ALBERT

Description: ABSTRACT A simple procedure, which enables accurate measurement of transmission electron microscopy (TEM)/STEM probe currents using an energy loss spectrometer drift tube is described. The currents obtained are compared with those measured on the fluorescent screen to enable the losses due to secondary and backscattered electrons to be determined. The current values obtained from the drift tube allow the correction of fluorescent screen current densities to yield true current. They also enable CCD conversion efficiencies to be obtained, which in turn allows images to be calibrated in terms of electron fluence. Using probes of known current in conjunction with a NiO reference specimen enables the X-ray detector solid angle to be determined. The NiO specimen also allows a wide range of other EDS detector parameters to be obtained, including the presence of ice and carbon contamination. A range of performance characteristics are reported for two large area EDS detector systems. Many of the measurements reported herein have been automated via the use of freely available scripts for DigitalMicrograph. Microsc. Res. Tech., 2015 . © 2015 Wiley Periodicals, Inc.

Description: The presence of denticles in the external surface, oral cavity, fins, and clasper of Elasmobranchii has been widely reported. These structures, called body denticles, may be observed on the body surface of sharks. Dermal and oral denticles are made up by a basal plate that is embedded in the dermis, forming a peduncle that grows from the base to the crown. These denticles may protect the skin against abrasion, and improve hydrodynamics and gill arches function. Rhizoprionodon lalandii is a widely distributed and very common species in Brazilian coastal areas. The aims of this study was to compare the morphology of oral and body denticles of R. lalandii to understand the implications of these structures in the behavior of these animals. Morphological analysis showed that there are differences between dermal and oral denticles, which are related to their role in different body regions. Body denticles have three cusps, and well-defined crests and ridges, and literature data suggest that suggest that hydrodynamics is their main function. Most of the oral denticles have only one cusp, and their morphology and distribution showed that their main functions are preparing food to be swallowed and protecting the oral cavity against abrasion. Microsc. Res. Tech., 2015 . © 2015 Wiley Periodicals, Inc.

Description: ABSTRACT Objectives This study investigated the effects of sliding on the ultrastructure of three representative esthetic superelastic 0.014 inch nickel-titanium (NiTi) archwires. Methods : Atomic force microscopy, scanning electron microscopy, and light microscopy were used to estimate the surface roughness of archwires and bracket systems. Energy-dispersive X-ray spectroscopy was used to estimate the molecular differences between coated and uncoated areas. A combination of four different types of 0.014 inch metallic wires and two different types of 0.022 inch × 0.028 inch conventional brackets were evaluated by in vitro sliding tests using a novel self-made tensile-strength tester with a miniature load cell and syringe pump. The NiTi wires included an uncoated NiTi archwire (CO group), epoxy resin-coated NiTi archwire (ER group), Teflon ® -coated NiTi archwire (TF group), and Ag/biopolymer-coated NiTi archwire (AG group). The brackets included contained stainless steel (SS) and ceramic (CE) brackets. Results : Both ER and TF wire groups exhibited less surface roughness than CO wire groups. The AG group showed the highest surface roughness compared with the others because of its silver particles ( P 〈0.001, ANOVA test). In vitro sliding tests led to a significant increase ( P  〈 0.001, ANOVA test) in the surface roughness of all 0.014 inch NiTi wires regardless of bracket type. The wire groups combined with SS brackets were rougher than those of CE brackets regardless of the coating materials because of exfoliation of the coating materials. The TF-SS group showed the highest increase (fivefold) in surface roughness compared to the others, while the ER groups showed the lowest increase (1.4-fold) in surface roughness compared with the others ( P  〈 0.001, ANOVA test). Conclusions : The results suggested that the sliding-driven surface roughness of superelastic NiTi archwires is directly affected by coating materials. Although the efficiency of orthodontic treatment was affected by various factors, epoxy resin-coated archwires were best for both esthetics and tooth movement when only considering surface roughness. Microsc. Res. Tech., 2015 . © 2015 Wiley Periodicals, Inc.

Description: ABSTRACT This study aimed to evaluate effectiveness and effects of bleaching with 35% hydrogen peroxide with and without calcium on color, micromorphology, and the replacement of calcium and phosphate on the enamel surface. Thirty bovine enamel blocks (5.0 × 5.0 mm) were placed into the following groups: G1: artificial saliva (control); G2: 35% hydrogen peroxide gel without calcium (Whiteness HP Maxx–FGM); and G3: 35% hydrogen peroxide gel with calcium (Whiteness HP Blue–FGM). Three color measurements were performed with a spectrophotometer: untreated (baseline), after performing staining, and after application of bleaching agents. Calcium deposition on the enamel was evaluated before and after the application of bleaching agents using energy-dispersive X-ray spectrometry. The enamel surface micromorphology was observed under scanning electron microscopy. The pH of each product was measured. The data were subjected to one-factor analysis of variance (ANOVA), and any differences were analyzed using Tukey's test ( P  〈 0.05). G3 showed greater variation in total color after the experiment than G2 and G1; there was no significant difference in calcium or phosphorus concentration before and after the experimental procedures; morphological changes were observed only in G2 and G3; and the pH values of the Whiteness HP Maxx and Whiteness HP Blue bleaching agents were 5.77 and 7.79, respectively. The 35% hydrogen peroxide with calcium showed greater bleaching potential, but the addition of calcium had no effect in terms of reducing morphological changes or increasing the calcium concentration on the enamel surface. Microsc. Res. Tech., 2015 . © 2015 Wiley Periodicals, Inc.

Description: ABSTRACT Previous articles have pointed out the presence of type III collagen within the extracellular structure of the parenchymatous organs. This study aimed to quantitatively characterize the collagen polymorphism at the capsule and parenchymal trabeculae of the largest lymphoid organ of the body i.e., the spleen, in mouse, rat, and rabbit models. Following a Picrosirius Red-Polarization procedure and computer assisted image analysis of paraffin sections, the results showed (1) a predominant and significantly higher amount of type III collagen in the trabeculae area compared to the capsule area in the three species, (2) no statistical difference among the three species concerning the parenchymal collagen polymorphism or the type I/type III collagen ratio, (3) a heterogeneous type I/type III collagen ratio varying from 0.86 (mouse) to 6.62 (rabbit) in the fibromuscular capsule region. A qualitative analysis corroborated these histomorphometric results. In conclusion, the spleen may be used as (1) a control tissue to qualitatively visualize type I and III collagen under polarization microscopy and to validate the quality of PSR staining (2) an aid to accurately calibrate the angle of polarization before quantitative measurements of type I and type III collagen. Among the studied species, the rabbit spleen appeared to be the most appropriate control tissue as it showed the highest amount of type I collagen in the capsule and a similarly high amount of type III collagen in the parenchymal trabeculae. Microsc. Res. Tech., 2015 . © 2015 Wiley Periodicals, Inc.

Description: ABSTRACT Physiological oral mechanical forces may play a role on the progression of enamel carious lesions to cavitation. Thus, the aim of this study was to describe, by 3D finite element analysis, stress, and strain patterns in sound and carious enamel after a simulated occlusal load. Micro-CT based models were created and meshed with tetrahedral elements (based on an extracted third molar), namely: a sound (ST) and a carious tooth (CT). For the CT, enamel material properties were assigned according to the micro-CT gray values. Below the threshold corresponding to the enamel lesion (2.5 g/cm 3 ) lower and isotropic elastic modulus was assigned ( E  = 18 GPa against E 1  = 80 GPa, E 2  =  E 3  = 20 GPa for sound enamel). Both models were imported into a FE solver where boundary conditions were assigned and a pressure load (500 MPa) was applied at the occlusal surface. A linear static analysis was performed, considering anisotropy in sound enamel. ST showed a more efficient transfer of maximum principal stress from enamel to the dentin layer, while for the CT, enamel layer was subjected to higher and concentrated loads. Maximum principal strain distributions were seen at the carious enamel surface, especially at the central fossa, correlating to the enamel cavity seen at the original micro-CT model. It is possible to conclude that demineralized enamel compromises appropriate stress transfer from enamel to dentin, contributing to the odds of fracture and cavitation. Enamel fracture over a dentin lesion may happen as one of the normal pathways to caries progression and may act as a confounding factor during clinical diagnostic decisions. Microsc. Res. Tech., 2015 . © 2015 Wiley Periodicals, Inc.

Description: ABSTRACT We have developed a real-time imaging technique for diagnosis of kidney diseases which is composed of two steps, staining renal cells safely with food dyes and optical sectioning of living renal tissue to obtain histological images by multiphoton microscopy (MPM). Here, we demonstrated that the MPM imaging with food dyes, including erythrosine and indigo carmine, could be used as fluorescent agents to visualize renal functions and structures such as glomerular bloodstreams, glomerular filtration, and morphology of glomeruli and renal tubules. We also showed that the kidneys of IgA nephropathy model-mice stained with the food dyes presented histopathological characteristics different from those observed in normal kidneys. The use of the food dyes enhances the quality of tissue images obtained by MPM and offers the potential to contribute to a clinical real-time diagnosis of kidney diseases. Microsc. Res. Tech., 2015 . © 2015 Wiley Periodicals, Inc.

Description: ABSTRACT Telenomus dendrolimusi Chu is a dominant egg parasitoid of Dendrolimus , which is the most serious conifer defoliator in China. However, the host locating mechanism of T. dendrolimusi is unclear. To take better advantage of this kind of parasitoid, it is necessary to clarify the basic knowledge of the organization of the wasp's sensory system. In this study, the external morphologies of the sensilla on the entire female and male T. dendrolimusi body were described using scanning electron microscopy. Antennae shape and sensilla types were sexually dimorphic. Ten segments and nine types of sensilla were found on the female antenna, including basiconic sensillum (BS), chaeticum sensillum (ChS), trichoid sensillum type 1 (TS1), trichoid sensillum type 2 (TS2), trichoid curvata sensillum (TCS), sickle-shaped sensillum (SSS), multiporous gustatory sensillum (MGS), campaniform sensillum (CS), and styloconica sensillum (STS). The distribution of the sensilla was directional on female antenna. The male antenna consisted of 12 segments, and 1–3 segments were similar to those of the female. Five types of sensilla were found on male antenna, including BS, ChS, TS1, TCS, and STS. The mouthparts, thoracic legs, eyes, mesonotum, and wings were all covered with longitudinally fluted TSs without hole. A tympana structure appeared on the thoracic legs. Special crevice-shaped structures were found on the abdomens of both T. dendrolimusi sexes. Possible sensilla functions are discussed according to their morphology. These results provide a solid basis for further researches about the parasitic mechanisms of T. dendrolimusi . Microsc. Res. Tech., 2015 . © 2015 Wiley Periodicals, Inc.

Description: Endothelial glycocalyx (GCX) is located on the apical surface of vascular endothelial cells and is composed of a negatively-charged network of proteoglycans and glycoproteins. The GCX plays an important role in maintaining the integrity of vascular walls and preventing leakage of plasma. Therefore, degradation of the GCX is believed to lead to pathological leakage of plasma. Because the GCX is a very thin layer, its ultrastructural image has been demonstrated on electron microscope. To explore the function of the GCX, it should be visualized by a microscope in vivo. Thus, we developed in vivo visualization technique of the GCX under fluorescence microscopy using a mouse dorsal skinfold chamber (DSC) model. To label and visualize the GCX, we used fluorescein isothiocyanate (FITC)-labeled lectin, which has a high specificity for sugar moieties. We examined the affinity of the different lectins to epivascular regions under an intravital fluorescent microscope. Among seven different lectins we examined, FITC labeled Triticum vulgaris (wheat germ) agglutinin (WGA) delineated the GCX most clearly. Binding of WGA to the GCX was inhibited by chitin hydrolysate, which contained WGA-binding polysaccharide chains. Furthermore, the septic condition attenuated this structure, suggesting structural degradation of endothelial GCX layer. In conclusion, FITC-labeled WGA lectin enabled visualization of endothelial GCX under in vivo fluorescence microscopy. Microsc. Res. Tech., 2015 . © 2015 Wiley Periodicals, Inc.

Description: ABSTRACT The purpose of this study was to analyze, by confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM), the morphology of sealant/enamel interface after surface treatment with Biosilicate. Before pits and fissures sealing, the occlusal surfaces of 10 sound human molars were sectioned perpendicularly at the fissures in order to obtain three slices for each tooth. Slices were randomly assigned into three groups ( n  = 10) according to sealing protocol: Group 1- Acid etching + Biosilicate + glass ionomer-based sealant (Clinpro XT Varnish, 3M ESPE); Group 2- Acid etching + glass ionomer-based sealant (Clinpro XT Varnish, 3M ESPE); Group 3- No sealing. All slices were subjected to thermal cycling (5,000 cycles; 5–55°C; dwell time: 30s). Half of the slices from each group ( n  = 5) were analyzed by CLSM and the other half by SEM. Groups 1 and 2 were also submitted to EDS analysis and their data were evaluated by Two-Way ANOVA e Tukey's test (α=5%). EDS data analysis showed higher amounts of silicon (Si) ions than calcium (Ca) ions in Group 1 ( P  〈 0.05); Group 2 presented higher amounts ( P  〈 0.05) of Ca ions than Si ions. It may be concluded that the use of Biosilicate for surface treatment did not affect the morphology of glass ionomer-based sealant/enamel interfaces. Microsc. Res. Tech., 2015 . © 2015 Wiley Periodicals, Inc.

Description: ABSTRACT Sjogren's syndrome (SS) is a progressive autoimmune condition mainly affecting the salivary and lacrimal glands with an incidence of primary SS between 1/100 and 1/1,000. SS implies an alteration in the epithelium and subepithelium innervation, with consequent reduction of corneal sensitivity. It is necessary to have noninvasive quantitative methods to characterize the status of the corneal nerve fibers of the patients in order to choose and follow the best therapy. Entropy (information dimension) of the nerve corneal fibers distribution observed by confocal microscopy was evaluated in patients with primary SS ( n  = 30, 6 males, 24 females, 21–81 years), diagnosed by biopsy of salivary gland and blood tests and in sex- age-matched healthy subjects ( n  = 12). Corneal nerve fiber density, Langerhans cell count, and cell density in the nerve plexus images were also evaluated. In selected patients salivary gland atrophy degree was also evaluated. Nerve corneal distribution observed by confocal microscopy is fractal. Entropy of the corneal nerve distribution statistically distinguishes between SS patients and healthy subjects: patients present a lower value of information dimension of the corneal nerve fibers distribution than healthy individuals ( P  〈 0.001). Percentage of grouped cases classified by entropy according to the subjects (selected patients vs. healthy) showed a 100% sensitivity and 96% specificity, P  〈 0.0001 with a low value of coefficient of variation among the individuals (6–7 times lower than the other morphometric indexes). Entropy correlated with the severity of the disease (salivary gland atrophy degree, P  〈 0.01). Evaluation of entropy of the corneal nerve distribution observed by a laser confocal microscopy appears to quantitatively and noninvasively characterize an aspect of the SS patients in relation to the recognition of an impairment of their ocular surface, giving us for the first time a method to objectively and precisely characterize the corneal innervation status in the SS patients. Microsc. Res. Tech., 2015 . © 2015 Wiley Periodicals, Inc.

Description: ABSTRACT Widefield frequency-domain fluorescence lifetime imaging microscopy (FD-FLIM) is a fast and accurate method to measure the fluorescence lifetime of entire images. However, the complexity and high costs involved in construction of such a system limit the extensive use of this technique. PCO AG recently released the first luminescence lifetime imaging camera based on a high frequency modulated CMOS image sensor, QMFLIM2. Here we tested and provide operational procedures to calibrate the camera and to improve the accuracy using corrections necessary for image analysis. With its flexible input/output options, we are able to use a modulated laser diode or a 20 MHz pulsed white supercontinuum laser as the light source. The output of the camera consists of a stack of modulated images that can be analyzed by the SimFCS software using the phasor approach. The nonuniform system response across the image sensor must be calibrated at the pixel level. This pixel calibration is crucial and needed for every camera settings, e.g. modulation frequency and exposure time. A significant dependency of the modulation signal on the intensity was also observed and hence an additional calibration is needed for each pixel depending on the pixel intensity level. These corrections are important not only for the fundamental frequency, but also for the higher harmonics when using the pulsed supercontinuum laser. With these post data acquisition corrections, the PCO CMOS-FLIM camera can be used for various biomedical applications requiring a large frame and high speed acquisition. Microsc. Res. Tech., 2015 . © 2015 Wiley Periodicals, Inc.

Description: ABSTRACT We use atomic force microscopy in conjunction with a fluorescence microscope capable of optical sectioning to acquire images of white blood cells while force is applied with the AFM tip. The indentation profile within the cell is compared to the profile of the AFM tip: examples are shown for indentations at the center of the cell which are reasonable matches to the tip profile, and an additional example is shown for an indentation that is on the tilted side of a highly rounded cell and that differs from the tip shape. We also demonstrate that the AFM tip can interact with internal cell structures, we show that the contact area between the cell and the substrate can increase under applied pressure, that the main body of the cell can fuse with the extended lamellipodium, and that the cell can be displaced laterally by the AFM tip. The features illustrated here are relevant to the interpretation of indentation experiments that measure cell elasticity properties, as is discussed briefly. Microsc. Res. Tech., 2015 . © 2015 Wiley Periodicals, Inc.

Description: ABSTRACT In this study, we report the distribution of orexin A (OXA), orexin B (OXB), and orexin receptor (OX2R) immunoreactive (ir) cells in the hypothalamus and gastrointestinal tract of Oncorhynchus mykiss fed diets with different dietary fatty acid compositions. Trout were fed five iso-energetic experimental diets containing fish oil, or one of four different vegetable oils (olive, sunflower, linseed, and palm oils) as the added dietary lipid source for 12 weeks. OXA, OXB, and OX2R immunoreactive neurons and nervous fibers were identified in the lateral and ventro-medial hypothalamus. OXA, OXB, and OX2R ir cells were found in the mucosa and glands of the stomach and in the mucosa of both the pyloric cecae and intestine. OX2R ir cells were localized in the mucosa layer of both the pyloric cecae and intestine. These immunohistochemical (IHC) results were confirmed via Western blotting. Antibodies against preproorexin (PPO) crossreacted with a band of ∼16 kDa in the hypothalamus, stomach, pyloric cecae, and intestine. Antibodies against OX2R crossreacted with a band of ∼38 kDa in the hypothalamus, pyloric cecae, and intestine. The presence and distribution of OXA, OXB, and OX2R ir cells in the hypothalamus and gastrointestinal tract did not appear to be affected by dietary oils. The presence of orexin system immunoreactive cells in the stomach, pyloric cecae, and intestine of rainbow trout, but not in the enteric nervous system, could suggest a possible role of these peptides as signaling of gastric emptying or endocrine modulation, implying a main local action played by orexins. Microsc. Res. Tech., 2015 . © 2015 Wiley Periodicals, Inc.

Description: ABSTRACT The pineal gland is an endocrine gland found in all mammals. This article describes the morphology of this important gland in two species of Caviideae, namely the yellow-toothed cavy and the red-rumped agouti. Ten adult animals of the two species used in current analysis were retrieved from the Center for the Multiplication of Wild Animals (CEMAS/UFERSA) and euthanized. The glands were removed and photographed in situ and ex situ. They were fixed in a paraformaldehyde solution 4% or glutaraldehyde 2.5% solution and submitted to routine histological techniques respectively for light and scanning electron microscopy. Macroscopically, the pineal gland with its elongated structure may be found between the cerebral hemispheres facing the rostral colliculi. Microscopically, pinealocytes and some glia cells were predominant. Contrastingly, to the cavy's pineal gland, a capsule covered the organ in the agouti, with the emission of incomplete septa to the interior, which divided it into two lobules. Light and scanning electron microscopes failed to show calcareous concretions in the pineal gland. Based on the topography of the cavy's and agouti's pineal gland, it may be classified as supra-callosum and ABC type. Microsc. Res. Tech., 2015 . © 2015 Wiley Periodicals, Inc.

Description: ABSTRACT Different therapeutic techniques have been developed for regeneration of articular cartilage injuries, but none has provided an optimal solution to their treatment. Human umbilical cord blood-mesenchymal Stem Cells (HUCB-MSCs) have been considered as promising alternative cell source for cartilage repair. Objectives: Examining the success rate of MSCs isolation from HUCB as well as chondrogenic differentiation potential of HUCB-MSCs in vitro. Materials and methods: 32 UCB samples were collected, in addition to 5 bone marrow (BM) and 5 peripheral blood (PB) samples, taken as reference controls. Samples were used for mononuclear cells isolation from which MSCs were expanded under complete aseptic conditions, were verified morphologically and through the presence of CD44 and CD105, and absence of CD34. Results: Success rate of UCB-MSCs isolation was (25%), a rate that was lower than those of PB (40%) and BM (80%). Accordingly, certain input parameters have been recommended for successful MSCs isolation from UCB. On selecting samples in which recommended parameters were fulfilled, success rate was increased to 72%. This was together with providing optimal experiment conditions; mainly type of expansion medium, success rate reached 80%. Then, successfully expanded MSCs were subjected to chondrogenic differentiation by culturing in pelleted micromass system in presence of transforming growth factor beta-1 and chondrogenic medium devoid of fetal bovine serum to evaluate their ability to undergo chondrogenesis. Differentiation was verified microscopically using special stains, and proved by reverse transcriptase-polymerase chain reaction for expression of aggrecan and collagen II genes. In conclusion, in vitro differentiation into chondrocytes is possible from HUCB-MSCs. Microsc. Res. Tech., 2015 . © 2015 Wiley Periodicals, Inc.

Description: Cardiovascular disease is the primary cause of morbidity and mortality in today's world. Due to the lack of healthy autologous vessels, more tissue-engineered blood vessels are needed to repair or replace the damaged arteries. Biomaterials play an indispensable role in creating a living neovessel with biological responses. Silk fibroin produced by silkworms possesses good cytocompatibility, tailorable biodegradability, suitable mechanical properties, and minimal inflammatory reactions. In addition, regenerated silk fibroin solutions can be processed into various forms of scaffolds such as films, fibers, tubes, and porous sponges. These features make silk fibroin a promising biomaterial for small-diameter vascular grafts. The present article focuses on the applications of silk fibroin for vascular regeneration. A brief overview of the properties of silk fibroin is provided, following which the current research status and future directions of the main types of silk fibroin scaffolds for vascular regeneration are reviewed and discussed. Microsc. Res. Tech., 2015 . © 2015 Wiley Periodicals, Inc.

Description: ABSTRACT Polarized microscopy provides unique information on anisotropic samples. In its most complete implementation, namely Mueller microscopy, this technique is well suited for the visualization of fibrillar proteins orientations, with collagen in the first place. However, the intrinsic optical anisotropy of unstained tissues has to be enhanced by Picrosirius Red (PR) staining to enable Mueller measurements. In this work, we compared the orientation mapping provided by Mueller and second harmonic generation (SHG) microscopies on PR stained samples of vaginal and uterine cervix tissues. SHG is a multiphoton technique that is highly specific to fibrillar collagen, and was taken as the “gold standard” for its visualization. We showed that Mueller microscopy can be safely used to determine collagen orientation in PR stained cervical tissue. In contrast, in vaginal samples, Mueller microscopy revealed orientations not only of collagen but also of other anisotropic structures. Thus PR is not fully specific to collagen, which necessitates comparison to SHG microscopy in every type of tissue. In addition to this study of PR specificity, we determined the optimal values of the staining parameters. We found that staining times of 5 min, and sample thicknesses of 5 µm were sufficient in cervical and vaginal tissues. Microsc. Res. Tech., 2015 . © 2015 Wiley Periodicals, Inc.

Description: Beet Armyworm, Spodoptera exigua is a herbivorous moth and a serious pest of many economically important plants, which are used as food sources. Because of rigorous standards of food quality, usage of synthetic insecticides in crop protection, against pests, is limited. Solanaceae plant extracts may be a relatively cheap source of efficient natural insecticides that can limit usage of synthetic substances. Their biological activity is not fully known. In particular, ultrastructural studies, using transmission electron microscopy, are not usual. In the present article we describe the effects of sublethal concentrations of tomato and potato leaf extracts against S. exigua . Acute lethal effects were not observed. Both extracts exerted similar effects within midgut and fat body cells. Midgut cells were not significantly altered while fat body cells showed prominent swelling of nuclear envelope and endoplasmic reticulum, vacuolization of mitochondria and fusion of fat droplets. These changes were much more intensive within groups exposed to potato than tomato extracts at highest concentration at least. Light microscopy was used to observe and document developmental alterations of S. exigua exposed to potato and tomato leaf extracts. Potato leaf extracts significantly decreased hatching success and caused morphological malformations of imagoes. Among them, malformations of wings were the most prominent. Interestingly, these effects were not observed within populations exposed to tomato extracts at highest concentration at least.

Description: Surface ultrastructure of the gills of the angler catfish Chaca chaca was investigated to unravel the adaptive modifications associated with the feeding ecology of the fish. The fish is often found in mud or in soft substrates where they remain buried both for protection and to feed. Gill rakers present on the gill arch in most fish species are absent in this fish. The absence of gill rakers are associated with the feeding habit of the fish and is considered to facilitate the swallowing of captured prey smoothly without any hindrance. Highly corrugated surface of the gill arch and gill filaments could be associated to retain water/mucus to prevent dessicassion of the fish. Papillae like epithelial protuberances each bearing a taste bud at its summit toward the pharyngeal side of the gill arch is associated with the sorting of the food. Large number of mucous goblet cells on the gill arch epithelium are considered to secret copious mucus to lubricate the prey for easy swallowing. In C. chaca the gill septa between gill filaments are reduced. This could enhance the flexibility and permit the free movement of the gill filaments. Extensive secondary lamellae and infrequent mucous goblet cells on secondary lamellae are associated to increase the surface area to enhance efficiency of gaseous exchange.

Description: In the present research, different adhesive techniques were used to set up fillings with composite resins. After the application of etch and rinse or self etch adhesive technique, marginal adaptation of composite fillings was estimated by the length of margins without gaps, and by the microretention of resin in enamel and dentin. The study material consisted of 40 extracted teeth. Twenty Class V cavities were treated with 35% phosphorous acid and restored after rinsing by Adper Single Bond 2 and Filtek Ultimate - ASB/FU 3M ESPE composite system. The remaining 20 cavities were restored by Adper Easy One - AEO/FU 3M ESPE composite system. Marginal adaptation of composite fillings was examined using a scanning electron microscope (SEM). The etch and rinse adhesive technique showed a significantly higher percentage of margin length without gaps (in enamel: 92.5%, in dentin: 57.3%), compared with the self-etch technique with lower percentage of margin length without gaps, in enamel 70.4% ( p  〈 .001), and in dentin-22.6% ( p  〈 .05). In the first technique, microretention was composed of adhesive and hybrid layers as well as resin tugs in interprismatic spaces of enamel, while the dentin microretention was composed of adhesive and hybrid layers with resin tugs in dentin canals. In the second technique, resin tugs were rarely seen and a microgap was dominant along the border of restoration margins. The SEM analysis showed a better marginal adaptation of composite resin to enamel and dentin with better microretention when the etch and rinse adhesive procedure was applied.

Description: The purpose of this study was to compare total-etch, self-etch, and selective etching techniques on the marginal microleakage of Class V composite restorations prepared by Er:YAG laser and bur. Class V cavities prepared on both buccal and lingual surfaces of 30 premolars by Er:YAG laser or bur and divided into six groups. The occlusal margins were in enamel, and the cervical margins were in cementum. Group-1: bur preparation(bp)+Adper Single Bond 2 (ASB)+Filtek Z550 (FZ); Group-2: laser preparation(lp)+(ASB)+(FZ); Group-3: bp + Clearfil S3 Bond Plus (CSBP)+(FZ); Group-4: lp+(CSBP) (FZ); Group-5: bp + acid etching+(CSBP)+(FZ); Group-6: lp + acid etching+(CSBP)+(FZ). All teeth were stored in distilled water at 37°C for 24 hr, and then thermocycled 1000 times (5–55°C). Five teeth from each group were chosen for the microleakage investigation, and two teeth for the scanning electron microscope evaluation. Teeth which were prepared for the microleakage test were immersed in .5% methylene blue dye for 24 hr. After immersion, the teeth were sectioned and observed under a stereomicroscope for dye penetration. Data were analyzed using Kruskal–Wallis and Mann–Whitney U tests ( p  〈 .05). More microleakage was observed in the cervical regions compared to the occlusal regions in Groups 3, 5, and 6, respectively ( p  〈 .05). There is no statistically significant difference in Groups 1, 2, and 4, in terms of cervical regions versus occlusal regions ( p  〉 .05). No significant differences were observed among any groups in terms of occlusal and cervical surfaces, separately ( p  〉 .05). Different etching techniques did not influence microleakage of Class V restorations prepared by Er:YAG laser and bur.

Description: Protein–membrane interactions are still an important topic of investigation. One of the suitable experimental techniques used by the scientific community to address such question is atomic force microscopy. In a previous work, we have reported that the binding mechanism between the cytolytic and antimicrobial protein (Cyt2Aa2) and lipid/cholesterol bilayers was concentration-dependent, leading to either the formation of holes in the bilayer or aggregates. Here we study such binding mechanism as a function of time at low protein concentrations (10 µg/mL). We demonstrate that although holes are formed during the first stages of the protein–lipid interaction, a reparation process due to molecular mobility in the bilayer leads to a homogenous and isotropic protein–lipid/cholesterol layer within 3 hr. The combination of imaging, force spectroscopy, and phase contrast delivered information about topography dynamics (molecular mobility), layer thickness, and mechanical properties of the protein–lipid/cholesterol system. These results highlight the importance of the observation time in (such type of) protein–lipid interactions (at low protein concentrations).

Description: Atomic force microscopy (AFM) is an enormous tool to observe nature in highest resolution and understand fundamental processes like friction and tribology on the nanoscale. Atomic resolution in highest quality was possible only in well-controlled environments like ultrahigh vacuum (UHV) or controlled buffer environments (liquid conditions) and more specified for long-term high-resolution analysis at low temperatures (∼4 K) in UHV where drift is nearly completely absent. Atomic resolution in these environments is possible and is widely used. However, in uncontrolled environments like air, with all its pollutants and aerosols, unspecified thin liquid films as thin as a single molecular water-layer of 200 pm or thicker condensation films with thicknesses up to hundred nanometer, have been a problem for highest resolution since the invention of the AFM. The goal of true atomic resolution on hydrophilic as well as hydrophobic samples was reached recently. In this manuscript we want to review the concept of ambient AFM with atomic resolution. The reader will be introduced to the phenomenology in ambient conditions and the problems will be explained and analyzed while a method for scan parameter optimization will be explained. Recently developed concepts and techniques how to reach atomic resolution in air and ultra-thin liquid films will be shown and explained in detail, using several examples.

Description: The visualization of taxonomically diagnostic features of individual pollen grains can be a challenge for many ecologically and phylogenetically important pollen types. The resolution of traditional optical microscopy is limited by the diffraction of light (250 nm), while high resolution tools such as electron microscopy are limited by laborious preparation and imaging workflows. Airyscan confocal superresolution and structured illumination superresolution (SR-SIM) microscopy are powerful new tools for the study of nanoscale pollen morphology and three-dimensional structure that can overcome these basic limitations. This study demonstrates their utility in capturing morphological details below the diffraction limit of light. Using three distinct pollen morphotypes ( Croton hirtus, Dactylis glomerata , and Helianthus sp.) and contrast-enhancing fluorescent staining, we were able to assess the effectiveness of the Airyscan and SR-SIM. We further demonstrate that these new superresolution methods can be easily applied to the study of fossil pollen material.

Description: Segmentation of objects from a noisy and complex image is still a challenging task that needs to be addressed. This article proposed a new method to detect and segment nuclei to determine whether they are malignant or not (determination of the region of interest, noise removal, enhance the image, candidate detection is employed on the centroid transform to evaluate the centroid of each object, the level set [LS] is applied to segment the nuclei). The proposed method consists of three main stages: preprocessing, seed detection, and segmentation. Preprocessing stage involves the preparation of the image conditions to ensure that they meet the segmentation requirements. Seed detection detects the seed point to be used in the segmentation stage, which refers to the process of segmenting the nuclei using the LS method. In this research work, 58 H&E breast cancer images from the UCSB Bio-Segmentation Benchmark dataset are evaluated. The proposed method reveals the high performance and accuracy in comparison to the techniques reported in literature. The experimental results are also harmonized with the ground truth images.

Description: In this paper, by applying the differential quadrature (DQ) method, a semi analytical model has been developed for atomic force microscope cantilever, and then by using the interfacial forces between the cantilever tip and imaged surfaces, a 2D model has been extracted for imaging nano-sized fine samples. By employing the present model, several simple and standard samples have been imaged, and finally the effects of the microcantilever's structural damping and its stiffness on the imaging results have been investigated. It has been observed that, through the control of damping, the quality of the acquired images is considerably improved. It has also been shown that the self-softening and self-hardening properties of cantilever have serious effects on the obtained images. The present model can be used to study the effects of different parameters on the process of imaging small-scale samples. Also, as one of its most important applications, this model can be used in common multiscale models for simulating and predicting the effects of large and small fields on each other.

Description: Three-dimensional optical super-resolution imaging is capable of providing 3D visualization of cellular structures in nanoscale detail. The past decade has witnessed the blossoming of 3D super-resolution imaging technologies. In this review, we comprehensively discuss and compare the imaging depth, resolution enhancement, and imaging speed of the existing 3D super-resolution imaging techniques.

Description: Reversible photoswitching has been proposed as a way to identify molecules that are present in small numbers over a large, non-switching, background. This approach, called optical-lock-in-detection (OLID) requires the deterministic control of the fluorescence of a photochromic emitter through optical modulation between a bright (on) and a dark state (off). OLID yields a high-contrast map where the switching molecules are pinpointed, but the fractional intensities of the emitters are not returned. The present work presents a modified OLID approach (quantitative OLID or qOLID) that yields quantitative information of the switching (f SW ) and non-switching (f NS ) components. After the validation of the method with a sample dataset and image sequence, we apply qOLID to measurements in cells that transiently express the photochromic protein EYQ1. We show that qOLID is efficient in separating the modulated from the non-modulated signal, the latter deriving from background/autofluorescence or fluorophores emitting in the same spectral region. Finally, we apply qOLID to Förster (Fluorescence) Resonance Energy Transfer (FRET) imaging. We here demonstrate that qOLID is able to highlight the distribution of FRET intensity in a sample by using a photochromic donor and a non-photochromic acceptor.

Description: Introduction Pregnancy-related deep vein thrombosis (DVT) is most common during the late phase of pregnancy and the first 6-weeks postpartum. Pregnancy-related DVT can have long-term complications, specifically post-thrombotic syndrome (PTS). Fibrin network ultrastructure is altered during pregnancy and post-partum. It is therefore essential to evaluate fibrin fiber diameter during and after pregnancy as this may provide insight into pregnancy-related DVT and subsequent PTS. Materials and methods The fibrin network ultrastructure of females during different phases of pregnancy was compared to that of non-pregnant females to assess possible changes to the fibrin network morphology and fibrin fiber diameter using scanning electron microscopy micrographs. Results The fibrin network arrangement was more densely packed during different phases of pregnancy, corresponding to earlier findings. Fibrin diameter decreased significantly during pregnancy, with the greatest decrease occurring during the late phase of pregnancy. The fractal dimensions of fibrin micrographs increased significantly during pregnancy compared to nonpregnant females. These changes are indicative of a simultaneous hypercoagulable and hypofibrinolytic state and correspond to the increased risk of DVT and subsequent development of PTS. Conclusion It is critical to identify “vulnerable” females with an inflammatory predisposition to prevent possible DVT and subsequent PTS. Modifiable risk factors like obesity and smoking should be addressed to alleviate the burden on the coagulation system. Morphological and viscoelastic techniques are crucial in assessing the coagulatory health of females during pregnancy.

Description: Objectives Chemotherapy targets rapidly dividing tissues in the body. It destroys the progenitor cells in gonads resulting in premature ovarian failure. Studies have suggested that bone marrow-derived stem cells can generate oocytes in chemotherapy treated female rats after transplantation. The present study aimed to assess mechanism of homing, the action of injected BM-MSCs on ovarian function after ovarian damage. Experimental design : Seventy two female albino rats were randomly allocated into Control and CTX group, The Experimental protocol was lasted for 12 weeks during which serum FSH and E2 were monitored twice at the end of the 2nd week (12 rats) and 8th week (6 rats). Stem cells identification and homing were evaluated by Flowcytometry and tagging of stem cells with iron oxide particles respectively. Also, histopathological examination was done to evaluate both degeneration (6 rats at 4th week) and regeneration (6 rats at 12th week) of ovarian tissue together with assessment of the levels of TNF-α in ovarian homogenate and IGF-I as a growth factor in ovarian tissue. Principal observations : Partial improvement of E2 and FSH levels as well as ovarian architecture. Elevation of ovarian TNF- α levels and of IGF-I immunohistochemical expressions in ovarian tissues of BM-MSCs injected rats were noticed following homing of BM- MSCs in the ovarian stroma in both control and chemotherapy groups. Conclusion : Injected BM- MSCs can home in the stroma of the injured ovaries. IGF-I and TNF- α may have a role in the attraction of stem cells in vivo.

Description: Lasers and photodynamic therapy have been considered a convergence treatment for onychomycosis, which is a fungal infection on the nail bed and nail plate. Laser therapies have shown satisfactory results without significant complications for onychomycosis; however, the mechanism of clearing remains unknown. In this work, we investigated changes in the chemical structure of nail keratin induced by Nd:YAG laser using Raman spectroscopy. Toe nails with onychomycosis were treated with 1064 nm Nd:YAG laser. After laser treatment, the disulfide band (490–590 cm −1 ) of nail keratin was rarely observed or was reduced in intensity. The amide I band (1500–1700 cm −1 ) also showed changes induced by the laser. The α-helical (1652 cm −1 ) structures dominated the β-sheet (1673 cm −1 ) in nontreated nail, but the opposite phenomenon was observed after laser treatment.

Description: This note reports on the proper correction of force data acquired with an atomic force microscope (AFM). The force–time representation is hereby used to obtain the correction factors for the overall offset and slope for a single force–time curve, as the initial force, F 0  =  F ( t 0 ), and the rate of change in the force per unit of time, dF/dt , respectively. The report shows that a complete set of force data, including the approach, delay and retraction regions, can be simultaneously corrected in the force–time representation by subtracting the line CL t = F 0  +  dF/dt·t to the experimental data. The method described here outperforms the one commonly employed in the correction of AFM force curves and highlights the convenience of using the force–time representation for force data processing wherein the artifactual behavior can be expressed as a single, differentiable function of time.

Description: To better understand the basis of urinary calculi formation, we studied the composition, microstructure, and element analysis of different types of urinary calculi. Sixty people with urinary stones in Shanxi province were selected randomly. The composition of urinary stones was analyzed using Fourier transform infrared spectroscopy. The microstructure of material components was observed by a scanning electron microscopy and the elemental distribution and composition were analyzed by an X-ray energy spectrometer. Furthermore, general information, BMI, history of medicine, chronic medical history, family history, and recurrence rates were collected. Female-to-male ratio was 1:2.5; median age was 43.2 years old. Of the patients, 13.3% were found definite family history and 46.6% of patients for recurrence history. It was found that mixed stones account for the largest proportion (65%), followed by calcium oxalate monohydrate calculi (26.67%). In mixed stones, the mixture of calcium oxalate monohydrate and hydroxyapatite had the largest proportion, accounting for 71.79%. Stones showed different microcosmic characteristics and element distribution. Stones varied widely in distribution, infrared spectrum, microstructure, and element composition, which provided an important basis for urinary calculi research regarding urinary stone formation.

Description: The spatial resolution of a stimulated emission depletion (STED) microscope is theoretically unlimited and practically determined by the signal-to-noise ratio. Typically, an increase of the STED beam's power leads to an improvement of the effective resolution. However, this improvement may vanish because an increased STED beam's power is often accompanied by an increased photobleaching, which worsen the effective resolution by reducing the signal strength. A way to lower the photobleaching in pulsed STED (P-STED) implementations is to reduce the peak intensity lengthening the pulses duration (for a given average STED beam's power). This also leads to a reduction of the fluorophores quenching, thus a reduction of the effective resolution, but the time-gated detection was proved to be successful in recovering these reductions. Here we demonstrated that a subnanosecond fiber laser beam (pulse width ∼600 ps) reduces the photobleaching with respect to a traditional stretched hundreds picosecond (∼200 ps) beam provided by a Ti:Sapphire laser, without any effective spatial resolution lost.

Description: This study describes the mucosa-associated lymphoid tissue (MALT) in odontocetes from the Brazilian coast and freshwater systems. Seven species were evaluated and tissue samples were analyzed by light, scanning and transmission electron microscopy, and immunohistochemistry. Laryngeal tonsil was a palpable oval mass located in the larynx, composed of a lymphoepithelial complex. Dense collections of lymphocytes were found in the skin of male fetus and calf. Clusters of lymphoid tissue were found in the uterine cervix of a reproductively active juvenile female and along the pulmonary artery of an adult female. Lymphoid tissues associated with the gastrointestinal tract were characterized by diffusely arranged or organized lymphocytes. The anal tonsil was composed of an aggregate of lymphoid tissue occurring exclusively in the anal canal, being composed of squamous epithelium branches. MALT was present in different tissues and organic systems of cetaceans, providing constant protection against mucosal pathogens present in their environment.

Description: ABSTRACT With the development of spherical aberration (Cs) corrected scanning transmission electron microscopy (STEM), high angle annular dark filed (HAADF) imaging technique has been widely applied in the microstructure characterization of various advanced materials with atomic resolution. However, current qualitative interpretation of the HAADF image is not enough to extract all the useful information. Here a modified peaks finding method was proposed to quantify the HAADF-STEM image to extract structural and chemical information. Firstly, an automatic segmentation technique including numerical filters and watershed algorithm was used to define the sub-areas for each atomic column. Then a 2D Gaussian fitting was carried out to determine the atomic column positions precisely, which provides the geometric information at the unit-cell scale. Furthermore, a self-adaptive integration based on the column position and the covariance of statistical Gaussian distribution were performed. The integrated intensities show very high sensitivity on the mean atomic number with improved signal-to-noise ( S/N ) ratio. Consequently, the polarization map and strain distributions were rebuilt from a HAADF-STEM image of the rhombohedral and tetragonal BiFeO 3 interface and a MnO 2 monolayer in LaAlO 3 /SrMnO 3 /SrTiO 3 heterostructure was discerned from its neighbor TiO 2 layers. Microsc. Res. Tech., 2016 . © 2016 Wiley Periodicals, Inc.

Description: ABSTRACT The ultrastructural study carried out on (a) oocytes of Armadillidium vulgare during vitellogenesis, (b) mature eggs taken from the ovaries during the parturial moult of the posterior half of the body, and (c) fertilized eggs collected within a few hours of their release into the brood pouch, has clearly demonstrated that before the fertilization the chorion is the only envelope present in the egg of oniscidean isopods. In the mature eggs, the chorion appears as a uniformly electron-dense lamina, about 0.4–0.5 µm thick, which does not show any specialized area. A second envelope, described by other authors as vitelline envelope, is formed above the oolemma only right after fertilization and appears separated from the chorion by a space full of liquid. The ways in which the genesis of this envelope is realized are not yet clear; it could be interpreted rather as a fertilization membrane. The investigations carried out with the aid of a battery of FITC-lectins have highlighted the presence at the chorion surface of unfertilized eggs of various saccharide residues distributed in uniform way. No significant change was observed in the pattern of lectins binding to the chorion of eggs taken from the brood pouch, thus demonstrating how, after the fertilization, no significant rearrangement in the distribution of saccharide residues present on the egg surface occurs in A. vulgare . The ways in which, therefore, the recognition, the binding and the entry of the peculiar sperm of oniscidean isopods into the egg occur, still remain all to be deciphered. Microsc. Res. Tech., 2016 . © 2016 Wiley Periodicals, Inc.

Description: ABSTRACT The opossum Monodelphis domestica presents movement of the temporomandibular joint (TMJ) reflecting adaptation to eating habits similar to movement in humans, but the structure of the TMJ is not yet known. Thus, nine young M. domestica , of both sexes were weighed, anesthetized with xylazine (10 mg kg −1 ), and ketamine (70 mg kg −1 ) and processed for: 1. The analyses of the macroscopic angioarchitecture after latex injection, as well as the topography of the TMJ; 2. The analysis of microvascularization after injection of Mercox resin and corrosion of soft tissue with NaOH using scanning electron microscopy and; 3. The histological evaluation of the TMJ with an optical microscope. Macroscopic analysis of the latex injected vessels revealed the distribution of the arteries from the common carotid artery, receiving branches of the superficial temporal and maxillary arteries. The mandibular condyle has the long axis in the lateral–lateral direction, and is convex in the anterior–posterior direction. Its topography was determined in relation to the eye and external acoustic meatus. With scanning electron microscopy, microvascularization consists of arterioles of varying diameter (85–15 µm) of the meandering capillary network in the retrodiscal region, and a network of straight capillaries in the TMJ anterior region. Via light microscopy the TMJ has similar histological features to those of humans. These macroscopic, microscopic and ultrastructural data from TMJ of the M. domestica could be a suitable model for TMJ physiology and pathophysiology studies for then speculate on possible human studies. Microsc. Res. Tech., 2016 . © 2016 Wiley Periodicals, Inc.

Description: Oracle Bone Inscriptions in the Shang dynasty (1600–1046 BC) are the earliest well-developed writing forms of the Chinese character system, and their carving techniques have not been studied by tool marks analysis with microscopy. In this study, a digital microscope with three-dimensional surface reconstruction based on extended depth of focus technology was used to investigate tool marks on the surface of four pieces of oracle bones excavated at the eastern area of Huayuanzhuang, Yinxu site(ca., 1319–1046 BC), the last capital of the Shang dynasty, Henan province, China. The results show that there were two procedures to carve the characters on the analyzed tortoise shells. The first procedure was direct carving. The second was “outlining design,” which means to engrave a formal character after engraving a draft with a pointed tool. Most of the strokes developed by an engraver do not overlap the smaller draft, which implies that the outlining design would be a sound way to avoid errors such as wrong and missing characters. The strokes of these characters have different shape at two ends and variations on width and depth of the grooves. Moreover, the bottom of the grooves is always rugged. Thus, the use of rotary wheel-cutting tools could be ruled out. In most cases, the starting points of the strokes are round or flat while the finishing points are always pointed. Moreover, the strokes should be engraved from top to bottom. When vertical or horizontal strokes had been engraved, the shell would be turned about 90 degrees to engrave the crossed strokes from top to bottom. There was no preferred order to engrave vertical or horizontal strokes. Since both sides of the grooves of the characters are neat and there exists no unorganized tool marks, then it is suggested that some sharp tools had been used for engraving characters on the shells. Microsc. Res. Tech., 2016 . © 2016 Wiley Periodicals, Inc.

Description: ABSTRACT Their aim was to examine whether microvascular leakage of endogenous albumin, a representative marker for blood–brain barrier (BBB) damage, was induced in the periventricular area of diabetic db/db mice because periventricular white matter hyperintensity formation in magnetic resonance images was accelerating in elderly patients with diabetes mellitus. Using light and electron microscopes, and semi-quantitative analysis techniques, immunoreactivity of endogenous albumin, indicating vascular permeability, was examined in the periventricular area and spinal cord of db/db mice and db/+m control mice. Greater immunoreactivity of albumin was observed in the vessel wall of the periventricular area of db/db mice than in controls. Additionally, weak immunoreactivity was observed in the spinal cord of both db/db mice and controls. The number of gold particles, indicating immunoreactivity of albumin, in the perivascular area of db/db mice was significantly higher than that of control mice, but there was no significant difference in the number of particles in the spinal cord between db/db mice and controls. These findings suggest that albumin microvascular leakage, or BBB breakdown, is induced in the periventricular area of diabetic mice. Microsc. Res. Tech., 2016 . © 2016 Wiley Periodicals, Inc.

Description: ABSTRACT Alzheimer's disease (AD) is the main cause of dementia in the elderly population. Over 30 million people worldwide are living with dementia and AD prevalence is projected to increase dramatically in the next two decades. In terms of neuropathology, AD is characterized by two major cerebral hallmarks: extracellular β-amyloid (Aβ) plaques and intracellular Tau inclusions, which start accumulating in the brain 15-20 years before the onset of symptoms. Within this context, the scientific community worldwide is undertaking a wide research effort to detect AD pathology at its earliest, before symptoms appear. Neuroimaging of Aβ by positron emission tomography (PET) is clinically available and is a promising modality for early detection of Aβ pathology and AD diagnosis. Substantive efforts are ongoing to develop advanced imaging techniques for early detection of Tau pathology. Here, we will briefly describe the key features of Tau pathology and its heterogeneity across various neurodegenerative diseases bearing cerebral Tau inclusions (i.e., tauopathies). We will outline the current status of research on Tau-specific PET tracers and their clinical development. Finally, we will discuss the potential application of novel super-resolution and label-free techniques for investigating Tau pathology at the experimental level and their potential application for AD diagnosis. Microsc. Res. Tech., 2016 . © 2016 Wiley Periodicals, Inc.

Description: ABSTRACT This review reports on the combined use of the atomic force microscopy (AFM) and several type of optical/fluorescence/laser scanning microscopy for investigating cells. It is shown that the hybrid systems of AFM with optical-derived microscopies enable to study in detail cell surface properties (such as topography), their mechanical properties (e.g., Young's modulus) mechanotransduction phenomena and allow to gain insight into biological-related pathways and mechanisms in the complex nanoworld of cells. Microsc. Res. Tech., 2016 . © 2016 Wiley Periodicals, Inc.

Description: ABSTRACT The study of phenotypic variation in plant pathogenesis provides fundamental information about the nature of disease resistance. Cellular mechanisms that alter pathogenesis can be elucidated with confocal microscopy; however, systematic phenotyping platforms—from sample processing to image analysis—to investigate this do not exist. We have developed a platform for 3D phenotyping of cellular features underlying variation in disease development by fluorescence-specific resolution of host and pathogen interactions across time (4D). A confocal microscopy phenotyping platform compatible with different maize–fungal pathosystems (fungi: Setosphaeria turcica , Cochliobolus heterostrophus , and Cercospora zeae-maydis ) was developed. Protocols and techniques were standardized for sample fixation, optical clearing, species-specific combinatorial fluorescence staining, multisample imaging, and image processing for investigation at the macroscale. The sample preparation methods presented here overcome challenges to fluorescence imaging such as specimen thickness and topography as well as physiological characteristics of the samples such as tissue autofluorescence and presence of cuticle. The resulting imaging techniques provide interesting qualitative and quantitative information not possible with conventional light or electron 2D imaging. Microsc. Res. Tech., 2016 . © 2016 Wiley Periodicals, Inc.

Description: ABSTRACT The description and application of a modified Scanning Electron Microscope preparation technique using hexamethyldisilazane for small parasitic copepods was demonstrated though a high resolution depiction of individuals of Ergasilus labracis sampled from three spined stickleback ( Gasterosteus aculeatus) in Bay D'Espoir, Newfoundland during summer 2015 and from archival samples retrieved from Atlantic salmon par ( Salmo salar ) stored at the Atlantic reference centre, St. Andrews, New Brunswick. The specimens were very well preserved showing high quality detail of important features and verifying those previously described using light microscopy by Hogans. Additionally the technique allowed excellent in situ demonstrations of mouth parts, swimming legs, and unusual and previously undescribed features of the second antenna including prominent striations and pore-like structures found to define the claw. It is thought that this technique will become a quick and efficient tool for describing important taxonomic features of small parasitic copepods like E. labracis or other similar small aquatic organisms. Microsc. Res. Tech., 2016 . © 2016 Wiley Periodicals, Inc.

Description: ABSTRACT The endometrial layer of the uterus is characterized by continuous cycle of cell growth and apoptosis in response to hormonal changes. Apoptosis is regulated by several apoptotic regulators, but their significance in involuting uterus has not been well understood. For that reason, aim of this study was to investigate possible role of apoptosis-related proteins (bax and survivin) and enzymes (caspase-3 and calpain-1) in the involuting uterus of the rat, using immunohistochemistry. Our results indicated cytoplasmic and nuclear immunostaining for bax, caspase-3, calpain-1 and survivin proteins were found in the endometrial epithelium and stromal cells such as fibroblasts, mast cells and macrophages, and blood vessels; however, calpain-1 immunoreactivity in the endometrial fibroblast was quite weak or absent. Supranuclear punctate bax immunolabelling was also observed in the endometrial fibroblasts and luminal and glandular epithelial cells from days 1st and 3rd following parturition, respectively. Although survivin was localized in the apical cytoplasm underneath the apical membrane of the luminal epithelium on the 1st and 3rd days, it was also localized in the apicolateral membrane and basal cytoplasm on the 10th and 15th days of involution. Immunostainigs demonstrated that expression patterns of all examined proteins varied with structural changes in the luminal epithelium, and number of immunopositive fibroblasts for bax, caspase-3 and survivin increased with advance of postpartum days and reached a maximum on postpartum days 10 and 15. These results suggest that the process of postpartum involution of endometrium may be regulated by apoptotic and non-apoptotic activity of bax, caspase-3, calpain-1, and survivin. Microsc. Res. Tech., 2016 . © 2016 Wiley Periodicals, Inc.

Description: A large scale isolation and purification of theaflavin (TF) and epigallocatechin (EGC) has been successfully developed by tannase-mediated biotransformation combining high-speed countercurrent chromatography. After tannase hydrolysis of a commercially available theaflavins extract (TE), the content of TF and EGC in tannase-mediated biotransformation product (TBP) achieved approximately 3 times enrichment. SEM studies revealed smooth tannase biotransformation and the possibility of recovery of the tannase. A single 1.5 hours' HSCCC separation for TF and EGC employing a two-phase solvent system could simultaneously produce 180.8 mg of 97.3% purity TF and 87.5 mg of 97.3% purity EGC. However, a preparative HPLC separation of maximum injection volume containing 120 mg TBP prepared 11.2 mg TF of 94.9% purity and 7.7 mg EGC of 89.9% purity. HSCCC separation demonstrated significant advantages over Prep HPLC in terms of sample loading size, separation time, environmental friendly solvent systems, and the production.

Description: Acute lymphoblastic leukemia (ALL) is a cancer that starts from the early version of white blood cells called lymphocytes in the bone marrow. It can spread to different parts of the body rapidly and if not treated, would probably be deadly within a couple of months. Leukemia cells are categorized into three types of L1, L2, and L3. The cancer is detected through screening of blood and bone marrow smears by pathologists. But manual examination of blood samples is a time-consuming and boring procedure as well as limited by human error risks. So to overcome these limitations a computer-aided detection system, capable of discriminating cancer from noncancer cases and identifying the cancerous cell subtypes, seems to be necessary. In this article an automatic detection method is proposed; first cell nucleus is segmented by fuzzy c-means clustering algorithm. Then a rich set of features including geometric, first- and second-order statistical features are obtained from the nucleus. A principal component analysis is used to reduce feature matrix dimensionality. Finally, an ensemble of SVM classifiers with different kernels and parameters is applied to classify cells into four groups, that is noncancerous, L1, L2, and L3. Results show that the proposed method can be used as an assistive diagnostic tool in laboratories.

Description: Perpendicular or parallel magnetic fields are used to magnetize the tips used in magnetic force microscopy (MFM). In this process, perpendicular or parallel magnetic dipole moments are produced on the tip plane, thus leading to the formation of polar magnetized tips (PM-tips) or longitudinally magnetized tips (LM-tips), respectively. The resolution of an MFM image of a magneto-optic disk is used for calibration of these tips, and the saturated magnetic fields of the PM- and LM-tips are found to be 2720 Oe and 680 Oe, respectively. Because both tips can simultaneously magnetize the sample during the scanning process when measuring a Co thin film, clear MFM images are captured, which enable the identification of magnetizable regions and the distribution of the magnetic domains on the sample surface. These results will be useful for improving the manufacturing processes required for soft nano-magnetic film production.

Description: Objectives The aim of this study was to create an analytical tool for the three-dimensional distribution of immunohistochemically stained cells in the arteriovenous (AV) loop model of the femoral vessels of rats that fuses two-dimensional histological slides into stacks. Methods: A total of 22 AV loops were implanted into male syngeneic Lewis rats by creating an arteriovenous fistula between the femoral artery and vein by interposing a femoral vein graft of the contralateral extremity. This fistula was embedded into an isolation chamber filled with a fibrin matrix. Specimens were explanted after 7 to 14 days, and the AV loop was processed using standard histological protocols. Immunohistochemical staining for HIF-1α and a counter staining with hematoxylin was performed. Various layouts with different cutting planes, regions of interest, and post-processing algorithms were evaluated. Results and observations: The proximal-to-distal cutting perpendicular to the vascular axis proved to be the best layout for mapping the three-dimensional constructs containing the AV loop. A semi-automatic algorithm for the differentiation of immunohistochemical positive and negative cells was developed. Conclusion: The newly established methods from this study constitute an excellent tool for the general evaluation of the AV loop model – particularly with regard to the three-dimensional distribution of immunohistochemical positive and negative cells.

Description: An algorithm, based on the wavelet scalogram energy, for automatically detecting events in force-extension AFM force spectroscopy experiments is introduced. The events to be detected are characterized by a discontinuity in the signal. It is shown how the wavelet scalogram energy has different decay rates at different points depending on the degree of regularity of the signal, showing faster decay rates at regular points and slower rates at singular points (jumps). It is shown that these differences produce peaks in the scalogram energy plot at the event points. Finally, the algorithm is illustrated in a tether analysis experiment by using it for the detection of events in the AFM force-extension curves susceptible to being considered tethers.

Description: Objective To evaluate the safety of rapid cryopreservation for human testicular tissues by comparing the meiotic recombination in the fresh and thawed testis after rapid freezing. Methods Twelve male patients with prostate cancer (PC) who had given birth to healthy children at youth and need to receive surgical removal of testicular tissue at present were selected in this study. Testicular tissues from 4 cases of PC patients were randomly divided into two parts, one part as fresh tissue and the other to receive rapid freezing treatment. Fidelity analysis for homologous genetic recombination and synapsis were performed by immunofluorescence after prepared by a micro-spreading technique. Results The average number of MLH1 foci per cell, autosomal synaptonemal complex (SC) containing 0∼5 MLH1 foci and percent of cells with one MLH1 focus on XY chromosome showed no difference between the fresh and frozen thawed testicular tissues from the same case ( P 〉0.05). And, no significant difference in the frequency of gaps and splits on SCs was observed in fresh and thawed spermatocytes ( P  〉 0.05). Conclusion Rapid cryopreservation showed little effect on the frequency of meiotic recombination and fidelity of synapsis in human spermatocytes from PC patients, and acted as an effective method for preserving male fertility.

Description: The Pap smear is the primary screening tool for invasive cervical cancer resulting from a persistent infection with oncogenic human papillomavirus (HPV); however, there are the problems such as the inability to distinguish between HPV infection and cervical dysplasia and a low sensitivity remain. We present preliminary findings of a label-free method to detect and classify HPV infection and cervical dysplasia using human cervical fluids. Three experimental groups, defined as normal, HPV-positive, and cervical dysplasia, were evaluated through their Raman spectral patterns for noise-independence, high reproducibility, and uniformity. Clinical diagnosis was performed through liquid-based cervical cytology, HPV test, and cervical histologic examination. Healthy cervical fluids showed a strong Raman intensity at 877 cm −1 (symmetric C–C stretching), and at 963 cm −1 (phosphate), compared to a reference Raman peak at 1003 cm −1 (phenylalanine symmetric ring breath). The HPV-positive cervical fluids showed a strong intensity of a Raman peak at 1448 cm −1 corresponding to C–H deformation vibration mode and the highest similarity between the central and ring zones among the three groups. The cervical dysplasia fluids showed the presence of strong peaks compared to the control and HPV-positive groups. In addition, different Raman spectra were acquired according to HPV type. Therefore, all ranges of cervical fluid-induced Raman spectra could be used to detect the presence of cervical pre-cancer. Raman peak-gated assessment provides a label-free and nondestructive tool for the clinical diagnosis of HPV infection and cervical precancerous changes.

Description: This study intensely focuses on to the localization and identification of crustacean hyperglycemic hormone (CHH) producing neurosecretory cells in the eyestalk of the blue swimmer crab Portunus pelagicus . Anti- Carcinus maenas -CHH was used to identify the location of CHH neurosecretory cells by immunohistochemistry. Ten pairs of eyestalks were collected from intact adult intermoult female crab and fixed in Bouin's fixative. Eyestalks were serially sectioned and stained with chrome-hematoxylin-phloxine stain. Histological studies show the presence of different types of neurosecretory cells namely A (multipolar), B (tripolar), C (bipolar), D (unipolar), E (oval), and F (spherical) in the medulla interna, externa, and terminalis regions based on their size, shape, and tinctorial properties. The neurohemal organ, sinus gland (SG) was observed laterally between medulla interna and terminalis regions. Immunohistochemical studies showed the presence of distinct CHH-like immunoreactivity in the optic ganglia. Divergent group of neurosecretory cells with varying degree of immunoreactivity with Anti- Carcinus maenas -CHH (low, moderate, and intense reactivity) were identified in medulla terminalis, medulla interna, medulla externa, and sinus gland. The present study maps the various types of neurosecretory cells in the optic ganglia and also shows the presence of CHH-like immunoreactivity in various regions of optic ganglia in P. pelagicus . The presence of these unique neurosecretory cell types with larger cell diameter in medulla terminalis, a region that bears the neurosecretory cell bodies, suggest high secretory activity.

Description: ABSTRACT We describe here an automated imaging system developed at the Center for High Throughput Minimally Invasive Radiation Biodosimetry. The imaging system is built around a fast, sensitive sCMOS camera and rapid switchable LED light source. It features complete automation of all the steps of the imaging process and contains built-in feedback loops to ensure proper operation. The imaging system is intended as a back end to the RABiT—a robotic platform for radiation biodosimetry. It is intended to automate image acquisition and analysis for four biodosimetry assays for which we have developed automated protocols: The Cytokinesis Blocked Micronucleus assay, the γ-H2AX assay, the Dicentric assay (using PNA or FISH probes) and the RABiT-BAND assay. Microsc. Res. Tech., 2015 . © 2015 Wiley Periodicals, Inc.

Description: ABSTRACT Little is known about the development of the olfactory organs of camel. In this study, prenatal development and neuronal differentiation of the vomeronasal organ (VNO) and the olfactory epithelium (OE) of the one-humped camel were studied by immunohistochemistry and lectin histochemistry. A neuronal marker, protein gene product (PGP) 9.5, but not a marker of fully differentiated olfactory receptor cells, olfactory marker protein, intensely labeled the olfactory receptor cells of the VNO and OE at 395 mm, 510 mm, and 530 mm fetal ages, indicating that the olfactory receptor cells are differentiated, but not fully matured both in the VNO and the OE. In 187 mm and 190 mm fetuses, PGP 9.5 yielded faint immunoreactive signals in the VNO, but not in the OE, although the presence of olfactory receptor cells were demonstrated in both tissues by intense WGA and LEL stainings. We conclude that the camel VNO and OE bear differentiated, but still immature receptor cells; in addition, the onset of neuronal differentiation seems to be somewhat earlier in the VNO than in the OE till half of the prenatal life. Microsc. Res. Tech., 2015 . © 2015 Wiley Periodicals, Inc.

Description: ABSTRACT The aim of this study was to evaluate the effects of different power parameters of an Erbium, Cromium: Yttrium, Scandium, Gallium, Garnet laser (Er,Cr:YSGG laser) on the morphology, attachment of blood components (ABC), roughness, and wear on irradiated root surfaces. Sixty-five incisive bovine teeth were used in this study, 35 of which were used for the analysis of root surface morphology and ABC. The remaining 30 teeth were used for roughness and root wear analysis. The samples were randomly allocated into seven groups: G1: Er,Cr:YSGG laser, 0.5 W; G2: Er,Cr:YSGG laser, 1.0 W; G3: Er,Cr:YSGG laser, 1.5 W; G4: Er,Cr:YSGG laser, 2.0 W; G5: Er,Cr:YSGG laser, 2.5 W; G6: Er,Cr:YSGG laser, 3.0 W; G7: scaling and root planning (SRP) with manual curettes. The root surfaces irradiated by Er,Cr:YSGG at 1.0 W and scaling with manual curettes presented the highest degrees of ABC. The samples irradiated by the Er,Cr:YSGG laser were rougher than the samples treated by the manual curette, and increasing the laser power parameters caused more root wear and greater roughness on the root surface. The Er,Cr:YSGG laser is safe to use for periodontal treatment, but it is not appropriate to use irradiation greater than 1.0 W for this purpose. Microsc. Res. Tech., 2015 . © 2015 Wiley Periodicals, Inc.

Description: ABSTRACT Intramandibular glands have been poorly studied in polymorphic ants, where the differences between castes were unsufficiently scrutinized. Leaf-cutting ants possess one of the most complex systems of communication and labor division, which is polymorphic well as age polyethism, and makes them an ideal model for the study of intramandibular glands. This study has investigated the occurrence of intramandibular glands in female castes and subcastes of Atta laevigata . The mandibles of the queen, medium, and minor workers, and soldiers were submitted to histological, histochemical, ultrastructural, and morphometric analyses. The class-3 gland cells and the epidermal gland with a reservoir were found in all the castes. The queens and soldiers showed a higher number of class-3 gland cells, distributed within the mandible as well as a greater gland size in comparison to the workers. The histochemical tests, periodic acid-Schiff (PAS), mercury-bromophenol, and Nile blue, were similar for the class-3 gland cells and epidermal glands with a reservoir. However, the tests evidenced differences between the castes, with carbohydrates strongly positive in all of them, whereas neutral lipids were found in the queen and soldiers. The protein was weakly positive in the queen, whereas in the soldier, medium, and minor workers these reactions were strongly positive in the intramandibular glands. Our findings in A. laevigata suggest that intramandibular glands are directly involved in labor division and consequently in chemical communication between the castes. Microsc. Res. Tech., 2015 . © 2015 Wiley Periodicals, Inc.

Description: ABSTRACT The influence of environmental factors on dimensional measurements of atomic force microscopy (AFM) was investigated experimentally. Measurements were taken with environmental control over a whole AFM chamber and a local sample chamber to highlight the influence of working conditions on the instrument itself. Both temperature and humidity were found to have a significant impact on pitch measurements of a two-dimensional grating. The effect of temperature on the behavior of the microscope itself is generally larger than the thermal expansion or contraction of the sample. The effect of humidity was further determined to be relevant to the scan direction and velocity. For precise AFM dimensional measurements, the possible influences of temperature and humidity must be carefully considered. Microsc. Res. Tech., 2015 . © 2015 Wiley Periodicals, Inc.

Description: ABSTRACT Using high resolution transmission electron microscopy with image simulation and Fourier analysis, the Li 1− x FePO 4 ( x  〈 0.01), Li 1− x FePO 4 ( x ∼ 0.5), and FePO 4 particles, prepared by charging or discharging the 053048 electrochemical cells (thickness: 5 mm, width: 30 mm, height: 48 mm) and dismantled inside an Ar-filled dry box, were investigated. The high resolution images reveal: (1) the solid solution of Li 1− x FePO 4 ( x  〈 0.01) contains some missing Li ions leading PO 4 group distorted around M1 tunnel of the unit cell; (2) the texture of the particles of Li 1− x FePO 4 ( x ∼0.5) has homogeneously distributed compositional domains of LiFePO 4 and FePO 4 resulting from spinodal decomposition which promote Li ion easily getting into the particle due to uphill diffusion, (3) the particles of FePO 4 formed in charging have heavily distorted lattice and contain some isolated LiFePO 4 , (4) interface between LiFePO 4 and FePO 4 and between amorphous and crystal region provides the lattice distortion of small polarons. Microsc. Res. Tech., 2015 . © 2015 Wiley Periodicals, Inc.

Description: ABSTRACT The aim of this study was to evaluate the presence of residues of sodium hypochlorite gel, chlorhexidine gel, and EDTA gel on dentinal walls after canal preparation through chemical SEM- elemental chemical microanalysis (EDS) analysis. Forty-eight single-rooted teeth were selected. They had their crowns sectioned and were instrumented with a reciprocating system. The canals were irrigated with 5 mL of saline solution during root canal preparation. After instrumentation, the root canals were irrigated with 3 mL 17% EDTA followed by 1 min of ultrasonic passive activation (3× 20 sec) to remove the smear layer, and then irrigated with 3 mL of saline solution. The specimens were randomized into three groups ( n  = 12) according to the chemical substance that filled the root canal for 30 min: GI: 5.5% sodium hypochlorite gel; GII: 2% chlorhexidine gel; GIII: 24% EDTA gel; Negative control group: no substance was used. Then, the root canals were irrigated with 6 mL of saline solution followed by 1 min of ultrasonic passive activation (3× 20 sec). After ultrasonic activation, the canals were irrigated with 2 mL saline. The roots were sectioned, and the percentage of each chemical element present in the samples was analyzed through chemical SEM-EDS microanalysis. All experimental groups showed a significantly higher percentage of chemical elements (Na and/or Cl) than the control group ( P  〈 0.03). This in vitro study has shown that, regardless of chemical solutions used even after the final irrigation protocol, chemical residues of different substances remained attached to the root canal walls. Microsc. Res. Tech., 2015 . © 2015 Wiley Periodicals, Inc.

Description: ABSTRACT In the past years, the self-assembly of specific proteins has been paid more and more attention due to their significant role in human health and fabrication of new materials. In this article, we explore the effect of reaction conditions on the self-assembly of natural silk sericin protein, including the molecular weight and the concentration of sericin, pH, and metal ions in the reaction system. The results indicate that all these factors, especially species and concentration of metal ions, could influence the self-assembly process of the silk sericin protein. A series of assemblies with various morphologies can be fabricated by modulating the reaction condition. The article may provide some clue for the understanding of the protein self-assembly in the body and a method to fabricate new organic materials with different morphology. Microsc. Res. Tech., 2016 . © 2016 Wiley Periodicals, Inc.

Description: ABSTRACT Phyllotreta striolata (Fabricius) is an important pest of Brassicaceae in Southeast Asia and North America. Using scanning electron microscopy, we observed the external structure, number, and distribution of the antennal sensilla in P. striolata females to discuss the putative function of these sensilla in host location and oviposition behaviors. The antenna of female P. striolata is filiform, composed of a scape, a pedicel, and a flagellum with 9 flagellomeres. Five types of sensilla were identified, including sensilla cheaetica, sensilla trichodea, Böhm bristles, sensilla auricillica, and sensilla basiconica (five subtypes, SB1–SB2). External structure and distribution of antennal sensilla are compared with data from other insect species. In addition, we discuss the possible functions of antennal sensilla based on their characteristics. Microsc. Res. Tech., 2016 . © 2016 Wiley Periodicals, Inc.

Description: ABSTRACT Growth-year authentication has extraordinary significance for plant growth, structure and development research, and has a wide range of applications in value assessment of economic crops. Panax ginseng is the most commonly used medicinal plant in Asian countries. The fix number of growth-year is an important quality evaluation which is difficult to be obtained accurately in current technical conditions. Preliminary authentication theory for growth-year has been described in previous studies using a short-lived perennial medicinal plant ( Paeonia lactiflora pall.) as the research material. In this research, we focused on the growth-year estimation in ginseng cultivars, and attempt to explore the age estimation method for vascular plants according to mathematical simulation of the root structure development. Micro data was obtained from 204 individuals of 3 different kinds of ginseng cultivars, which have a series of gradient age and a clear growth record. Outer diameter of the vascular cambium (b) and the radius of cross section (r) were measured with ordinary stereo microscope. We further designed and established two different kinds of authentication model based on the taproot structure development for growth year authentication ( P and M = K*X 1 a 1 X 2 a 2 ). Moreover, the models were applied to identify the growth year of ginseng without damage using Micro-CT or DEI reconstruction. A potential method, have been recently described, the age of ginseng can be analyzed by telomere length and telomerase activity. However, we found that there are different results indicated in other species. We concluded that microscopic methods perceived currently were provided a more effective means for growth-year authentication. Microsc. Res. Tech., 2016 . © 2016 Wiley Periodicals, Inc.

Description: ABSTRACT In this contribution, they have attempted to develop a labeling technique for in vivo imaging of functionally active plasmid DNA in cyanobacterial cells through its decoration with semiconductor quantum dots (Qdots) as fluorescent nanoprobes. For that purpose biotinylated plasmid slr2060 DNA was conjugated with Qdots-streptavidine. The intact DNA was visualized in a single green color by light microscopy. These Qdots-DNA conjugates were capable of expressing the acyltransferase enzyme. Qdots-DNA conjugates and confocal microscope imaging technique were adopted to visualize the gene transport across the membrane of the live cyanobacteria cell in real time. Long-term kinetic study enabled to reveal the steps of extracellular and intracellular microenvironment for plasmid transportation into the live cell. To confirm these processes a confocal microscope and indicator plate assay test were applied in tandem. In this contribution, Qdots-labeled plasmid DNA was utilized for the first time for long-term intracellular imaging studies in cyanobacteria species PCC6803. The results showed that the Qdots-labeled plasmid DNA detection could be used as a powerful labeling technique for visualization of exogenous DNA entry and tracking into living cells by confocal microscopy. Microsc. Res. Tech., 2016 . © 2016 Wiley Periodicals, Inc.

Description: ABSTRACT The present study was designed to investigate the relationships between clinicopathological findings and the resorptive conditions of root apices of teeth with periodontitis. The samples included 21 root apices with large periapical radiolucent lesions. The preoperative computed tomography (CT) and intraoperative findings were correlated with the presence, extension, and the progression pattern of periapical resorption using a scanning electron microscope. The subjects' age, gender, chief complaint, type of tooth, percussion test results, size of periapical lesion using CT, and intraoperative findings were recorded. All apicoectomies were performed under an operative microscope for endodontic microsurgery. A significant large size was observed in cystic lesions compared with granulomatous lesions. The cementum surface at the periphery of the lesion was covered with globular structures (2–3 μm in diameter). Cementum resorption started as small defect formations at the surface. As the defect formation progressed, a lamellar structure appeared at the resorption area, and the size of globular structures became smaller than that of globules at the surface. Further resorption produced typical lacuna formation, which was particularly observed in fracture cases. The most morphologically severe destructive pattern of dentin resorption was observed in large cystic lesions. This study is the first report to elucidate the relationships between three clinical types of undesirable periapical lesions: (1) undertreatment, (2) periapical fracture, (3) macro-level resorption, and the microstructure of external root resorption including from small defects at the cementum surface to a significant destructive pattern inside the dentin. Microsc. Res. Tech., 2016 . © 2016 Wiley Periodicals, Inc.

Description: In this work, we present the method followed to construct a pseudophase diagram of two phospholipids: 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphoethanolamine and 1-palmitoyl-2-oleoyl- sn -glycero-3-phospho-(1′- rac -glycerol). Two different techniques, DSC and AFM, have been used based in the determination of the onset ( T onset ) and completion ( T offset ) temperatures of the gel-to-liquid crystalline phases ( L β L α ), the first from the endotherms from liposomes and the second from the topographic images of supported lipid bilayers. The features of both phase diagrams are discussed emphasizing the influence of Ca 2+ presence and the substrate (mica) on the transition undergone by the phospholipid mixture. Microsc. Res. Tech., 2016 . © 2016 Wiley Periodicals, Inc.

Description: ABSTRACT The micro-X-ray fluorescence by synchrotron radiation (μ-XRF) is a method to determine the composition of tissues without destroying the samples. However, this technique has never been used for the analysis of mesenchymal stem cells (MSC). This study compared different protocols for fixing, storing, preserving, and establishing the correct numbers of dental derived MSC submitted to μ-XRF analysis. Stem cells were obtained from human dental tissue. After cell expansion, and MACS isolation, the samples were fixed and the following quantities of cells 1 × 10 4 to 1 × 10 7 were divided in two groups: G1 : fixed in 4% paraformaldehyde diluted in phosphate-buffered saline solution, and G2: fixed in 4% paraformaldehyde diluted in MilliQ water. The G1 cells showed precipitation of chemical components from the solution resulting in the formation of salt crystals while G2 cells were clear and almost transparent in the sample holder. With regards to cells concentration, the best results occurred when four droplets of 1 × 10 7 cells were analyzed. This work shows that to identify and study the distribution of trace elements in MSC by μ-XRF, the best protocol is fixation in 4% paraformaldehyde diluted with MilliQ water at 4°C and a concentration of four incremental droplets of 1 × 10 7 cells. Microsc. Res. Tech., 2016 . © 2016 Wiley Periodicals, Inc.

Description: ABSTRACT Objectives We sought to devise and test a multifunctional contrast dye agent for X-ray based digital radiography (DR) or computer tomography (CT), magnetic resonance imaging (MRI), and colored staining in ex vivo validation part of animal experiments. Materials and Methods: The custom-formulated contrast dye namely red iodized oil (RIO) was prepared by solubilizing a lipophilic dye Oil Red O in iodized poppy seed oil (Lipiodol or LPD) followed by physicochemical characterizations. To explore and test the utility of RIO, normal rats ( n  = 10) and rabbits ( n  = 10) with myocardial infarction (MI) were euthanized by overdose of pentobarbital for infusion of RIO through catheterization. The bodies and/or excised organs including heart, liver, spleen, kidneys, pancreas, and intestines of the rats and rabbits were imaged at clinical mammography, CT and MRI units. These images were qualitatively studied and quantitatively analyzed using Wilcoxon Rank test with a P value 〈 0.05 being considered of a statistically significant difference. Imaging findings were verified by histomorphology. Results : All experimental procedures were carried out successfully with the use of RIO. T1 and T2 relaxation time was 234.2 ± 2.6 ms and 141.9 ± 3.0 ms for RIO, close to that of native LPD. Proton ( 1 H) NMR spectroscopy revealed almost identical profiles between RIO and native LPD. The clinical mammography unit, 128-slice CT scanner and 3.0T MRI magnet were well adapted for the animal experiments. Combined use of RIO with DR, MRI, CT and histology enabled microangiography of the organs, 3D visualization of rat pancreas, validation of in vivo cardiac quantification of MI and cause determination of the rabbit death after coronary occlusion. RIO appeared as red droplets and vacuoles in vessels by frozen and paraffin sections. Image analysis showed the superiority of DR images, which provided better overall image quality (4.35 ± 0.49) for all analyzed liver vessel segments. MRI images revealed moderate to good overall image quality ratings (3.45 ± 0.52). Comparing the signal intensities of vessel and liver with different MRI sequences, all P values were 〈0.01. Conclusions: RIO proved to be a multifunctional contrast dye, which could be applied as an imaging biomarker for tissue vascularity or blood perfusion, for visualization of organ anatomy and for ex vivo validation of in vivo animal experiments. Microsc. Res. Tech., 2016 . © 2016 Wiley Periodicals, Inc.

Description: ABSTRACT Nitric oxide (NO) is produced by nitric oxide synthases (NOSs) and plays an important role in all levels of reproduction from the brain to the reproductive organs. Recently, it has been discovered that all germ cells and Leydig cells in the cat testis exhibit stage-dependent nuclear and cytoplasmic endothelial (eNOS) and inducible (iNOS)-NOS immunoreactivity and cytoplasmic nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) reactivity. As a continuation of this finding, in this study, cellular localization of NADPH-d and immunolocalization and expression of all three NOS isoforms were investigated in the intratesticular (tubuli recti and rete testis), and excurrent ducts (efferent ductules, epididymal duct and vas deferens) of adult cats using histochemistry, immunohistochemistry and western blotting. NADPH-d activity was found in the midpiece of the spermatozoa tail and epithelial cells of all of ducts, except for nonciliated cells of the efferent ductules. Even though the immunoblotting results revealed similar levels of nNOS, eNOS and iNOS in the caput, corpus and cauda segments of epididymis and the vas deferens, immunostainings showed cell-specific localization in the efferent ductules and region- and cell-specific localization in the epididymal duct. All of three NOS isoforms were immunolocalized to the nuclear membrane and cytoplasm of the epithelial cells in all ducts, but were found in the tail and the cytoplasmic droplets of spermatozoa. These data suggest that NO/NOS activity might be of importance not only for the functions of the intratesticular and excurrent ducts but also for sperm maturation. Microsc. Res. Tech., 2016 . © 2016 Wiley Periodicals, Inc.

Description: ABSTRACT The Atomic Force Microscopy (AFM) technique appears as a central tool for the characterization of DNA adsorption onto lipid interfaces. Regardless of the huge number of surveys devoted to this issue, there are still fascinating phenomena in this field that have not been explored in detail by AFM. For instance, adsorption of DNA onto like-charged lipid surfaces mediated by cations is still not fully understood even though it is gaining popularity nowadays in gene therapy and nanotechnology. Studies related to the complexation of DNA with anionic lipids as a non-viral gene delivery vehicle as well as the formation of self-assembled nanoscale DNA constructs (DNA origami) are two of the most attractive systems. Unfortunately, molecular mechanisms underlying the adsorption of DNA onto anionic lipid interfaces remain unclear so far. In view of that, AFM becomes an appropriate technique to provide valuable information to understand the adsorption of DNA to anionic lipid surfaces. As a second part of this review we provide an illustrative example of application of the AFM technique to probe the DNA adsorption onto a model lipid monolayer negatively charged. Microsc. Res. Tech., 2016 . © 2016 Wiley Periodicals, Inc.

Description: ABSTRACT Core Facilities (CF) for advanced light microscopy (ALM) have become indispensable support units for research in the life sciences. Their organizational structure and technical characteristics are quite diverse, although the tasks they pursue and the services they offer are similar. Therefore, throughout Europe, scientists from ALM-CFs are forming networks to promote interactions and discuss best practice models. Here, we present recommendations for ALM-CF operations elaborated by the workgroups of the German network of ALM-CFs, German Bio-Imaging (GerBI). We address technical aspects of CF planning and instrument maintainance, give advice on the organization and management of an ALM-CF, propose a scheme for the training of CF users, and provide an overview of current resources for image processing and analysis. Further, we elaborate on the new challenges and opportunities for professional development and careers created by CFs. While some information specifically refers to the German academic system, most of the content of this article is of general interest for CFs in the life sciences. Microsc. Res. Tech., 2016 . © 2016 THE AUTHORS MICROSCOPY RESEARCH AND TECHNIQUE PUBLISHED BY WILEY PERIODICALS, INC.

Description: ABSTRACT Puberty is characterized by psychosomatic alterations, whereas chronic ethanol consumption is associated with morphophysiological changes in the male reproductive system. The purpose of this study was to show the toxic effects on testis and epididymal morphophysiology after ethanol administration during peripuberty. To this end, male Wistar rats were divided into two groups: ethanol (E) group: received a 2 g dose of ethanol/kg in 25% (v/v); and control (C) group: received the same volume of filtered water; both were treated by gavage for 54 days. On the 55th day of the experiment, epididymis, and testis were collected for sperm count, histopathology, mast cell count, and morphometry. The vas deferens was collected for sperm motility analysis. The femur and testicle were used for cytogenetic analysis. Ethanol exposure caused reduction in daily sperm production (DSP) and in sperm motility, multinucleated cells or those having no chromosomal content, and late chromosome migrations. No changes were observed in the number of chromosomes in the mitotic analysis. However, some alterations could be seen in meiocytes at different stages of cell division. Stereological analysis of the epididymis indicated reorganization of its component in the 2A and 5A/B regions. The epididymal cauda had greater recruitment, and both degranulated and full mast cells showed an increase in the initial segment, in the ethanol group. In conclusion, ethanol administration during the pubertal phase affects epididymis and testis in adult rats, as indicated mainly by our new findings related to mast cell number and meiotic impact. Microsc. Res. Tech., 2016 . © 2016 Wiley Periodicals, Inc.

Description: ABSTRACT We study the feasibility of volume imaging from a few angular views/scans in a light sheet fluorescence microscopy. Two-dimensional (2D) images (angular views) were acquired at an angular separation of 10° and volume images were constructed with merely 18, 9, and 6 views. We study the structural changes in a 5-day old Zebrafish embryo labeled with Phalloidin TRITC that binds to F-Actin of embryo cell. To collect the data, the specimen is rotated (for varying sampling angles Δθ) with respect to a fixed vertical axis passing through the volume-of-interest (yolk sac). In the proposed realization of selective plane illumination microscopy (SPIM) technique, the translation is completely avoided. Analysis shows rich structural information with marginal reduction in contrast. Comparison with the state-of-the-art SPIM shows appreciable volume reconstruction (from an order less 2D scans) that may be good enough for rapid monitoring of macroscopic specimens. Microsc. Res. Tech., 2016 . © 2016 Wiley Periodicals, Inc.

Description: ABSTRACT We describe the use of a rabbit maxillary sinus model, characterized by thin osseous tissue and low bone density, for the evaluation of surface-treated implants by histologically and histomorphometrically comparing the osseointegration patterns depending on the surface treatment methods. Twenty rabbits were randomly assigned to two groups of 10 animals, one receiving 5 × 3 mm customized implants (machined, MA or sandblasted and acid etched, SLA) placed in sinus and the other receiving implants placed in a tibia. Histological observation of the implant placed in sinus shows relatively more active new bone formation, characterized by trabecular bone pattern underneath the cortical bone in sinus as compared with that in tibia. Histomorphometric analysis in the rabbits receiving implants in a tibia, the NBIC (%) associated with the SLA surface implant was greater than that associated with the MA implant at 2 weeks (55.63 ± 8.65% vs. 47.87 ± 10.01%; P 〉 0.05) and at 4 weeks (61.76 ± 9.49% vs. 42.69 ± 10.97%; P 〈 0.05). Among rabbits receiving implants in a sinus, the NBIC (%) associated with the SLA surface implant was significantly greater than that associated with the MA surface implant both at 2 weeks (37.25 ± 7.27% vs. 20.98 ± 6.42%; P 〈 0.05) and at 4 weeks (48.82 ± 6.77% vs. 31.51 ± 9.14%; P 〈 0.05). As a result, we suggest that the maxillary sinus model is an appropriate animal model for assessing surface-treated implants and may be utilized for the evaluation of surface-treated implants in poor bone quality environment. Microsc. Res. Tech., 2015 . © 2015 Wiley Periodicals, Inc.

Description: ABSTRACT The region in tendons that surrounds bone extremities adapts to compression forces, developing a fibrocartilaginous structure. During maturation, changes occur in the amount and organization of macromolecules of the extracellular matrix of tendons, changing the tissue morphology. To study the effect of maturation on tendons, Pedrês chickens were sacrificed at 1, 5, and 8 months old and had the calcaneal tendon (CT) divided into proximal region, submitted to tension/compression forces ( p ), and distal region submitted to tension force ( d ). Morphological analysis of the p region showed the presence of fibrocartilage in all ages. In the central part of the fibrocartilage, near a diminishment of the metachromasy, there was also a development of a probable fat pad that increased with the maturation. The activity of MMP-2 and MMP-9 was higher at 5 and 8 months old, in both regions, compared with 1-month-old animals. In SDS-PAGE analysis, components with electrophoretic migration similar to decorin and fibromodulin increased with maturation, particularly in the d region. The Western blotting confirmed the increased amount of fibromodulin with maturation. In conclusion, our results show that process of maturation leads to the appearance of a probable fat pad in the center of the fibrocartilage of CT, with a reduced amount of glycosaminoglycans and an increased activity of MMPs. Microsc. Res. Tech., 2015 . © 2015 Wiley Periodicals, Inc.

Description: ABSTRACT The objective of this study was to characterize the three-dimensional (3D) surface micromorphology of the ceramics produced from nanoparticles of alumina and tetragonal zirconia (t-ZrO 2 ) with addition of Ca +2 for sintering improvement. The 3D surface roughness of samples was studied by atomic force microscopy (AFM), fractal analysis of the 3D AFM-images, and statistical analysis of surface roughness parameters. Cube counting method, based on the linear interpolation type, applied for AFM data was used for fractal analysis. The morphology of non-modified ceramic sample was characterized by the rather big (1–2 μm) grains of α-Al 2 O 3 phase with a habit close to hexagonal drowned in solid solution of t-ZrO 2 with smooth surface. The pattern surfaces of modified composite content a little amount of elongated prismatic grains with composition close to the phase of СаСеAl 3 О 7 as well as hexahedral α-Al 2 O 3 -grains. Fractal dimension, D , as well as height values distribution have been determined for the surfaces of the samples with and without modifying. It can be concluded that the smoothest surface is of the modified samples with Ca +2 modifier but the most regular one is of the non-modified samples. A connection was observed between the surface morphology and the physical properties as assessed in previous works. Microsc. Res. Tech., 2015 . © 2015 Wiley Periodicals, Inc.

Description: ABSTRACT The present study aims at characterizing the three-dimensional (3-D) morphology of a Co–Cr–Mo dental alloy surface as a result of three different procedures used for polishing it. The sample surface morphology of the sampled surface was examined employing atomic force microscopy (AFM), statistical surface roughness parameters, and fractal analysis. An extra-hard dental alloy of cobalt–chromium–molybdenum (Co–Cr–Mo) (Wironit ® , from BEGO, Bremen, Germany) was prepared and moulded. Different polishing treatments were carried out on three groups of six samples each—a total of 18 samples. The first group contained six electropolished (EP) samples. The second group containing six samples went through a mechanical polishing process employing green rubber discs and a high shine polishing paste applied by a rotating black brush (BB). The third group comprising six samples as well went through a mechanical polishing process by means of green rubber discs, high shine polishing paste, and a rotating deer leather brush (DL). Fractal analysis on the basis of a computational algorithm applied to the AFM data was employed for the 3-D quantitative characterization of the morphology of the sampled surfaces. The fractal dimension D (average ± standard deviation) of 3-D surfaces for BB samples (2.19 ± 0.07) is lower than that of the DL samples (2.24 ± 0.08), which is still lower than that of the EP samples (2.27 ± 0.09). The results indicated the BB samples as presenting the lowest values of statistical surface roughness parameters, thus the best surface finish, while the EP samples yielded the highest values. Microsc. Res. Tech., 2015 . © 2015 Wiley Periodicals, Inc.

Description: ABSTRACT The regeneration of the tail in lizards is likely sustained by stem/progenitor cells located in the stump after amputation of the tail. This microscopic and ultrastructural study shows the localization of 5-bromo-deoxy-uridine (5BrdU)-long retaining labeled cells in different tissues of the tail stump. These putative stem/progenitor cells are sparsely detected in the epidermis of scales, adipose tissue, intermuscle connective septa, myosatellite cells, and perichondrion of the vertebrae. Most of 5BrdU-labeled cells are present in the bone marrow of vertebrae as hemocytoblasts and reticulate cells, whereas more numerous myelocytes and polychromatophilic erythroblasts show a variable level of nuclear labeling. 5BrdU and tritiated-thymidine labeled and unlabeled hemopoietic cells are seen in circulating vessels and in the blastema where their maturation is completed. This observation indicates that the entire differentiation span of both white and red blood cells, at least during tail regeneration, lasts longer than 4 weeks. Labeled polychromatophilic erythroblasts and heterophilic and basophilic myelocytes are present in the synusoidal vessels of the regenerating tail. This study indicates that extravasating blood cells involved in immunity make large part of the forming blastema cell population, but are replaced by mesenchymal cells of different origin. The presence of long retaining labeled cells in tissues of the tail stump is likely connected to the production of blastema mesenchymal cells. Although no direct cell-lineage study has been done, histological, immunocytochemical, and autoradiographic studies have indicated that it is from these tissues that proliferating cells appear mainly localized after tail amputation and blastema formation. Microsc. Res. Tech., 2015 . © 2015 Wiley Periodicals, Inc.

Description: ABSTRACT Friedreich's ataxia (FRDA) is an autosomal recessive inherited disorder involving progressive damage to the central and peripheral nervous systems and cardiomyopathy. FRDA is caused by the silencing of the FXN gene and reduced levels of the encoded protein, frataxin. Frataxin is a mitochondrial protein that functions primarily in iron-sulfur cluster synthesis. Skin disorders including hair abnormalities have previously been reported in patients with mitochondrial disorders. However, to our knowledge, ultra-structural hair alterations in FRDA were not demonstrated. The purpose of this study was to determine ultra-structural alterations in the hairs of FRDA patients as well as carriers. Hair specimen from four patients, who are in different stages of the disease, and two carriers were examined by scanning electron microscope. Thin and weak hair follicles with absence of homogeneities on the cuticular surface, local damages of the cuticular layer, cuticular fractures were detected in both carriers and patients, but these alterations were much more prominent in the hair follicles of patients. In addition, erosions on the surface of the cuticle and local deep cavities just under the cuticular level were observed only in patients. Indistinct cuticular pattern, pores on the cuticular surface, and presence of concavities on the hair follicle were also detected in patients in later stages of the disease. According to our results, progression of the disease increased the alterations on hair structure. We suggest that ultra-structural alterations observed in hair samples might be due to oxidative stress caused by deficient frataxin expression in mitochondria. Microsc. Res. Tech., 2015 . © 2015 Wiley Periodicals, Inc.

Description: The purpose of this study is to determine the possible changes in the localization of the four Epidermal Growth Factor Receptors and three ligands in quail lungs from the first day of hatching until the 125th after hatching using immunohistochemical methods. Immunohistochemical results demonstrated that four EGFRs and their ligands are chiefly located in the cytoplasm of cells. Additionally, ErbB4, AREG, and NRG1 are localized to the nucleus and nucleolus, but EGF is present in the nucleolus. ErbB2 was also found in the cell membrane. In the epithelium of secondary bronchi, the goblet cells only exhibited ErbB1 and ErbB2, whereas the basal and ciliated cells exhibited EGFRs and ligands immunoreactivity. The atrial granular cells displayed moderate levels of ErbB1–ErbB3 and EGF and strong levels of ErbB4, AREG, and NRG1 immunoreactivity. While the squamous atrial cells and squamous respiratory cells of air capillaries and endothelial cells of blood capillaries exhibited moderate to strong ErbB2, ErbB4, AREG, and NRG1 immunoreactivity, they had negative or weak ErbB1, ErbB3, and EGF immunoreactivity. The expression levels of ErbB2–ErbB4, EGF, AREG, and NRG1 were also detected in fibroblasts. Although ErbB2 was highly expressed in the bronchial and vascular smooth muscle cells, weak expression of ErbB1, ErbB3, AREG and EGF and moderate expression of ErbB4 and NRG1 were observed. Macrophages were only negative for ErbB1. In conclusion, these data indicate that the EGFR-system is functionally active at hatching, which supports the hypothesis that the members of EGFR-system play several cell-specific roles in quail lung growth after hatching. Microsc. Res. Tech., 2015 . © 2015 Wiley Periodicals, Inc.

Description: ABSTRACT An easy and low-cost method to elaborate a container to dehydrate nematodes and other meiofauna in order to process them for scanning electron microscopy (SEM) is presented. Illustrations of its elaboration, step by step, are included. In addition, a brief methodology to process meiofauna, especially nematodes and kinorhynchs, and illustrations are provided. With this methodology it is possible to easily introduce the specimens, to lock them in a closed chamber allowing the infiltration of fluids and gases (ethanol, acetone, carbon dioxide) but avoiding losing the specimens. After using this meiofauna basket for SEM the results are efficient. Examples of nematode and kinorhynch SEM pictures obtained using this methodology are also included. Microsc. Res. Tech., 2015 . © 2015 Wiley Periodicals, Inc.

Description: ABSTRACT The effect of nonweight-bearing exercise on osteoporotic bones remains controversial and inconclusive. The purpose of this study was to evaluate the effects of swimming on osteoporotic tibias of rats submitted to hindlimb suspension. Initially, 20 Wistar rats were used to confirm a significant bone loss following 21 days of unloading. Thirty rats were then divided into 3 groups and followed during 51 days: CON (nonsuspended rats), S + WB (suspended rats for 21 days and then released for regular weight-bearing) and, S + Swim (suspended rats for 21 days and then released from suspension and submitted to swimming exercise). We observed that swimming exercise was effective at fully recovering the bone deterioration caused by suspension, with significant increments in BMD, bone strength and bone volume. On the other hand, regular weight-bearing failed at fully restoring the bone loss induced by unloading. These results indicate that swimming exercise may be a potential tool to improve bone density, strength, and trabecular volume in tibias with bone loss induced by mechanical unloading in suspended rats. We conclude that this modality of activity could be beneficial in improving bone mass, strength, and architecture in osteoporotic individuals induced by disuse, such as bed rest or those exposed to microgravity, who may not be able to perform weight-bearing exercises. Microsc. Res. Tech., 2015 . © 2015 Wiley Periodicals, Inc.

Description: ABSTRACT The endodermal cells of the human yolk sac (YS) produce non-nucleated erythrocytes (NNEs) and numerous serum proteins that are transiently storage within the YS cavity. After their transfer via the vitelline duct to the embryo gastrointestinal lumen, the nutrients’ final fate is unknown. With the aim of investigate how erythroid cells and nutrients are conveyed to embryo circulation, we studied, using a morphological and immunohistochemical approach, the embryo anatomy and the serum protein α-fetoprotein (AFP) presence, in 15 human embryos and their YS, collected from tubal pregnancies from 4 to 8 wpf. We observed at 5 wpf, a strong AFP staining in the endodermal cells of the YS, thereafter AFP was only present in the YS cavity and the gastrointestinal lumen. During 7 wpf, AFP expression declined and disappeared, concomitant with YS regression. Between 5 and 7 wpf, NNEs were observed in the gastrointestinal cavity, where they accumulate in the stomach. Here, the cells were attached to the endodermal epithelial cells or were free in the lumen. By scanning electron microscopy, we identified signs of NNEs phagocytized by endodermal cells. Those NNEs free in the lumen, after hemolysis, were probably removed by endocytosis (cell debris). Taking all together, we postulate that after reaching the endodermal epithelial cells of the stomach, nutrients are transferred to the embryo by a phagocytic/endocytic mechanism that is operative until the end of 6 wpf. After absorption, NNEs are probably degraded within phagosomes, nutrients delivered to the cell cytoplasm and then transported towards the embryonic circulation. Microsc. Res. Tech., 2015 . © 2015 Wiley Periodicals, Inc.

Description: ABSTRACT Cell adhesion forces are typically a mixture of specific and nonspecific cell-substrate and cell–cell interactions. In order to resolve these phenomena, Atomic Force Microscopy appears as a powerful device which can measure cell parameters by means of manipulation of single cells. This method, commonly known as cell-probe force spectroscopy, allows us to control the force applied, the area of interest, the approach/retracting speed, the force rate, and the time of interaction. Here, we developed a novel approach for in situ cantilever cell capturing and measurement of specific cell interactions. In particular, we present a new setup consisting of two different half-surfaces coated either with recrystallized SbpA bacterial cell surface layer proteins (S-layers) or integrin binding Fibronectin, on which MCF-7 breast cancer cells are incubated. The presence of a clear physical boundary between both surfaces benefits for a quick detection of the region under analysis. Thus, quantitative results about SbpA-cell and Fibronectin-cell adhesion forces as a function of the contact time are described. Additionally, the importance of the cell spreading in cell–cell interactions has been studied for surfaces coated with two different Fibronectin concentrations: 20 μg/mL (FN20) and 100 μg/mL (FN100), which impact the number of substrate receptors. Microsc. Res. Tech., 2016 . © 2016 Wiley Periodicals, Inc.

Description: Imaging of soft matter with atomic force microscopy (AFM) is challenging due to tip-induced deformation, which convolutes with the measurement. The challenges are generally more serious in liquid environments due to a severe loss of sensitivity of the vibrating microcantilever to external forces, as well as due to the presence of undesirable mechanical resonances when piezoelectric excitation systems are used. Furthermore, the choice of imaging parameters can have a significant impact on the quality of the results, such that the customary practices used for tuning the cantilever are not always appropriate. Here we explore the influence of the chosen drive frequency on the imaging of ultrasoft matter, using surface gas nanobubbles on gold-coated glass and highly oriented pyrolytic graphite (HOPG) as a test platform. We carry out single- and multifrequency AFM experiments using both the traditional amplitude-peak method and the recently proposed phase-slope-peak method for tuning the cantilever, as well as piezoelectric and photothermal excitation, providing an extensive discussion on the factors governing the level of tip-induced sample deformation and the quality of the phase contrast obtained for each of the methods. The general conclusion is that there is no “one-size-fits-all” approach for tuning the cantilever for low-impact tapping-mode AFM, although rational optimization of the imaging process is generally possible, whereby the choice of the drive frequency plays a prominent role. The physical insight and guidelines provided here can be extremely useful for the gentle imaging of a wide range of biological and other soft materials in liquid environments.

Description: This study evaluated the effect of Er,Cr:YSGG laser on the root canal dentin after luting a fiber post. Twenty-four bovine teeth roots were prepared using NiTi instruments and filled with Sealer 26 and gutta-percha. Post spaces were prepared and roots were distributed according to dentin treatment ( n  = 8): 2.5%NaOCl (group control), Er,Cr:YSGG laser (1.5 W, 20 Hz, 20 s) (group test 1) or 2.5%NaOCl + Er,Cr:YSGG laser (group test 2). Fiber posts were luted using adhesive cement (Rely X U200, 3M) and roots were prepared to confocal laser scanning microscopy (CLSM) and scanning electron microscope (SEM). The morphology of interface, thickness of cement, and the gaps and tags were analyzed. Non-parametrical data for thickness of cement were submitted to Friedman and Kruskall–Wallis tests ( α  = 0.05) and parametrical data for gaps to ANOVA ( α  = 0.05). CLSM of the cement thickness and gaps revealed no significant difference in surface treatment (NaOCl, Er,Cr:YSGG laser and NaOCl + Er,Cr:YSGG laser) ( p  〉 .05) and canal thirds (cervical, middle, and apical) ( p  〉 0.05). SEM showed tags and a residual layer of cement adhered to dentin, mainly in laser-irradiated specimens. The pretreatment of root canal with Er,Cr:YSGG laser previously to luting the fiber post with a self-adhesive cement did not influence the cement thickness and gaps but affected the dentin interaction.

Description: In this study, we have analyzed the viability and cell growth, as well as, the mineralization of extracellular matrix (ECM) by alizarin red and von Kossa staining of calvaria-derived osteogenic cultures, treated with TGF-β1 alone or associated with Dex comparing with acid ascorbic (AA) + β-glicerophosphate (βGP) (positive mineralization control). The expression of the noncollagenous proteins bone sialoprotein (BSP), osteopontin (OPN) and fibronectin (FN) were evaluated by indirect immunofluorescence. In addition, the main ultrastructural morphological findings were assessed by transmission electron microscopy. Osteogenic cells were isolated of calvaria bone from newborn (2-day-old) Wistar rats were treated with TGF-β1 alone or with dexamethasone for 7, 10, and 14 days. As positive mineralization control, the cells were supplemented only with AA+ βGP. As negative control, the cells were cultured with basal medium (α-MEM + 10%FBS + 1%gentamicin). The treatment with TGF-β1, even when combined with Dex, decreased the viability and cell growth when compared with the positive control. Osteoblastic cell cultures were positive to alizarin red and von Kossa stainings after AA + βGP and Dex alone treatments. Positive immunoreaction was found for BSP, OPN and FN in all studied treatments. Otherwise, when the cell cultures were supplemented with TGF-β1 and TGF-β1 + Dex, no mineralization was observed in any of the studied periods. These present findings suggest that TGF-β1, in the studied in vitro doses, inhibits the proliferation and differentiation of osteoblastic cells by impairment of nodule formation.

Description: Airborne particulate matter (PM) has been included among the most important air pollutants by governmental environment agencies and academy researchers. The use of terrestrial plants for monitoring PM has been widely accepted, particularly when it is coupled with SEM/EDS. Herein, Tillandsia stricta leaves were used as monitors of PM, focusing on a comparative evaluation of Environmental SEM (ESEM) and High-Pressure SEM (HPSEM). In addition, specimens air-dried at formaldehyde atmosphere (AD/FA) were introduced as an SEM procedure. Hydrated specimen observation by ESEM was the best way to get information from T. stricta leaves. If any artifacts were introduced by AD/FA, they were indiscernible from those caused by CPD. Leaf anatomy was always well preserved. PM density was determined on adaxial and abaxial leaf epidermis for each of the SEM proceedings. When compared with ESEM, particle extraction varied from 0 to 20% in air-dried leaves while 23–78% of particles deposited on leaves surfaces were extracted by CPD procedures. ESEM was obviously the best choice over other methods but morphological artifacts increased in function of operation time while HPSEM operation time was without limit. AD/FA avoided the shrinkage observed in the air-dried leaves and particle extraction was low when compared with CPD. Structural and particle density results suggest AD/FA as an important methodological approach to air pollution biomonitoring that can be widely used in all electron microscopy labs. Otherwise, previous PM assessments using terrestrial plants as biomonitors and performed by conventional SEM could have underestimated airborne particulate matter concentration.

Description: Rice false smut, caused by the fungal pathogen Villosiclava virens , is one of the most important rice diseases in the world. Previous studies reported that the pathogen has less number of cell wall-degraded genes and attacks dominantly rice stamen filaments and extends intercellularly. To reveal why the fungus infects plant stamen filaments, inoculation test on barley was carried out with the similar protocol to rice. The experimental results showed that the fungus could penetrate quickly into barley stamen filaments and extends both intracellularly and intercellularly, usually resulting in severe damage of the stamen filament tissues. It also attacked young barley lodicules and grew intercellularly by chance. The light microscopic observations found that the epidermal and cortex cells in barley stamen filaments arranged loosely with very thick cell walls and large cell gaps. Cellulose microfibrils in barley stamen filament cell walls arranged very sparsely so that the cell walls looked like transparent. The cell walls were very soft and flexible, and often folded. However, V. virens extended dominantly in the noncellulose regions and seemed never to degrade microfibrils in barley and rice cell walls. This suggested that the unique structures of rice and barley stamen filaments should be fit for their function of elongation in anthesis, and also endow with the susceptibility to the fungus, V. virens . Microsc. Res. Tech., 2016 . © 2016 Wiley Periodicals, Inc.

Description: For tobacco mosaic virus (TMV) as a model virus, this article shows typical issues of scanning soft biological matter by atomic force microscopy (AFM). TMV adsorbed on chemically different flat surfaces, gold, mica, and APDMES-functionalized silicon, is studied in air and aqueous environment. In air, the TMV particles arrangement shows some variety, depending on the substrate. The height of TMV is reduced to 13.7, 15.8, and 15.6 nm, for gold, APDMES, and mica, respectively while the width is about ∼30 nm due to the influence of the tip radius. In aqueous solution, the surface charges of the virus and the solid support play an important role in the virus adsorption process. While deposition on negatively charged mica is favored only at low pH values, it is shown that positively charged APDMES functionalized silicon can be a suitable substrate to work with at neutral pHs. The effects of cantilever oscillation's free amplitude ( A 0 ) and the amplitude set-point ( A ) are also assessed here. While high A 0 prompt reversible deformation of TMV in measurements performed in air, irreversible damage of the virus in liquid conditions (water) is observed using stiff cantilevers (0.35 N m −1 ) and high A 0 (81 nm), leading to a 6 nm reduction in the height of TMV after the first scan. Finally, low values of the amplitude set-point ( A / A 0  = 0.3), which means applying higher forces to the sample, also brings the damage of TMV virus assemblies, reducing its monolayer roughness to 0.3 nm.

Description: The aim of this study was to evaluate the effect of initial NaOCl on the decalcification and erosion ability of EDTA and QMix. Sixty-maxillary-incisors were bisected longitudinally and the tooth-halves were used. The experiment was conducted in two-sets. In set-I, 80-tooth halves were treated in the presence or absence of initial NaOCl and EDTA. In set-II, 40-tooth halves were immersed in NaOCl and QMix. After each treatment, calcium-ion release was determined with flame photometry. The erosion was imaged using SEM. Initial NaOCl led to concentration- and time-dependent increase in calcium removal effect of 17% EDTA ( p  〈 .05). The rate of calcium removal and root canal wall erosion was considerably more severe with the use of 5% NaOCl for 3 min ( p  〈 .05). QMix as a final solution showed less decalcification and erosion than 17% EDTA when used 5% NaOCl as an initial irrigant ( p  〈 .05). Optimizing the concentration and application time of NaOCl can decrease the decalcification effect of chelating agents.

Description: In this study, we investigate the biochemical characteristics of oxidative stress in age-related macular degeneration (AMD) and diabetic retinopathy (DR) by analyzing aqueous humors. Nondiabetic cataract aqueous humor was used as the control. The level of oxidative damage was evaluated based on changes in Raman spectral intensity. Seven prominent peaks were detected at 1002, 1043, 1062, 1352, 1419, 1454, and 1656 cm −1 . We proposed four multimodal biomarkers to distinguish these peaks based on the ratios of Raman intensities in two wavelengths, including CHO (C–O stretching or C–O–H bending modes), AG (adenine and guanine), PRO-AG (protein and AG), and PHEα (phenylalanine symmetric ring breath and amide I α-helix) markers. The presence of oxidative damage was detected by CHO and AG markers associated with C–O stretching, C–O–H bending modes in carbohydrates (1043 cm −1 ), and the nucleic acids adenine and guanine (1352 cm −1 ), respectively. DR-related oxidative damage was identified by PRO-AG and PHEα markers associated with adenine, guanine, and protein components (1419 and 1454 cm −1 ) and amide I α-helix protein structure (1656 cm −1 ), respectively. AMD-related oxidative damage was identified by four biomarkers. Four multimodal biomarkers with simple linear threshold values achieved high sensitivity of 100% and high specificity of 100% for classifying oxidative stress-induced AMD and DR diseases. Therefore, Raman-based label-free optical detection is effective for detecting the presence of age-related or diabetic oxidative damage in aqueous humor.

Description: The increasing recognition that tissue elasticity is an important regulator of cell behavior in normal and pathologic conditions such as fibrosis and cancer has driven the development of cell culture substrata with tunable elasticity. Such development has urged the need to quantify the elastic properties of these cell culture substrata particularly at the nanometer scale, since this is the relevant length scale involved in cell-extracellular matrix (ECM) mechanical interactions. To address this need, we have exploited the versatility of atomic force microscopy to quantify the elastic properties of a variety of cell culture substrata used in mechanobiology studies, including floating collagen gels, ECM-coated polyacrylamide gels, and decellularized tissue sections. In this review we summarize major findings in this field from our group within the context of the state-of-the-art in the field, and provide a critical discussion on the applicability and complementarity of currently available cell culture assays with tunable elasticity. In addition, we briefly describe how the limitations of these assays provide opportunities for future research, which is expected to continue expanding our understanding of the mechanobiological aspects that support both normal and diseased conditions.

Description: The aim of this study was to assess the effect of time and the ultrasonic activation of ethylenediaminetetraacetic acid (EDTA) in removing the smear layer of at root canal. A total of 60 bovine inferior incisors were transversely sectioned and instrumented; A smear layer was added in the root dentin surface. The specimen were then divided into six groups according to the following final irrigation protocols: Group (G) 1: conventional irrigation with sodium hypochlorite (NaOCl); G2: conventional irrigation with NaOCl + EDTA for 20 s; G3: conventional irrigation with NaOCl + EDTA for 60 s; G4: passive ultrasonic irrigation (PUI) with NaOCl; G5: PUI with NaOCl + PUI with EDTA for 20 s; G6: PUI + NaOCl and PUI with EDTA for 60 s. Samples were analyzed by scanning electron microscope (SEM) at ×500 increase. The images were measured by three blinded evaluators and classified in scores ranging from 1 to 5. The results were analyzed by the Pearson correlation test and the ANOVA nonparametric Kruskal–Wallis and Dunn's tests were used for the comparisons. Irrigated samples only with NaOCl (G1 and G4) had higher scores when compared with other groups. G5 and G6, irrigated with 17% EDTA and ultrasonic irrigation had lower scores. However, there was no statistically significant difference in G2 and G3, where there was no irrigation. It was concluded that irrigation with 17% EDTA for 20 s promoted the effective removal of smear layer, regardless of ultrasonic irrigation.

Description: Bites by Bothrops snakes normally induce local pain, haemorrhage, oedema and myonecrosis. Mammalian isolated nerve-muscle preparations exposed to Bothrops venoms and their phospholipase A 2 toxins (PLA 2 ) can exhibit a neurotoxic pattern as increase in frequency of miniature end-plate potentials (MEPPs) as well as in amplitude of end-plate potentials (EPPs); neuromuscular facilitation followed by complete and irreversible blockade without morphological evidence for muscle damage. In this work, we analysed the ultrastructural damage induced by Bothrops jararacussu and Bothrops bilineatus venoms and their PLA 2 toxins (BthTX-I and Bbil-TX) in mouse isolated nerve-phrenic diaphragm preparations (PND). Under transmission electron microscopy (TEM), PND preparations previously exposed to B. jararacussu and B. bilineatus venoms and BthTX-I and Bbil-TX toxins showed hypercontracted and loosed myofilaments; unorganized sarcomeres; clusters of edematous sarcoplasmic reticulum and mitochondria; abnormal chromatin distribution or apoptotic-like nuclei. The principal affected organelles, mitochondria and sarcoplasmic reticulum, were those related to calcium buffering and, resulting in sarcomeres and myofilaments hypercontraction. Schwann cells were also damaged showing edematous axons and mitochondria as well as myelin sheath alteration. These ultrastructural changes caused by both of Bothrops venoms and toxins indicate that the neuromuscular blockade induced by them in vitro can also be associated with nerve and muscle degeneration.