Wallis, Mann–Whitney) showed homogeneous adhesive interface formed and sealer tags in all groups with significant statistical differences with AH Plus, regardless of PBS immersion. AH Plus was superior to the other sealers for both BS and quality of interface formation. Immersion in PBS did not interfere on BS or adhesive interface of the sealers tested. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT Purpose This study used five clinical assessments and Raman spectroscopy to investigate the age- and hyperglycemia-related properties of the murine retina over an eight-week experimental period. Method Acute hyperglycemia and chronic hyperglycemia were assessed with blood glucose (BG) and glycated hemoglobin (HbA1c) levels, respectively. Changes in the retinal thickness and neovascularization were evaluated with optical coherence tomography and fluorescein angiography (FAG). Histological changes in the retina were studied after periodic acid-Schiff (PAS) staining. Raman spectroscopy was used to examine the molecular structures and chemical compositions of the retinal tissues. Results The young hyperglycemic group had acute hyperglycemia with a BG level of 576 ± 22 mg/dL and HbA1c of 5.9 ± 0.6%, while the aged hyperglycemic group displayed chronic hyperglycemia with a BG level of 607 ± 28 mg/dL and HbA1c of 11.2 ± 1.5%. Aged hyperglycemic retinas showed an insignificant (5%) decrease in thickness and no presence of vascular leaky lesions with FAG. There was no histological evidence of the retinal neovascularization with PAS staining of these aged hyperglycemic retinas. In the aged group, Raman intensities assigned to the C–C symmetric breathing of the aromatic ring in phenylalanine (1003 cm −1 ), the NH 2 amide III α-helix deformation in the protein structure (1265 cm −1 ), and the C=O stretching vibration of amide I α-helix structure in collagen (1657 cm −1 ) were all decreased. Conclusion These decreased Raman intensities indicate elevated reactive oxygen species (ROS) and oxidative damage to proteins such as those involved in cell apoptosis. A decrease in these ROS-related Raman peaks indicates an aging effect on the retina. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT This study analyzed the quality of obturation and physical properties of MTA Fillapex and AH Plus sealer. A sample of 30 human maxillary central incisors were instrumented with Protaper until a F5 (50/05) file. Both sealers were mixed with Rhodamine-B dye to allow visualization on a confocal laser-scanning microscope (CLSM). Next, the canals were filled using the single cone technique. After setting, all samples were sectioned at 2, 4, and 6 mm from the apex. CLSM was used to analyze the gaps and sealer penetration into the dentinal tubules. All samples were scanned 10 µm below the dentin surface and images were recorded at 100× magnification using the fluorescent mode. Additionally, the solubility, flowability and setting time of the sealers were evaluated. All the measured quantities of the examined materials were evaluated for significant differences by means of statistical analysis. The CLSM analysis of the MTA Fillapex showed the highest percentage of gaps at all sections (P = 0.0001). Physical tests revealed adequate properties for both sealers except for a higher solubility of the MTA Fillapex (P = 0.0001). The MTA Fillapex presented flowability and intratubular penetration similar to the AH Plus. Nevertheless, the MTA Fillapex sealer presented a higher solubility and considerable quantity of gaps between the sealer/dentin interface in relation to the AH Plus sealer. Clinicians must take into consideration, the quality of endodontic sealers as it is essential in the outcome of the root canal filling. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT Macro and microdissection methods, conventional histology and immunohistochemical procedures were used to investigate the nasal cavity and turbinate complex in fetal and adult sheep, with special attention to the ethmoturbinates, the vestibular mucosa, and the septal mucosa posterior to the vomeronasal organ. The ectoturbinates, which are variable in number and size, emerge and develop later than the endoturbinates. The olfactory sensory epithelium is composed of basal cells, neurons, and sustentacular cells organized in strata, but numerous different types are distinguishable on the basis of their thickness and other properties; all variants are present on the more developed turbinates, endoturbinates II and III. Mature neurons and olfactory nerve bundles express olfactory marker protein. We found no structure with the characteristics that in mouse define the septal organ or the ganglion of Grüneberg. Our results thus suggest that in sheep olfactory sensory neurons are exclusively concentrated in the main olfactory epithelium and (to a lesser extent) in the vomeronasal organ. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT With the application of the elements of all major super-resolution techniques including stimulated emission depletion, structure illumination microscopy, and photo-activated localization microscopy, the incoherent crossed standing-wave microscopy achieves parallel super-resolution imaging. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT The Sarcophagidae are one of the most numerous groups of Diptera in the world, consisting of many species of forensic interest, whose immature stages are useful in the estimation of postmortem interval. The immature stages of some species of forensic importance still remain unknown or undescribed, like in the case of Sarcophaga ( Liopygia ) cultellata Pandellé, 1986, a species restricted to the Iberian Peninsula, south of France and north of Italy, which shares a ecological niche with species of the same subgenus, e.g., Sarcophaga ( Liopygia ) argyrostoma (Robineau-Desvoidy, 1830) and Sarcophaga ( Liopygia ) crassipalpis Macquart, 1839, making it necessary to lay the groundwork for a proper specific differentiation before it can be successfully applied in forensic practice. This study provides the first micromorphological description of all preimaginal stages of S . ( L .) cultellata using light microscopy and scanning electron microscopy (SEM), the results of which allow the morphology of the main features to be followed during the immature life cycle. We propose a combination of features for distinguishing Liopygia from other sarcophagid subgenera, based on the current level of morphological knowledge of immature stages. S . ( L .) cultellata can be differentiated from S . ( L .) argyrostoma and S . ( L .) crassipalpis in every immature stage by both light microscopy and SEM. The presence of tegumental warts and a fan-shaped anterior spiracle with a single row of 15–18 respiratory papillae allow distinguishing the third instar larvae of S . ( L .) cultellata from other Sarcophaga species described hitherto by SEM. Identification keys based on light microscopy observations are provided, covering all the immature stages of Liopygia subgenus occurring in the Iberian Peninsula. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT The Caribbean bivalves Codakia orbicularis (Linné, 1758) and C. orbiculata (Montagu, 1808) live in seagrass beds of Thalassia testudinum and harbor intracellular sulfur-oxidizing gamma-proteobacteria. These bacterial symbionts fix CO 2 via the Calvin Benson cycle and provide organic compounds to the bivalve. During experimentally induced starvation, no reduced sulfur compounds and no organic particle food are available; the symbionts could be considered as the sole nutrient source of the host bivalve. A previous study has shown that the intracellular bacterial population decreased considerably during starvation and that bacterial endosymbionts were not released by the bivalves. In this study, the activity of two lysosomal marker enzymes (acid phosphatase and arylsulfatase) was detected using cytochemical experiments coupled with energy-dispersive X-ray transmission electron microscopy during sulfide and organic particle starvation. The degradation of bacterial endosymbionts began after 2 weeks of starvation in C. orbiculata and after 3 weeks in C. orbicularis . Degradation processes seem to be continuous over several months and could be responsible for the disappearance of the bacterial endosymbionts within the gills during starvation. These data suggest that the host use symbionts as a nutrient source to survive a hunger crisis. The carbon transfer from the symbionts to the host could be flexible and could consist in transfer of organic matter, “milking,” under normal feeding conditions and digestion of the symbionts under starved conditions. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT In this study, we aimed to describe the morphological characteristics of the lingual papillae in two species of Otariidae family by stereomicroscopy and scanning electron microscopy. We used tongues of two South American Otariidae species. The tongues were elongated and terminated in bifid apex and there was no median sulcus on the dorsal lingual surface. The most numerous type of lingual papilla was filiform in the South American fur seal (SASL) and entire dorsal lingual surface was covered by these filiform papillae but the dorsal surface of the tongue of the South American sea lion was covered by numerous polygonal projections, which were different in size. Fungiform papillae were detected in only SASL and they randomly distributed on the lingual apex and body, and some fungiform papillae were collected into twosome or threesome groups on the posterior part of the lingual body. Circumvallate papilla was found in the center of the lingual radix of South American sea lion. Thread-like conical papillae were common for both species and they located on the lingual radix. We determined that lingual surface morphology was completely different in each species, although they were members of the same family, Otariidae. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT Accurately accessing changes in the intracellular volumes (or numbers) of peroxisomes within a cell can be a lengthy task, because unbiased estimations can be made only by studies conducted under transmission electron microscopy. Yet, such information is often required, namely for correlations with functional data. The optimization and applicability of a fast and new technical proceeding based on catalase immunofluorescence was implemented herein by using primary hepatocytes from brown trout ( Salmo trutta f. fario ), exposed during 96 h to two distinct treatments (0.1% ethanol and 50 µM of 17α-ethynylestradiol). The time and cost efficiency, together with the results obtained by stereological analyses, specifically directed to the volume densities of peroxisomes, and additionally of the nucleus in relation to the hepatocyte, were compared with the well-established 3,3'-diaminobenzidine cytochemistry for electron microscopy. With the immuno technique it was possible to correctly distinguish punctate peroxisomal profiles, allowing the selection of the marked organelles for quantification. By both methodologies, a significant reduction in the volume density of the peroxisome within the hepatocyte was obtained after an estrogenic input. The most interesting point here was that the volume density ratios were quite correlated between both techniques. Overall, the immunofluorescence protocol for catalase was evidently faster, cheaper and provided reliable quantitative data that discriminated in the same way the compared groups. After this validation study, we recommend the use of catalase immunofluorescence as the first option for rapid screening of changes of the amount of hepatocytic peroxisomes, using their volume density as an indicator. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT Decalcification of mineralized tissues is an essential step during tissue processing in the routine histopathology. The time required for complete decalcification, and the effect of decalcifier on cellular and tissue morphology are important parameters which influence the selection of decalcifying agents. In this study, we compared a decalcifying solution (ETDA) composed of both acid and chelating agents to a classical and well-known decalcifying agent (EDTA). To this purpose, the optic density of bone radiographs, residual calcium analysis, bone sample weight, and histological and immunohistochemical analysis were performed. Our data suggest that, similarly to EDTA, the ETDA solution completely removes the calcium ions from the samples enabling easy sectioning. However, unlike the EDTA, this agent takes much less time. Furthermore, both agents showed comparable decalcification efficacy, and similarly, they did not produce cellular, tissue or antigenicity impairments. Therefore, ETDA may be a suitable option when it is necessary an association between a rapid and complete removal of calcium minerals, and a suitable preservation of structure and antigenicity of tissues. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT The organization of the egg capsule of the euholognathan stonefly, which represents the family Taeniopterygidae (Nemuroidea) was investigated using light and electron microscopy techniques. The presence of a complex, multilayered egg capsule, composed of a vitelline envelope, multilayered chorion, and extrachorion is described. The morphology of the eggshell of Brachyptera risi was compared with that of euholognthan and systellognathan egg coverings and the ground plan of the egg capsule in Plecoptera was discussed. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT There are no studies on stem cells (SCs) and development and differentiation (DD) of the human adrenal glands. The SCs in DD of the adrenal glands were herein investigated histochemically and immunohistochemically in 18 human embryonic adrenal glands at gestational week (GW) 7–40. At 7 GW, the adrenal glands were present, and at 7 GW, numerous embryonic SCs (ESCs) are seen to create the adrenal cortex. The ESCs were composed exclusively of small cells with hyperchromatic nuclei without nucleoli. The ESCs were positive for neural cell adhesion molecule, KIT, neuron-specific enolase, platelet-derived growth factor receptor-α, synaptophysin, and MET. They were negative for other SC antigens, including chromogranin, ErbB2, and bcl-2. They were also negative for lineage antigens, including cytokeratin (CK)7, CK8, CK18, and CK19, carcinoembryonic antigen, carbohydrate antigen 19-9, epithelial membrane antigen, HepPar1, mucin core apoprotein (MUC)1, MUC2, MUC5AC, and MUC6, and cluster differentiation (CD)3, CD45, CD20, CD34, and CD31. The Ki-67 labeling index (LI) was high (Ki-67 LI = around 20%). α-Fetoprotein was positive in the ESCs and adrenal cells. The ESC was first seen in the periphery of the adrenal cortex at 7–10 GW. The ESC migrates into the inner part of the adrenal cortex. Huge islands of ESC were present near the adrenal, and they appeared to provide the ESC of the adrenal. At 16 GW, adrenal medulla appeared, and the adrenal ESCs were present in the periphery or the cortex, in the cortical parenchyma, corticomedullary junctions, and in the medulla. The adrenal essential architecture was established around 20 GW; however, there were still ESCs. At term, there are a few ESCs. These data suggest that the adrenal glands were created by ESCs. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT A mesiodens is the most common type of supernumerary tooth present in conjunction to normal dentition. A mesiodens may commonly occur in the central region of the upper or lower jaw. A mesiodens is different from normal teeth in terms of structure and shape. The aim of this study is to evaluate the micro- and nanoscale structural properties of mesiodens dentin by combined small- and wide-angle X-ray scattering (SAXS/WAXS) and Fourier transform infrared (FTIR) spectroscopy. Five freshly extracted, noncarious mesiodens and five normal dentin disks prepared from human incisor teeth were compared. Using FTIR, the phosphate-to-amide I, carbonate-to-phosphate, and carbonate-to-amide I band area ratios and the crystallinity index were quantified. SAXS/WAXS were used to study the nanostructure of mesiodens. An increase in the mineral content in the mesiodens dentin with respect to the normal group was found. Crystallinity was also significantly increased and the protein content decreased in the mesiodens dentin compared with that of normal dentin. SAXS/WAXS results revealed that mesiodens dentin has a more calcified tissue. Further, SAXS analysis revealed a nonuniform distribution of dentin fibrils in mesiodens. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT “Snow lotus” is a famous Chinese Materia Medica derived from species of the genus Saussurea (Compositae). To differentiate three representative easily-confused snow lotus herbs, namely, Saussurea involucrata (Kar. et Kir.) Sch.-Bip, Saussurea laniceps Hand.-Mazz., and Saussurea medusa Maxim., macroscopic features of the three herbs were systemically observed, and microscopic features were compared by using ordinary light microscopy, polarized light microscopy and scanning electron microscopy (SEM). The results indicate that, as for macroscopic identification, capitula situation and arrangement, and as for microscopic identification, pollen grains, nonglandular hairs, glandular hairs, and cells of inner surface of the microdiodange can be used to authenticate the three snow lotus herbs. Comprehensive table comparing the characteristics were presented in this study. SEM has been found to provide a number of unique characteristics of pollen grains. Based on the observation of pollen grains, evolution sequence of the three species was speculated. The present method was proven to be efficient, convenient, simple, and reliable, which was successfully applied to the authentication of three snow lotus herbs. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT Calcium hydroxide [Ca(OH) 2 ] residues in root canals may compromise sealing of filling and endodontic treatment success. The aim of this study was to compare the efficacy of using rotary instrument associated with EndoActivator, EndoVac, passive ultrasonic irrigation (PUI), and conventional needle irrigation (CNI), in Ca(OH) 2 removal from root canal, by means of scanning electron microscopy (SEM) images. Sixty-six human canines were prepared with the Protaper system up to F5 and filled with Ca(OH) 2 . After 7 days, Ca(OH) 2 was removed with rotary instrument F5 associated with the irrigation techniques used in each group ( n = 15): GI (CNI), GII (EndoVac), GIII (EndoActivator) and GIV (PUI). In all groups 15 mL of 2.5% NaOCl and 3 mL of 17% EDTA were used for Ca(OH) 2 removal. The Ca(OH) 2 residues was evaluated by SEM in the middle and apical third using a system of scores. The results were analyzed by the Kruskal-Wallis and Dunn tests ( α = 0.05). None of the techniques completely removed the Ca(OH) 2 from root canals. There was no difference between EndoActivator, EndoVac and PUI ( P 〉 0.05), but the three techniques removed more Ca(OH) 2 than the CNI ( P 〈 0,05), in the middle and apical thirds of the root canal. It was concluded that the rotary instrument combined with EndoActivator, EndoVac, and PUI was shown to be more efficient than the rotary instrument combined with the CNI in removing Ca(OH) 2 from the root canal. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT Introduction Hormonal fluctuations may influence fibrin structure. During the menstrual cycle, plasma fibrinogen levels change, mainly due to the variations of estrogen. Throughout the menstrual cycle estrogen levels peak twice, first during the mid-follicular phase and then a lower second peak during the luteal phase. Materials and methods In order to investigate the possible changes in the fibrin network throughout the menstrual cycle, the fibrin network ultrastructure of six healthy female participants were studied at different intervals in the menstrual cycle where differences in estrogen levels are prevalent. Blood plasma smears were prepared for scanning and transmission electron microscopy analysis. Results The external and internal structure of the fibrin fibers showed different morphologies throughout the menstrual cycle. The fibrin fibers were smooth during days 1–5. However, during days 12–14 of the menstrual cycle the fibrin fiber morphology started to change, becoming less smooth. During the luteal phase of the cycle (days 20–25), the network appears sticky, where the minor, thin fibers are more prominent between the thick fibers when compared to the menstrual phase. Conclusion The two estrogen peaks of the menstrual cycle coincide with the changes seen in the current qualitative research, where the fibrin morphology changes during the same time as the estrogen peaks occur. Purified fibrinogen confirmed that it is indeed estrogen that causes the altered fibrin network morphology. This research is the first to show ultrastructural changes in fibrin fiber morphology resulting from estrogen changes during the menstrual cycle. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT The plantaris longus tendon (PLT) in bullfrog develops a fibrocartilage-like tissue in the area that is functionally subject to compressive forces. The aim of this study was to analyze the modifications of the pressure-bearing region in bullfrog PLT with different ages (7, 180, and 1,080 days after the end of metamorphosis) using histomorphometric, ultrastructural and biochemical methods. Weak basophilia and cells with a fibroblastic phenotype were observed in the compression region at 7 days of age. On the other hand, a large area of intense tissue basophilia associated with a chondroblast-like cell population was noted at the other ages. Collagen fibers exhibited a three-dimensional network-like arrangement at all ages. The number of connective tissue cells increased between 7 and 180 days of age and was reduced in older animals. The 180-day-old animals presented a well-developed pericellular matrix rich in proteoglycans. The mean diameter of collagen fibrils increased from 7 to 180 days and was the same at 1,080 days. Glycosaminoglycan content was higher in 7-day-old animals. A higher amount of hydroxyproline was observed at 180 and 1,080 days. The swelling test showed a significant increase of wet weight in 7-day-old animals. In conclusion, the alterations that occur in the pressure-bearing of bullfrog PLT are the result of physiological alterations of the animal with the maturation and aging. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT Dynamic changes in the spatial distribution of chloroplasts are essential for optimizing photosynthetic capacity under changing light conditions. Light-induced movement of chloroplasts has been widely investigated, but most studies were conducted on isolated tissues or protoplasts. In this study, a two-photon microscopy (TPM) system was adapted to monitor the intracellular 3-dimensional (3D) movements of chloroplasts in intact leaves of plants during dark to light transitions. The TPM imaging was based on autofluorescence of chlorophyll generated by a femto-second Ti:Sapphire laser. All chloroplasts did not exhibit the same motion in response to irradiation variation. In the sub-epidermal mesophyll cells, chloroplasts generally moved away from the surface following blue light treatment, however many chloroplasts did not show any movement. Such spatial heterogeneity in chloroplast motility underlines the importance of monitoring intracellular orientation and movement of individual chloroplasts across intact leaves. Our investigation shows that the 3D imaging of chloroplasts using TPM can help to understand the changes in local photosynthetic capacity in intact leaves under changing environmental conditions. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT The aim of this study was to evaluate the effectiveness of 17% ethylene-diamine-tetra-acetic acid (EDTA) used alone or associated with 2% chlorhexidine gel (CHX) on intracanal medications (ICM) removal. Sixty single-rooted human teeth with fully formed apex were selected. The cervical and middle thirds of each canal were prepared with Gates Glidden drills and rotary files. The apical third was shaped with hand files. The specimens were randomly divided into two groups depending on the ICM used after instrumentation: calcium hydroxide Ca(OH) 2 +CHX or Ca(OH) 2 +sterile saline (SS). After seven days, each group was divided into subgroups according to the protocol used for ICM removal: instrumentation and irrigation either with EDTA, CHX+EDTA, or SS (control groups). All specimens were sectioned and processed for observation of the apical thirds by using scanning electron microscopy. Two calibrated evaluators attributed scores to each specimen. The differences between the protocols for ICM removal were analyzed with Kruskal-Wallis and Mann-Whitney U tests. Friedman and Wilcoxon signed rank tests were used for comparison between the score of debris obtained in each root canal third. Remains of Ca(OH) 2 were found in all specimens independently of the protocol and ICM used ( P  〉 0.05). Seventeen percent EDTA showed the best results in removing ICM when used alone ( P  〈 0.05), particularly in those associated with CHX. It was concluded that the chelating agent 17% EDTA significantly improved the removal of ICM when used alone. Furthermore, the type of the vehicle associated with Ca(OH) 2 also plays a role in the ICM removal. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT This article reports the results of a pilot project using Synchrotron Radiation µCT (computer-aided tomography) to examine the distribution of silica within phytoliths from rice husks. Experiments indicate that computed tomography can be used to show how silica accumulates and is distributed in a distinctive zigzag pattern of long epidermal cells that are characteristic of phytoliths from rice husks. This method will help us to understand why the dry ashing method produced much more zigzag pattern of long cells phytoliths from rice husk than did the acid extraction method. Besides, the zigzag morphological pattern exhibited by long epidermal cells is characteristic of this species which makes it useful in the identification of rice husks from archaeological contexts and indicating heating process. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT The purpose of this study was to elucidate the micro CT findings of tumoral vessels supplied by portal circulation during establishment of hepatic metastasis of colorectal cancer in a mouse model. Hepatic metastases were induced in 15 BALB/c mice through the injection of murine colonic adenocarcinoma tumor cells into the mesenteric vein. Micro-CT imaging of the tumoral vessels was obtained to clarify the microvascular architecture. We evaluated the sinusoidal structure, diameter of the tumoral vessels (DTV) and blood vessel density (BVD) according to tumor sizes ranging from 201 to 3,000 µm in diameter. A total of 116 tumors were observed on day 15 after cell injection. The mean diameter of a normal hepatic sinusoid was 11.7 ± 2.0 µm on micro CT. The DTV supplied by the portal vein of tumors measuring 1,001–1,500 µm in diameter was greater than that of tumors 200–1,000 µm in diameter. The mean BVD from the portal vein gradually decrease according to size of tumor from 201 to 3,000 µm in diameter ( r 2  = −0.584, P  〈 0.01). The characteristics of tumoral vessels supplied by portal circulation during establishment of hepatic colorectal metastases were well visualized with micro-CT imaging. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT Several scientific reports have shown the effects of Er:YAG laser irradiation on enamel morphology. However, there is lack of information regarding the morphological alterations produced by the acid attack on the irradiated surfaces. The aim of this study was to evaluate the morphological changes produced by acid dissolution in Er:YAG laser irradiated dental enamel. Forty-eight enamel samples were divided into four groups ( n  = 12). GI (control); Groups II, III, and IV were irradiated with Er:YAG at 100 mJ (12.7 J/cm 2 ), 200 mJ (25.5 J/cm 2 ), and 300 mJ (38.2 J/cm 2 ), respectively, at 10 Hz without water irrigation. Enamel morphology was evaluated before-irradiation, after-irradiation, and after-acid dissolution, by scanning electron microscopy (SEM). Sample coating was avoided and SEM analysis was performed in a low-vacuum mode. To facilitate the location of the assessment area, a reference point was marked. Morphological changes produced by acid dissolution of irradiated enamel were observed, specifically on laser-induced undesired effects. These morphological changes were from mild to severe, depending on the presence of after-irradiation undesired effects. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT The concerns over health and workplace hazards of formalin fixative, joined to its cross-linking of molecular groups that results in suboptimal immunohistochemistry, led us to search for an innovative safe fixative. Shellac is a natural material which is used as a preservative in foods and pharmaceutical industries. This study was undertaken to evaluate the fixation adequacy and staining quality of histopathological specimens fixed in the “shellac alcoholic solution” (SAS), and also to determine the validity of immunohistochemical staining of SAS-fixed material in comparison to those fixed in formalin. Fresh samples from 26 cases from various human tissues were collected at the frozen section room of King Abdulaziz University Hospital, and fixed in SAS fixative or in neutral buffered formaldehyde (NBF) for 12, 18, 24, and 48 h, and processed for paraffin sectioning. Deparaffinized sections were stained with hematoxylin and eosin (H&E) and immunostained for different antigens. The tissues fixed in SAS for 〉18 h showed best staining quality of H&E comparable to NBF-fixed tissues. Comparison of the immunohistochemical staining of different tissues yielded nearly equivalent readings with good positive nuclear staining quality in both fixatives. These findings support the fixation and preservation adequacy of SAS. Furthermore, it was concluded that the good staining quality obtained with SAS-fixed tissues, which was more or less comparable with the quality obtained with the formalin fixed tissues, supports the validity of this new solution as a good innovative fixative. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT Aquatic mammals underwent morphological and physiological adaptations due to the transition from terrestrial to aquatic environment. One of the morphological changes regards their vision since cetaceans' eyes are able to withstand mechanical, chemical, osmotic, and optical water conditions. Due to insufficient information about these animals, especially regarding their sense organs, this study aimed to describe the morphology of the Humpback whale ( Megaptera novaeangliae) eyeball. Three newborn females, stranded dead on the coast of Sergipe and Bahia, Brazil, were used. Samples were fixed in a 10% formalin solution, dissected, photographed, collected, and evaluated through light and electron microscopy techniques. The Humpback whale sclera was thick and had an irregular surface with mechanoreceptors in its lamina propria. Lens was dense, transparent, and ellipsoidal, consisting of three layers, and the vascularized choroid contains melanocytes, mechanoreceptors, and a fibrous tapetum lucidum. The Humpback whale eyeball is similar to other cetaceans and suggests an adaptation to diving and migration, contributing to the perception of differences in temperature, pressure, and lighting. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT The aim of this study was to evaluate the erosion-inhibiting effect of two toothpastes on the development of erosion-like lesions, by a confocal laser scanning microscope (CLSM). Forty human enamel blocks were divided into five groups ( n  = 8), in accordance to evaluate the GC MI Paste Plus and Oral B with stannous fluoride, applied as slurries and associated with toothbrush. Specimens were submitted to an erosion challenge from citric acid (0.5%, pH = 2.8), for 5 min, six times a day, alternating in artificial saliva immersions. Reference group was not exposed to treatment. Part of specimens (Groups 02 and 03) was exposed twice daily just to slurries, for 2 min, therefore specimens from Groups 04 and 05 were also abraded, for 30 s. The enamel surfaces were morphological characterized using CLSM images, with mineral loss being measured using the resulting 3D images referenced to an un-challenged portion of the sample. Step values were compared using the one-way ANOVA test. CLSM was shown to be a viable, noncontact, and simple technique to characterize eroded surfaces. The statistical difference in the step size was significant between the groups ( P  = 0.001) and using multiple comparisons a statistically significant protective effect of toothpastes was shown when these were applied as slurries. Although groups submitted to tooth brush showed mineral loss similar to reference control group, due to the damages of abrasion associated. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT Over the last decade, we have witnessed an outburst of many different optical techniques aimed at breaking the diffraction limit of light, providing super-resolution imaging on intact fixed cells. In parallel, single-molecule detection by means of fluorescence has become a common tool to investigate biological interactions at the molecular level both in vitro and in living cells. Despite these advances, visualization of dynamic events at relevant physiological concentrations at the nanometer scale remains challenging. In this review, we focus on recent advancements in the field of nanophotonics toward nanoimaging and single-molecule detection at ultrahigh sample concentrations. These approaches rely on the use of metal nanostructures known as optical antennas to localize and manipulate optical fields at the nanometer scale. We highlight examples on how different optical antenna geometries are being implemented for nanoscale imaging of cell membrane components. We also discuss different implementations of self-standing and two-dimensional antenna arrays for studying nanoscale dynamics in living cell membranes as well as detection of individual biomolecular interactions in the µM range for sensing applications. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT This study assessed the influence of different endodontic chemical substances on the adhesion of the Epiphany SE/Resilon system (with and without resinous solvent) to radicular dentin walls, using the push-out test and scanning electron microscopy (SEM). Forty-eight root canals of human canines were prepared biomechanically with ProTaper rotary files (crown-down technique) and the radicular dentin was treated with either 17% EDTA, 2% chlorhexidine gel (CHX) or 2.5% NaOCl (control). The root canals were filled with Resilon cones and Epiphany SE sealer with and without resinous solvent. Six groups of eight canals each had their roots sectioned transversally to obtain 1-mm thick slices. Data were subjected to statistical analysis by ANOVA and Tukey's tests. The specimens treated with 17% EDTA (1.59 ± 0.91) presented higher bond strength (P 〈 0.05) than those treated with 2.5% NaOCl (0.93 ± 0.27) and 2% CHX (0.92 ± 0.22). Significantly higher bond strength (P 〈 0.05) was observed when the Epiphany SE was prepared with (1.37 ± 0.78) than without (0.92 ± 0.33) solvent. Adhesive failures were predominant in all groups. SEM analysis showed greater homogeneity of the filling mass when the solvent was added to the sealer. Treatment of root canal walls with 17% EDTA, and addition of a resinous solvent to Epiphany SE produced the highest adhesion to radicular dentin. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT The mucosal pellicle is defined as the protein film adsorbed onto oral mucosa. This study aimed at characterizing the ultrastructure of human epithelial buccal cells and localizing salivary mucins MUC5B, a major constituent of the mucosal pellicle. Cells were sampled from the buccal surface and prepared for Transmission Electron Microscopy using high-pressure freezing/cryosubstitution followed by immunogold labelling of MUC5B. Morphologically, cells were visualized as typical cells of the superficial layer of a squamous nonkeratinized epithelium with a partly degraded plasma membrane. The outer surface of the plasma membrane was lined with a biological material of medium electron density. MUC5B were detected in the extracellular space, and particularly in the vicinity of the plasma membrane, sometimes onto fibrils protruding from the membrane. This area was, therefore, considered as constituting the mucosal pellicle, which appeared as a mixed film of both salivary and epithelial components. The distribution of gold particles suggested that the surface of the pellicle was not uniform, and that the film thickness could reach up to 100 nm. This work showed the feasibility of visualizing and characterizing the mucosal pellicle directly on human epithelial buccal cells sampled in a noninvasive manner. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT Coherent anti-Stokes Raman scattering (CARS) microscopy is a powerful tool for chemical analysis at a subcellular level, frequently used for imaging lipid dynamics in living cells. We report a high-power picosecond fiber-based laser and its application for optical parametric oscillator (OPO) pumping and CARS microscopy. This fiber-based laser has been carefully characterized. It produces 5 ps pulses with 0.8 nm spectral width at a 1,030 nm wavelength with more than 10 W of average power at 80 MHz repetition rate; these spectral and temporal properties can be slightly modified. We then study the influence of these modifications on the spectral and temporal properties of the OPO. We find that the OPO system generates a weakly spectrally chirped signal beam constituted of 3 ps pulses with 0.4 nm spectral width tunable from 790 to 930 nm optimal for CARS imaging. The frequency doubling unconverted part is composed of 7–8 ps pulses with 0.75 nm spectral width compatible with CARS imaging. We also study the influence of the fiber laser properties on the CARS signal generated by distilled water. In agreement with theory, we find that shorter temporal pulses allow higher peak powers and thus higher CARS signal, if the spectral widths are less than 10 cm −1 . We demonstrate that this source is suitable for performing CARS imaging of living cells during several hours without photodamages. We finally demonstrate CARS imaging on more complex aquatic organisms called copepods (micro-crustaceans), on which we distinguish morphological details and lipid reserves. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT The aim of the study was to assess the penetrability of two endodontic sealers (AH Plus and MTA Fillapex) into dentinal tubules, submitted to endodontic treatment and subsequently to endodontic retreatment. Thirty ex vivo incisors were prepared using ProTaper rotary system up to F3 instrument and divided in three groups according to the endodontic sealer used for root canal filling: AH Plus (AHP), MTA Fillapex (MTAF), and control group (CG) without using EDTA previously to the root canal filling. Rhodamine B dye (red) was incorporated to the sealers in order to provide the fluorescence which will enable confocal laser scanning microscopy (CLSM) assessment. All specimens were filled with gutta-percha cones using the lateral compaction technique. The specimens were submitted to endodontic retreatment using ProTaper Retreatment system, re-prepared up to F5 instruments and filled with gutta-percha cones and the same sealer used during endodontic retreatment. Fluorescein dye (green) was incorporated to the sealer in order to distinguish from the first filling. The roots were sectioned 2 mm from the apex and assessed by CLSM. No difference was found between the two experimental groups ( P 〉 0.05). On the other hand, in the control group the sealers were not capable to penetrate into dentinal tubules after endodontic treatment ( P 〉 0.05). In retreatment cases, none of the sealers were able to penetrate into dentin tubules. It can be concluded that sealer penetrability is high during endodontic treatment. However, MTA Fillapex and AH Plus do not penetrate into dentinal tubules after endodontic retreatment. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT The prostate is a mammalian gland that shows a complex process of organogenesis. Here, a morphological study to characterize the organogenesis of the ventral prostate lobe in male gerbils was conducted. The urogenital sinus (UGS) was dissected out and processed for paraffin embedding. Histological sections were subjected to cytochemical, immunofluorescence, immunohistochemical, and three-dimensional reconstruction techniques. We found that the first ventral buds emerged from the ventral urethral epithelium between the days 20 and 21 of prenatal life, reaching the ventral mesenchymal pad and initiating the branching process on the first day of postnatal life. The buds presented a V-shaped elongation, suggesting that the smooth muscle layer (SML) plays an important role during budding events. Indeed, whereas the androgen receptor (AR) was preferentially found in the UGS mesenchyme (UGM), estrogen receptor alpha (ERα) was localized in both the UGM and in the emerging buds. This study characterized the morphological aspects of the budding process in a different rodent from rat and mice, serving as a new model for future studies on developmental biology of the prostate. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT Super-resolution fluorescence microscopy is still a developing field. One of the limitations has been that standard labeling assays, which had been developed for conventional imaging, must be adjusted and optimized for each super-resolution method. These methods are more sensitive to noise, and require more intense labeling than conventional microscopy, which is not always trivial to achieve. Here, we describe the use of stimulation-emission depletion (STED) microscopy to locate messenger RNAs (mRNAs) in single neurons with high spatial precision. We address several technical difficulties we encountered in using fluorescent in situ hybridization (FISH) for STED imaging. We optimized the experimental protocol to detect mRNAs and proteins simultaneously, by performing FISH and immunostaining on the same samples. We tested our imaging approach in primary hippocampal neurons, studying the mRNAs of three important presynaptic proteins (synaptobrevin, synaptotagmin, and synaptophysin). Our approach allowed us to relate changes in mRNA levels and localization to neuronal physiology, under different activity regimes and also during neuronal development. We conclude that FISH can be performed efficiently using super-resolution techniques. This should contribute significantly to the clarification of the molecular mechanisms that govern mRNA distribution and dynamics within cells. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT A marker's dark triplet state is of great importance in fluorescence microscopy: It serves as a means to switch off fluorescent markers and is thus the enabling element for several super-resolution methods. On the other hand, intersystem-crossing to the electronic dark triplet state strongly reduces the fluorescence yield in conventional fluorescence microscopy. The ability to determine the kinetic parameters of transitions into the triplet state is thus of great importance and because fluorescence correlation spectroscopy (FCS) can be applied without disturbing the system under study, it is one of the preferred methods to do so. However, conventional FCS observations of triplet dynamics suffer from bias due to the spatially inhomogeneous irradiance profile of the excitation laser. Herein, we present a novel method to correct this bias and verify it by analyzing both Monte Carlo simulated and experimental data of the organic dye Rhodamine 110 in aqueous solution for both continuous-wave and pulsed excitation. Importantly, our approach can be readily generalized to most other FCS experiments that determine intensity dependent kinetic parameters. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT Despite the great number of studies devoted to neural stem/progenitor cell biology, the ultrastructural characteristics of these cells in vitro have not been fully studied. To determine the fine structure of hippocampal neural progenitor cells (NPCs) in culture, mouse fetal hippocampi (E18) were extracted, dissected, and cells were expanded as adherent monolayer culture. Electron microscopy revealed that NPCs had an immature phenotype, with a high nuclear/cytoplasmic ratio, small and scant organelles, underdeveloped endoplasmic reticulum, and many free ribosomes and polysomes. Our results may contribute to a better understanding of the fine structure and physiology of hippocampal NPCs in vitro. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT Yuzhu (Polygonati Odorati Rhizoma), Kangdingyuzhu (Polygonati Prattii Rhizoma), and zhugenqiyuzhu (Disporopsis Fuscopictae Rhizoma) are of the same family, but of different genera. They have all often used in Chinese Materia Medica (CMM) as Polygonati Odorati Rhizoma in China market. Three species of CMM are confused. For better application, we need to identify these plants accurately. This study use pharmacognosy technique and GC-MS analysis, three species of CMM were authenticated. In macroscopic characteristics, the fruit of Polygonati Odorati Rhizoma is blue-black, while the other two are maroon and dark purple orderly. Nodes of Polygonati Odorati Rhizoma are upward and light uplift, about 1 cm spacing, while the other are not. As for microscopic characteristics, the cortex of Polygonati Odorati Rhizoma only occupies about 1/5 of the radius of the transverse section with inconspicuous endodermis, which is much smaller than others. The type of vascular bundles of Polygonati Odorati Rhizoma is closed collateral, but the other is amphivasal. Raphides of calcium oxalate are scattered, but Raphides of the other two are like brooms and neat rows. GC–MS analysis of essential oil could provide different characteristics to distinguish three species. Twenty-three compounds were identified from essential oil of Polygonati Odorati Rhizoma and the main components were n -hexadecanoic acid (49.45%), while n -hexadecanoic acid of the other two are 23.92% and 9.45%. The content of n -hexadecanoic is strongly different. This research was aimed to establish a method by pharmacognosy and GC–MS analysis to identify three CMM and for providing scientifical data to ensure accuracy of origin of three species. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT Purpose : Cervical cancer is caused by persistent infection with high-risk types of human papillomavirus (HPV). The aim of this study was to investigate the potential of Raman spectroscopy as a stand-alone analytical tool for clinical diagnosis of HPV infection using human cervical fluids. Method : A drop-coating deposition surface-enhanced Raman scattering (DCD-SERS) method was identified as the most effective method of biochemical analysis in cervical biofluids. Results : Using a 2-µL sample, the proposed DCD-SERS method yielded Raman spectra with high reproducibility, noise-independence, and uniformity. Additionally, the produced spectra were independent of the volume of fluid used and detection zone analyzed within the central and the ring zones. The optical detection of HPV infection in cervical fluids could be detected accurately in the central zone. Different Raman spectra were obtained according to HPV type. In particular, HPV-16 and HPV-18, which are the major risk factors for cervical cancer, showed a distinct spectral difference in cervical fluids, with a similar pattern for high Raman bands at 〉1,000 cm −1 but a different pattern for low Raman bands at 〈1,000 cm −1 . Therefore, all ranges of HPV-gated DCD-SERS could be used to detect the presence of HPV infection. Conclusions : Raman spectroscopy provides a good alternative method for early clinical diagnosis of HPV infection, and we are hopeful that this proposed method of HPV assessment will be approved for use in obstetrics and gynecology clinics. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT Dystrophin-deficient muscles have repeated cycles of necrosis and regeneration, being susceptible to injury induced by muscle contractions. Some studies have demonstrated that tendons are also affected in mdx mice, based especially on the changes in biomechanical properties arising from the respective linked muscles. However, most studies have focused only on alterations in the myotendinous junction. Thus, the purpose of this work was to study biochemical and morphological alterations in the Achilles tendons of 60-day-old mdx mice. Hydroxyproline quantification, showed higher collagen concentration in the mdx mice as compared with the control. No difference between the tendons of both groups was found in the noncollagenous proteins dosage, and in the amount of collagen type III detected in the western blotting analysis. The zymography for gelatinases detection showed higher amounts of metaloproteinase-2 (active isoform) and of metalloproteinase-9 (latent isoform) in the mdx mice. Measurements of birefringence, using polarization microscopy, showed higher molecular organization of the collagen fibers in the tendons of mdx mice in comparison to the control group, with presence of larger areas of crimp. Ponceau SS-stained tendon sections showed stronger staining of the extracellular matrix in the mdx groups. Toluidine blue-stained sections showed more intense basophilia in tendons of the control group. In morphometry, a higher number of inflammatory cells was detected in the epitendon of mdx group. In conclusion, the Achilles tendon of 60-day-old mdx mice presents higher collagen concentration and organization of the collagen fibers, enhanced metalloproteinase-2 activity, as well as prominent presence of inflammatory cells and lesser proteoglycans. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT Iodine imparts strong contrast to objects imaged with electrons and X-rays due to its high atomic number (53), and is widely used in liquid form as a microscopic stain and clinical contrast agent. We have developed a simple technique which exploits elemental iodine's sublimation-deposition state-change equilibrium to vapor stain specimens with iodine gas. Specimens are enclosed in a gas-tight container along with a small mass of solid I 2 . The bottle is left at ambient laboratory conditions while staining proceeds until empirically determined completion (typically days to weeks). We demonstrate the utility of iodine vapor staining by applying it to resin-embedded tissue blocks and whole locusts and imaging them with backscattered electron scanning electron microscopy (BSE SEM) or X-ray microtomography (XMT). Contrast is comparable to that achieved with liquid staining but without the consequent tissue shrinkage, stain pooling, or uneven coverage artefacts associated with immersing the specimen in iodine solutions. Unmineralized tissue histology can be read in BSE SEM images with good discrimination between tissue components. Organs within the locust head are readily distinguished in XMT images with particularly useful contrast in the chitin exoskeleton, muscle and nerves. Here, we have used iodine vapor staining for two imaging modalities in frequent use in our laboratories and on the specimen types with which we work. It is likely to be equally convenient for a wide range of specimens, and for other modalities which generate contrast from electron- and photon-sample interactions, such as transmission electron microscopy and light microscopy. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT The ability of bone repair scaffolds to form bone is traditionally evaluated using cell culture and animal experiments. Mouse calvarial organ culture maintains the natural cell-to-cell and cell-to-matrix relationships as well as the anatomical order, and this model has been used to study the biological behavior of intramembranous bones. The aim of this study was to evaluate the potential of mouse calvarial organ culture to be used as an in vitro model to study the bone regenerative ability of bone repair polymer scaffolds. Critical size defects (CSD) were created in the parietal bones. Electrospun poly(ε-caprolactone) scaffolds were placed into one group of defects. The remaining defects served as a control. The bones were cultured for 38 days and analyzed with μCT, phase-contrast microscopy, dissecting microscopy, scanning electron microscopy, and energy-dispersive X-ray analyses. This organ culture technique is easily available and could permit researchers to quickly establish a valuable database of candidate bone repair scaffolds. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT In this letter, a shape from focus (SFF) method is proposed that utilizes the guided image filtering to enhance the image focus volume efficiently. First, image focus volume is computed using a conventional focus measure. Then each layer of image focus volume is filtered using guided filtering. In this work, the all-in-focus image, which can be obtained from the initial focus volume, is used as guidance image. Finally, improved depth map is obtained from the filtered image focus volume by maximizing the focus measure along the optical axis. The proposed SFF method is efficient and provides better depth maps. The improved performance is highlighted by conducting several experiments using image sequences of simulated and real microscopic objects. The comparative analysis demonstrates the effectiveness of the proposed SFF method. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT The use of non-standard low-temperature conditions in environmental scanning electron microscopy might be promising for the observation of coniferous tissues in their native state. This study is aimed to analyse and evaluate the method based on the principle of low-temperature sample stabilization. We demonstrate that the upper mucous layer is sublimed and a microstructure of the sample surface can be observed with higher resolution at lower gas pressure conditions, thanks to a low-temperature method. An influence of the low-temperature method on sample stability was also studied. The results indicate that high-moisture conditions are not suitable for this method and often cause the collapse of samples. The potential improvement of stability to beam damage has been demonstrated by long-time observation at different operation parameters. We finally show high applicability of the low-temperature method on different types of conifers and Oxalis acetosella . Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT This review reports on the use of the atomic force microscopy in the investigation of the mechanical properties of cells. It is shown that the technique is able to deliver information about the cell surface properties (e.g., topography), the Young modulus, the viscosity, and the cell the relaxation times. Another aspect that this short review points out is the utilization of the atomic force microscope to investigate basic questions related to materials physics, biology, and medicine. The review is written in a chronological way to offer an overview of phenomenological facts and quantitative results to the reader. The final section discusses in detail the advantages and disadvantages of the Hertz and JKR models. A new implementation of the JKR model derived by Dufresne is presented. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT Our article presents the results of the analyses we performed by corroborating the Optical Microscopy, Scanning Electron Microscopy, Energy Dispersive Xray Analysis and micro Fourier Transformed InfraRed Analysis techniques to identify the archaeo-metallurgic casting and ornamentation procedure of a decorated disk-butted axe, which was discovered recently east of the Carpathian mountains, in the Moldavian Plateau. There are few known axes of that type found (A1, according to the usual typologies), as they are specific to the Middle Bronze Age period west of the Carpathians—the Wietenberg, Suciu de Sus, and Otomani-Füzesabony cultures. The experimental data on the item under study revealed the fact that after casting it in molds made from porous silicone-based stone, the object was coated with a thin layer, by immersing it in a lightly fusible tin alloy, whose main alloy component was copper and arsenic and iron as secondary components. After refining the shiny white layer, they applied a beautiful decoration pattern made by incision and engraving. This battle axe was an indication of higher status, such items usually being owned by community leaders. This important fact proves that the ancient metallurgic craftsmen were able to elaborate and manufacture various alloys from which they made beautiful objects and it also offers a new insight into the social and symbolic function of certain antique bronze items. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT Enlightened by the principle of scanning probe microscopy or atomic force microscope (AFM), we proposed a novel surface topography imaging system based on the scanning of a piezoelectric unimorph cantilever. The height of sample surface can be obtained by recording the cantilever's strain using an ultra-sensitive strain gauge and the Z-axis movement is realized by electric bending of the cantilever. This system can be operated in the way similar to the contact mode in AFM, with the practical height detection resolution better than 100 nm. Imaging of the inner surface of a steel tube and on a transparent wing of a honey bee were conducted and the obtained results showed that this proposed system is a very promising solution for in situ topography mapping. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT Background: The molecular mechanisms of ductal plate (DP) development and differentiation (DD) in human fetal livers (HFLs) are unclear. Materials and Methods: The author immunohistochemically investigated expressions of NCAM, KIT, KIT, PDGFRA, and neuroendocrine antigens in 32 HFLs. Results: The processes of human intrahepatic bile duct (IBD) DD could be categorized into four stages: DP, remodeling DP, remodeled DP, and mature IBD. NCAM was always expressed in DP and remodeling DP, but not in remodeled DP and mature IBD. The biliary elements were positive for cytokeratin (CK)7, 8, 18, and 19. The hepatoblasts were positive for CK8 and CD18, but negative for CK7 and CK19; however, periportal hepatoblasts showed biliary-type CKs (CK7 and CK19). NCAM was always expressed in DP and remodeling DP, but not in remodeled DP and mature IBD. KIT was occasionally (12/32 cases) expressed in DP and remodeling DP, but not in remodeled DP and mature IBD. NCAM expression was also seen in some hepatoblasts and hematopoietic cells and neurons. KIT was also expressed in some hepatoblasts, hematopoietic cells, and mast cells. MET and PDGFRA were strongly expressed in DP, remodeling DP, remodeled DP, and mature IBD. MET and PDGFRA were also strongly expressed in hepatoblasts and hematopoietic cells. MET and PDGFRA were not expressed in portal mesenchyme, portal veins, sinusoids, and hepatic veins. DP showed immunoreactive chromogranin, synaptophysin, neuron-specific enolase (NSE), and CD56. Expressions of chromogranin and CD56 were infrequently seen in remodeling DP. No expressions of these four neuroendocrine antigens were seen in remodeled DP and mature IBD. The nerve fibers were consistently positive for chromogranin, synaptophysin, NSE, and CD56 in the portal mesenchyme in the stages of remodeling DP, remodeled DP, and mature IBDs. Conclusions: The data suggest that NCAM, KIT/stem cell factor-signaling, NSE, hepatocyte growth factor/MET signaling, PDGFα/PDGFRA signaling, chromogranin, synaptophysin, and CD56 play important roles in DD of biliary cells of HFL. They also suggest that the DP cells having neuroendocrine molecules give rise to hepatic stem/progenitor cells. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT The extra-cellular matrix of fertilized eggs in the bufonid toads Bufo bufo and Bufotes balearicus was studied to clear the relationships between structural and molecular diversity. Histochemical (PAS, AB pH 2.5 and pH 1.0, Beta-elimination PAS) and lectin-histochemical (Con A, WGA, Succinyl-WGA, PNA, RCA-1, DBA, SBA, AAA, UEA-I, LTA) techniques were used and the observations were made under light and electron microscopy. Both species present a fertilization envelope (FE) and two jelly layers (J 1 and J 2 ). The fibers of J 2 are shared among the eggs of a clutch in a jelly ribbon. The FE of both species presents neutral glycoproteins, mostly N -linked. In B. bufo there are also residuals of mannose and/or glucose and N -acetylglucosamine. In the FE fibers run parallel to egg's surface or are in bundles or looser hanks with no clear orientation. The J 1 layer of both species presents sialosulfoglycoproteins, mostly O -linked, with lactosaminylated, galactosaminylated, glycosaminylated, and fucosylated residuals. A lower amount of galactosaminylated residuals is observed in B. balearicus in respect to B. bufo , whereas the opposite is seen in the amount of fucosylated residuals. The J 2 layer is similar in composition to J 1 but in B. balearicus there are no glucosaminylated residuals. J layers present fibers and granules that reduce towards J 2 . Several microorganisms, in particular blue algae, are observed in the J 2 layer of both species. In respect to other species, B. bufo and B. balearicus have a lower number of jelly layers, but a comparable number of glycan types. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT Backgrounds: The fetal development of extrahepatic bile ducts (EBD) is unkown. Materials and Methods: Development of EBD was examined by immunohistochemistry in 16 fetuses of 7–40 gestational week (GW). Gall bladder (GB) was not investigated. Results: At seven GW, a hepato-pancreatic bud (HPB) was seen near the hepatic hilus. At eight GW, embryonic EBD, GB and pacreas developed from HPB. Portal veins (PV) and hepatic arteries (HAs) were present in EBD at eight GW. Liver parenchyma was already present in seven GW. At eight GW, EBD at porta hepatis (PH) was already established; PH EBD was derived from ductal plate (DP). The distal and middle EBD gradually develeped and took shape of EBD at nine GW. In PH, cystic and hepatic ducts developed from DP at eight GW. EBD developed further, accompanying many nerve fibers (NF) at PH and distal and middle EBD. Apparent PV and HA were seen around 12 GW. Around 20 GW, HA and capillaries proliferated, giving rise to peribiliary capillary plexus (PCP) in all parts of EBD. EBD grew gradually further, and around 30 GW extrahepatic peribiliary glands (EPG) emerged from EBD but not from cystic duct. Around 36 GW, exocrine pancreatic acinar cells emerged from remodeled DP at PH. At term (40 GW), EBD was established but was as yet immature. Numerous NF were present around EBD. Histochemically, EBD epithelium had no mucins at 7–12 GW but contained neutral and acidic mucins at 23–40 GW. EPG had abundant neutral and acidic mucins. Immunohistochemically, alpha-fetoprotein (AFP) was consistently positive in the epithelial and mesenychyma. The NF and muscles of HPB present at seven GW were positive for neural cell adhesion molecule (NCAM), neuron-specific enolase (NSE), platelet-derived growth factor receptor-α (PDGFRA), and KIT, but they disappeared in nine GW. Expressions of cytokeratin (CK) seven and CK19 in EBD and EPG were slight or none, while expression of CK8 was moderate, and that of CK18 was strong. NF were positive for NCAM, NSE, synaptophysin, and chromogranin, and PDGFRA. MUC1 and MUC6 apomucins were noted in EBD and EPG. EPG contained numerous endocrine cells positive for chromogranin, synaptophysin, NCAM and NSE. A few endocrine cells positive for these antigens were seen in EBD. Numeous KIT-positive stem cells (SC) were seen in PH, EBD, PV, HA, PCP, and EPG. NCAM-positive and bcl-2-positive SC were also located in these structures. Epithelial cells of EBD and EPG showed expressions of MET, PDGFRA, CA19-9, MUC1, MUC2, MUC6, KIT, bcl-2, and ErbB2. No expressions of HepPar1, carcinoembryonic antigen (CEA), and epithelial membrane antigen (EMA) were noted. Conclusions: Although the findings have limitatios because this study of humans are descriptive one, the present data suggest that the processes of the development and differentiation of EBD system may be associated with EBD SC, CK prolifes, SFC/KIT signaling, HGF/MET signaling, PDGRa/PDGFRA signaling, fibroblast growth factor/ErbB2 signaling, neuroendocrine lineage, NF differentiation, pancreatic aninar cell differentiation, PCP differentiation, MUC apomucins differentiation, and expressions of AFP and CA19-9. HepPar1, EMA and CEA were not involved in them. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT Human adipose derived stem cells have shown chondrogenic differentiation potential in cartilage tissue engineering in combination with biomimetic materials. In this study, the chondrogenic potential of a porous gelatin based scaffold genipin (GNP) crosslinked was investigated in human mesenchymal stem cells obtained from adipose tissue. Cells were cultured up to 4 weeks on the scaffold and on monolayer, MTT assay was performed to evaluate cell viability, light, and transmission electron microscopy were carried out to demonstrate cell proliferation, scaffold adhesion, and cell colonization inside the porous architecture of the biomaterial. The expression of chondrogenic markers such as SOX9, collagen type II, aggregan, and versican was investigated by Real Time PCR. Results showed an high cell viability, adhesion, and colonization of the scaffold. Real Time PCR data demonstrated an upregulation of all the chondrogenic markers analyzed. In conclusion, 3D gelatin GNP crosslinked porous scaffold provides an improved environment for chondrogenic differentiation of stem cells compared with cell monolayer culture system. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT The aim of the present research was to investigate the ultrastructural aspects and the immunoexpression of receptor activator of NFκB ligand (RANKL) and osteoprotegerin (OPG) on experimental periodontal disease of alendronate (ALN)-treated rats. Male Wistar rats received daily injections of 2.5 mg/kg body weight of ALN during 7 days previously and 7, 14, and 21 days after the insertion of a 4.0 silk suture into the gingival sulcus around the right upper second molar. Specimens were fixed in 0.1% glutaraldehyde + 4% formaldehyde under microwave irradiation, decalcified in 4.13% EDTA and paraffin embedded for TRAP histochemistry and immunohistochemistry for RANKL and OPG, or embedded in Spurr epoxy resin for TEM analysis. ALN reduced the activity of osteoclasts and significantly decreased the resorption of the alveolar crest. In the control group the alveolar crest appeared resorbed by TRAP-positive osteoclasts, which presented ultrastructural features of activated cells. The immunoexpression of RANKL was not inhibited by the drug; however, the expression of OPG was increased in the treated animals. The alveolar crest of ALN-treated specimens at 21 days showed signs of osteonecrosis, like empty osteocyte lacunae, the exposed bone regions and bacterial infection. The results showed that ALN treatment in individuals with periodontal disease represents a risk of osteonecrosis because of the reduced activity of osteoclasts resultant of the increased immunoexpression of OPG. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT A novel approach for studying the adsorption and evaporation processes of molecularly thin methanol film by the modified surface forces apparatus (M-SFA) is reported. This method can be used precisely to measure the thickness, morphology, and mechanical properties of the film confined between two mica surfaces in a real-time manner at gas atmosphere. By observing the adsorption and evaporation processes of the methanol molecule, it is found that the first adsorbed layer of the methanol film on the mica surface behaves as a solid-like structure. The thickness of this layer is measured to be about 3.2 Å, approximately equal to the diameter of a methanol molecule. Besides, this first adsorbed layer can carry normalized loads of more than 5.6 atm due to the carrying capacity conserved by the bond of mica-OH. The outer layers of the methanol film are further adsorbed with the increase of the exposure time, which are liquid-like and can be easily eliminated out from the substrate. The present study suggests that the interacting mode between hydroxy and mica is of great potential in material science and biomedical systems. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT Background : Nothing is known about huge clusters (HC) of embryonic stem cells (ESC) in human fetal organs (HFO). Aim : To know the status of HC-ESC in HFO. Methods : Morphology and immunohistochemistry (IHC) in 32 HFO of 7–40 gestational weeks (GW). Results : HC-ESC were seen in many HFO including central nervous system, spinal cords, spine, soft tissue, bone, skin, thyroid, lung, liver, pancreas, gall bladder, extrahepatic bile duct, adrenal, kidney, bladder, foregut, midgut, hindgut, female and male genital organs, and neurons. HC-ESC's were composed of two populations depending on constituting cells. One were large cells with ample acidophilic cytoplasms with vesicular nuclei and nucleoli. The other were small cells with scant cytoplasm with hyperchromatic nuclei without nucleoli, resembling lymphocytes. The HC-ESC were frequently showed neuronal differentiation. HC-ESC were positive for NCAM, synaptophysin, NSE, chromogranin, PDGFRA, AFP, ErbB2, bcl-2, KIT, MET. They were negative for CD45, CD3, CD20, EMA, CEA, CA19-9, cytokeratin (CK) 7, CK8, CK18, CK19, MUC1, MUC2, MUC5AC, and MUC6. The mean Ki-67 labeling index (LI) was 13% ± 7%. HC-ESC showed a little glycogen but lacked mucins. These HC-ESC were seen in 7–25 GW, and they were rarely seen in 26–40 GW. Conclusions : The morphology, IHC, and ontogeny of HC-ESC were described. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT We present a femtosecond-laser based nanoprocessing system for transient optical cell membrane poration to allow the introduction of foreign molecules into the interior of a cell with very high throughput. In the setup, cells flow through a micro-flow tube for spatial confinement and are simultaneously targeted by fs laser radiation. Beam-shaping generates a focal geometry along a line which is scanned across the micro-flow cell to increase the number of reachable cells. Successful cell membrane poration was observed indirectly by cell transfection even with cell-light interaction times in the millisecond range. The system was characterized by experiments with Chinese hamster ovary cells regarding cell viability, the uptake of extrinsic molecules and cell transfection efficiency. The continuous flow of cells enables a tremendous increase of cell throughput compared to previous nonflow approaches by treating millions of cells, although with only limited efficiency. The setup opens the possibility to realize a completely automated high-throughput laser-assisted cell-poration system which could be integrated in lab-on-a-chip devices. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT Composite resin is a dental material susceptible to color change over time which limits the longevity of restorations made with this material. The influence of light curing units and different fluoride mouthrinses on superficial morphology and color stability of a nanofilled composite resin was evaluated. Specimens ( N  = 150) were prepared and polished. The experimental groups were divided according to the type of light source (halogen and LED) and immersion media (artificial saliva, 0.05% sodium fluoride solution-manipulated, Fluordent Reach, Oral B, Fluorgard). Specimens remained in artificial saliva for 24-h baseline. For 60 days, they were immersed in solutions for 1 min. Color readout was taken at baseline and after 60 days of immersion. Surface morphology was analyzed by Scanning Electron Microscopy (SEM) after 60 days of immersion. Color change data were submitted to two-way Analysis of Variance and Tukey tests ( α  = 0.05). Surface morphology was qualitatively analyzed. The factor light source presented no significant variability ( P  = 0.281), the immersion media, significant variability ( P  〈 0.001) and interaction between factors, no significant variability ( P  = 0.050). According to SEM observations, no difference was noted in the surface of the specimens polymerized by different light sources, irrespective of the immersion medium. It was concluded that the light source did not influence the color stability of composite, irrespective of the immersion media, and among the fluoride solutions analyzed, Fluorgard was the one that promoted the greatest color change, however, this was not clinically perceptible. The immersion media did not influence the morphology of the studied resin. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT The control growth of the cubic meta-stable nitride phase is a challenge because of the crystalline nature of the nitrides to grow in the hexagonal phase, and accurately identifying the phases and crystal orientations in local areas of the nitride semiconductor films is important for device applications. In this study, we obtained phase and orientation maps of a metastable cubic GaN thin film using precession electron diffraction (PED) under scanning mode with a point-to-point 1 nm probe size beam. The phase maps revealed a cubic GaN thin film with hexagonal GaN inclusions of columnar shape. The orientation maps showed that the inclusions have nucleation sites at the cubic GaN {111} facets. Different growth orientations of the inclusions were observed due to the possibility of the hexagonal {0001} plane to grow on any different {111} cubic facet. However, the generation of the hexagonal GaN inclusions is not always due to a 60° rotation of a {111} plane. These findings show the advantage of using PED along with phase and orientation mapping, and the analysis can be extended to differently composed semiconductor thin films. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT Currently, the necessity of controlling infestation by ticks, especially by Rhipicephalus sanguineus , has led researchers and public health managers around the world to search for new and more efficient control methods. This way, we can highlight neem ( Azadirachta indica A. Juss) leaf, bark, and seed extracts, which have been very effective on tick control, and moreover causing less damage to the environment and to the host. This study showed the potential of neem as a control method for R. sanguineus through morphological and morphometric evaluation of the integument and synganglion of females, in semiengorged stage. To attain this, routine techniques of optical microscopy, scanning electron microscopy and morphometry of the cuticle and subcuticle of the integument were applied. Expressive morphological alterations were observed in both organs, presenting a dose-dependent effect. Integument epithelial cells and nerve cells of the synganglion showed signs of cell vacuolation, dilated intercellular boundaries, and cellular disorganization, alterations not previously reported in studies with neem. In addition, variations in subcuticle thickness were also observed. In general, the effects of neem are multiple, and affect the morphology and physiology of target animals in various ways. The results presented in this work are the first evidence of its effects in the coating and nervous system of ticks, thus allowing an indication of neem aqueous extracts as a potential control method of the brown dog tick and opening new perspectives on acaricide use. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT Aims : To evaluate the influence of a fluorescent dye (rhodamine B) on the physical and mechanical properties of three different luting cements: a conventional adhesive luting cement (RelyX ARC, 3M/ESPE), a self-adhesive luting cement (RelyX U-200, 3M/ESPE), and a self-etching and self-adhesive luting cement (SeT PP, SDI). Materials and Methods : The cements were mixed with 0.03 wt% rhodamine B, formed into bar-shaped specimens (n = 10), and light cured using an LED curing unit (Radii, SDI) with a radiant exposure of 32 J/cm 2 . The Knoop hardness (KHN), flexural strength (FS), and Young's modulus (YM) analyses were evaluated after storage for 24 h. Results : Outcomes were subjected to two-way ANOVA and Tukey's test (P = 0.05) for multiple comparisons. No significant differences in FS or YM were observed among the tested groups (P ≥ 0.05); the addition of rhodamine B increased the hardness of the luting cements tested. Conclusion : The addition of a fluorescent agent at 0.03 wt% concentration does not negatively affect the physical–mechanical properties of the luting cement polymerization behavior. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT Optimal resolution by stimulated emission depletion (STED) microscopy requires precise alignment of the donut-shaped depletion focus to the excitation focus. In this article, we demonstrate that fluorescence lifetime distribution can be implemented to align the STED system. Different from the traditional aligning methods in which a scattering imaging module is often equipped, the lifetime-based method is free from probable mismatches between the scattering mode and the fluorescent mode, drift errors caused by separate imaging and complex fitting methods. Based on this method, a spatial resolution of 38 nm by time-gated detection has been achieved. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT Non-alcoholic fatty liver disease (NAFLD) is a clinical-pathological syndrome that includes a wide spectrum of morphological alterations. In research, animal models are crucial in evaluating not only the pathogenesis of NAFLD and its progression, but also the therapeutic effects of various agents. Investigations on the ultrastructural features of NAFLD in humans are not copious, due to the difficulty to obtain human samples and to the long time of NAFLD to evolve. Translational comparative studies on the reliability of animal models in representing the histopathologic picture as seen in humans are missing. To overcome this lack of investigations, we compared the ultrastructural NAFLD features of an animal model versus human. Sprague-Dawley rats were fed with a high fat diet (HFD) for 1–4 weeks, while control rats were fed with a standard diet. Human specimens were collected from patients with diagnosed fatty liver disease, undergoing liver biopsies or surgery. Rat and human samples were examined by light microscopy and by transmission and high resolution scanning electron microscopy. The present work demonstrated that NAFLD in animal model and in human, share overlapping ultrastructural features. In conclusion, animal HFD represent an appropriate tool in studying the pathogenesis of NAFLD. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT Although several treatments for tendon lesions have been proposed, successful tendon repair remains a great challenge for orthopedics, especially considering the high incidence of re-rupture of injured tendons. Our aim was to evaluate the pharmacological potential of Aloe vera on the content and arrangement of glycosaminoglycans (GAGs) during tendon healing, which was based on the effectiveness of A. vera on collagen organization previously observed by our group. In rats, a partial calcaneal tendon transection was performed with subsequent topical A. vera application at the injury site. The tendons were treated with A. vera ointment for 7 days and excised on the 7 th , 14 th , or 21 st day post-surgery. Control rats received ointment without A. vera . A higher content of GAGs and a lower amount of dermatan sulfate were detected in the A. vera -treated group on the 14 th day compared with the control. Also at 14 days post-surgery, a lower dichroic ratio in toluidine blue stained sections was observed in A. vera -treated tendons compared with the control. No differences were observed in the chondroitin-6-sulfate and TGF-β1 levels between the groups, and higher amount of non-collagenous proteins was detected in the A. vera -treated group on the 21 st day, compared with the control group. No differences were observed in the number of fibroblasts, inflammatory cells and blood vessels between the groups. The application of A. vera during tendon healing modified the arrangement of GAGs and increased the content of GAGs and non-collagenous proteins. Microsc. Res. Tech. 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT Low voltage transmission electron microscopy (LVTEM) with accelerating voltages as low as 5 kV was applied to cell biology. To take advantage of the increased contrast given by LVTEM, tissue preparation was modified omitting all heavy metals such as osmium, uranium, and lead from the fixation, on block staining and counterstaining. Nonstained ultra-thin tissue sections (40 nm thick) generated highly contrasted images. While the aspect of the cells remains similar to that obtained by conventional TEM, some new substructures were revealed. The pancreatic acinar cells granules present a heterogeneous matrix with partitions corresponding to segregation of their different secretory proteins. Microvilli display their core of microfilaments anchored to the dense top membrane. Mitochondria revealed the presence of distinct particles along their cristea membranes that may correspond to the ATP synthase complexes or oxysomes. The dense nuclear chromatin displays a honey-comb appearance while distinct beads aligned along thin threads were seen in the dispersed chromatin. These new features revealed by LVTEM correlate with structures described or predicted through other approaches. Masking effects due to thickness of the tissue sections and to the presence of heavy metals must have prevented their observation by conventional TEM. Furthermore, the immunogold was adapted to LVTEM revealing nuclear lamin-A at the edge of the dense chromatin ribbons. Combining cytochemistry with LVTEM brings additional advantages to this new approach in cell biology. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT Delia platura (Meigen) is a phytophagous fly that can cause significant crop losses. To obtain a better understanding of the external morphology of this species, adult D . platura is studied using scanning electron microscopy. Organs or structures that are important for taxonomy, such as the compound eyes, spiracles, pulvilli, wings, and genitalia are highlighted to complement previous description based on light microscope. Mesothoracic and metathoracic spiracles of D. platura that provide efficiency in preventing entrance of fine materials or dust into the tracheal system are morphologically different. In addition, the elongate-oval pulvillus is densely covered with tenent setae with spoon-like tip, which can increase the number of contact points for attachment to a surface. Four types of sensilla are observed on the male genitalia of D. platura including: trichoid sensilla, chaetic sensilla, three subtypes of campaniform sensilla, and basiconic sensilla. Long bristles and microtrichiae are observed on the female genitalia of D. platura . The possible function of sensilla located in the genitalia of D. platura is discussed. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT Transverse and longitudinal sectioning of undecalcified cortical bone is a commonly employed technique for investigating the lamellar structure of the osteons. Since a flat surface is required, the specimen has to be grinded and then polished. Whereas the smear of debris and inorganic/organic deposits left by these treatments cannot be removed by ultrasonication alone, a chemical treatment of the specimen surface with either a basic or an acid etching solution is currently employed. A further effect of the latter can be the enhancement of the lamellar bone pattern. The kind of etching solution, its pH, the concentration of etchants, and the contact time significantly affect the sectioned surface when it is observed with scanning electron microscopy (SEM). The etching procedures can severely influence the obtained images. Homogeneous cortical bone specimens were sampled from the first metatarsal of two fresh human subjects. One or two cut surfaces were exposed to different acid and basic solutions in bonded conditions. Considering the type of chemical agents, the solution pH, and the exposure time of the specimens, the effects of several etching media have been investigated and compared. Strong etching, either acid or basic produced surface decalcification and severe damage of the collagen matrix, compromising any morphological or morphometric analysis. Weak acid etching (for example citric and acetic acid), even though causing distinctive alteration of the sample, enhanced the visibility of the lamellar pattern, while the polyphosphate treatment of the surface decalcified a thin layer matrix, ensuring a good visibility of fibrils and avoiding rough distortions. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT The ionic liquids (ILs) are recognized as the potential solvents for the pretreatment of lignocellulosic materials before biomass conversion. However, little knowledge of how the cell wall of biomass responds to the IL locally and dynamically during the pretreatment is available. In the current work, the process of IL pretreatment of poplar using 1-ethyl-3-methylimidazolium acetate ([C 2 mim][OAc]) was real-time monitored on a cellular level by employing confocal Raman microscopy. The results showed that the biomass dissolution during the IL pretreatment can be clearly divided into two stages: (1) slow penetration of IL, and (2) rapid dissolution of lignin and carbohydrates. In this case, the onset of the dissolution of these compositions occurred only after the cell wall of biomass swelled to a certain extent. Because the first stage was a slow process which determined the process reaction rate, it can be deduced that enhancing the penetration capacity of IL was crucial for improving the pretreatment efficiency. Based on the obtained results, a model was proposed to better understand how the plant cell wall responds to the IL before, during, and after pretreatment. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT Lavandula dentata L. is an aromatic plant used in folk medicine for different purposes and, for this reason, phytochemical surveys have been carried out in the search for bioactive substances aiming to support its uses. Since there is little knowledge on the structural aspects of L. dentata , this work has studied the anatomical characters of the leaf and stem using light and scanning electron microscopy, in order to assist the species identification. As a result, there are different types of trichomes: capitate glandular with uni- or bicellular head, peltate glandular with multicellular head, and branched non-glandular. The leaf is hypostomatic showing diacytic stomata. The epidermis is uniseriate and coated with striate cuticle. The mesophyll is dorsiventral and the midrib is concave–convex and traversed by a single collateral vascular bundle. The stem is quadrangular and has alternating strands of collenchyma and cortical parenchyma as well as a typical endodermis in the cortex. The phloem and xylem cylinders are traversed by narrow rays and there is an incomplete sclerenchymatic sheath adjoining the phloem. These results are a novelty for the species and contribute to distinguish it from other lavenders. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT Observation of sperm development and determination of their morphological characteristics are very important to the understanding of phylogenetic relationships and the study of sperm function during fertilization. Although ultrastructural studies of sperm development in the testes of the fruit fly Drosophila have been performed, there are few reports describing electron microscopic morphology of mature sperm, that is, those released from the testes to the seminal vesicles. Here, we present the first report of the sagittal organization of Drosophila sperm head and neck regions by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The head and tail structures of a mature sperm, for example, the acrosome, nucleus, and flagellum, were easy to distinguish by the morphological characteristics of the sperm surface by SEM. The morphological relationships between the surface and internal structures of mature sperm were confirmed by observing longitudinal sections with TEM. Our approach overcame the technical difficulties involved in sample preparation for electron microscopic observation of the Drosophila mature sperm head, and therefore, this study serves as an important foundation for future genetic dissection of sperm ultrastructure and function in male sterile mutants. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT The study focused on the description of pig gallbladder angioarchitecture, with particular emphasis on the specifics of the course of blood vessels in individual layers of the gallbladder wall. Furthermore, the vascular systems of the pig gallbladder were analyzed in terms of the adaptation of this organ to changes in its volume during cyclical bile storage and discharge. The gallbladder is supplied by the cystic artery, which in the pig represents a mixed pinnate and bipinnate pattern of branching. The light microscopic and scanning electron microscopic observations of three-dimensional vascular corrosion casts showed the presence of two main complex vascular networks in the wall of the gallbladder, one located in the subserosal and the other in the mucosa. The unique features in the pig, connected with the size of the gallbladder, is the well-developed horizontal venous plexus under folds of the mucosa, which is a voluminous reservoir of fluids absorbed from bile and vascular networks around mucous glands. Superficial blood vessels of the gallbladder run in vascular pairs or triads, where a single artery runs between two veins. The structures of blood flow control, that is, venous valves, were observed only in venules of the subserosal plexus. Spatial arrangement of the vascular network in the pig gallbladder shows functional plasticity during changes in gallbladder volume. The course of superficial blood vessels in the well-filled gallbladder is arcuate, while in the empty gallbladder it is undulated or spiral. In the mucosal and intramural vessels the direction of blood vessels may change from perpendicular to oblique. Microsc. Res. Tech. 00:000–000, 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT In this article a study of the distribution of heavy metals in Cupressus lindleyi breaching-leaves was done in Taxco, Guerrero. At the same, heavy metals micro-localization was conducted in the breaching-leaves to understand the structural changes provoked by mining waste on plants. The most abundant contaminants in soils, tailings and different plant organs (roots, stems, and leaves) were Zn, Mn, and Pb. Nevertheless, As was more accumulated in the stem and breaching-leaves. The translocation factor and the bio-concentration factor were less than 1. The structural changes observed were the great accumulation of starch grains and phenolic compounds in the palisade parenchyma, changes in the hypodermis cell wall and necrotic zones in the palisade parenchyma. The distribution of heavy metals in breaching-leaves tissues was homogeneous in most of the elements. These results showed that C. lindleyi is a species that can be employed in phytostabilization of contaminated zones with mining waste because it is a native plant that does not require a lot of conditions for its development. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT Introduction: Normal pregnancy is characterized by significant alterations in the haemostatic system accompanied by an augmented risk of thrombosis. Materials and methods: The fibrin network ultrastructure of different phases of pregnancy, namely early pregnancy (week 8–14), late pregnancy (week 36–40) as well as post-partum (week 6–8 after birth) were compared with nonpregnant fibrin networks as well as each other to establish whether differences in fibrin network morphology exist during pregnancy. Scanning electron microscopy was employed to analyse fibrin network morphology. Results: The fibrin networks from all phases of pregnancy appeared similar to each other, exhibiting prominent coagulant formation, an increase in the formation of minor, thin fibers, and the presence of granular globules. All three phases, however, differ from the typical fibrin network ultrastructure exhibited by the fibrin networks from nonpregnant individuals. The increase in estrogen associated with pregnancy may cause the increase in coagulation factors and ultimately the prothrombotic state characteristic of pregnancy. Conclusions: Since no differences were apparent between the different phases of pregnancy it suggests that activation of the coagulation system commences with pregnancy and this pro-thrombotic state continues till at least 8 weeks after birth. These results may shed light on possible pathological mechanisms employed in the development of abnormal or ailing pregnancy. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT This study follows the left ventricular (LV) hypertrophy in rats undergoing aerobic training alone (A), resistance training alone (R), or combined resistance and aerobic training (RA) (usually referred as concurrent training) program. A sedentary control group (C) was included. LV remodeling was evaluated using electron and light microscopy. The LV weight to body weight (LVW: BW) increased 11.4% in A group, 35% in the R group, and 18% in the RA group compared to the C group. The LV thickness increased 6% in the A group, 17% in the R group, and 10% in the RA group. The LV internal diameter increased 19% in the A group, 3% in the R group, and 8% in the RA group compared with the C group. The cross-sectional area of cardiomyocyte increased by 1% with the A group, 27% with R group, and 12% with RA training. The capillary density increased by 5.4% with A training, 11.0% with R training, and 7.7% with RA training compared with the C group. The volume fraction of interstitial collagen increased by 0.4% with training A, increased by 2.8% with R training, and 0.9% with RA training. In conclusion, except for the LV internal diameter, which increased more in the A group, the cardiac parameters increased more in the R group than in the other groups and in RA group than in A group. Collagen density increased from 5.4 ± 0.8% in the C group to 5.8 ± 0.6% in the A group (n. s.) ( P 〉 0.05), to 8.2 ± 0.7% in the R group ( P  〈 0.05), and to 6.3 ± 0.4% in the RA group ( P  〈 0.05). These results demonstrate a significant increase for collagen content in the LV with R and RA exercise, but the increase was higher with R training alone than with RA training. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT Visualization of bacteria in living plant cells and tissues is often problematic due to lack of stains that pass through living plant cell membranes and selectively stain bacterial cells. In this article, we report the use of 3,3′-diaminobenzidine tetrachloride (DAB) to stain hydrogen peroxide associated with bacterial invasion of eukaryotic cells. Tissues were counterstained with aniline blue/lactophenol to stain protein in bacterial cells. Using this staining method to visualize intracellular bacterial ( Burkholderia gladioli ) colonization of seedling roots of switch grass ( Panicum virgatum ), we compared bacterial free seedling roots and those inoculated with the bacterium. To further assess application of the technique in multiple species of vascular plants, we examined vascular plants for seedling root colonization by naturally occurring seed-transmitted bacteria. Colonization by bacteria was only observed to occur within epidermal (including root hairs) and cortical cells of root tissues, suggesting that bacteria may not be penetrating deeply into root tissues. DAB/peroxidase with counter stain aniline blue/lactophenol was effective in penetration of root cells to selectively stain bacteria. Furthermore, this stain combination permitted the visualization of the bacterial lysis process. Before any evidence of H 2 O 2 staining, intracellular bacteria were seen to stain blue for protein content with aniline blue/lactophenol. After H 2 O 2 staining became evident, bacteria were often swollen, without internal staining by aniline blue/lactophenol; this suggests loss of protein content. This staining method was effective for seedling root tissues; however, it was not effective at staining bacteria in shoot tissues due to poor penetration. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT This article proposes an innovative methodology which employs nondestructive techniques to assess the effectiveness of new formulations based on ionic liquids, as alternative solvents for enzymes (proteases), for the removal of proteinaceous materials from painted surfaces during restoration treatments. Ionic liquids (ILs), also known as “designer” solvents, because of their peculiar properties which can be adjusted by selecting different cation-anion combinations, are potentially green solvents due totheir low vapour pressure. In this study, two ionic liquids were selected: IL1 (1-butyl-3-methylimidazolium tetrafluoroborate ([BMIM][BF 4 ])) and IL2 (1-ethyl-3-methylimidazolium ethylsulphate ([EMIM][EtSO 4 ])). New formulations were prepared with these ILs and two different proteases (E): one acid (E1—pepsin) and one alkaline (E2—obtained from Aspergillus sojae ). These formulations were tested on tempera and oil mock-up samples, prepared in accordance with historically documented recipes, and covered with two different types of protein-based varnishes (egg white and isinglass—fish glue). A noninvasive multiscale imaging methodology was applied before and after the treatment to evaluate the cleaning's effectiveness. Different microscopic techniques—optical microscopy (OM) with visible and fluorescent light, scanning electron microscopy (SEM) and atomic force microscopy (AFM)—together with Matrix-Assisted Laser Desorption/Ionization—Time of Flight Mass Spectrometry (MALDI-TOF MS) were applied on areas cleaned with the new formulations (IL + E) and reference areas cleaned only with the commercial enzyme formulations (gels). MALDI-TOF proved particularly very useful for comparing the diversity and abundance of peptides released by using different enzymatic systems. Microsc. Res. Tech., 2014 . © 2014 Wiley Periodicals, Inc.