ALBERT

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 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 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.

Description: ABSTRACT Observation of heat-deproteinized cortical bone specimens in incident light enabled the high definition documentation of the osteonal pattern of diaphyseal Haversian bone. This prompted a study to compare these images with those revealed by polarized light microscopy, carried out either on decalcified or thin, undecalcified, resin-embedded sections. Different bone processing methods can reveal structural aspects of the intercellular matrix, depending on the light diffraction mode: birefringency in decalcified sections can be ascribed to the collagen fibrils orientation alone; in undecalcified sections, to both the ordered layout of collagen and the inorganic phase; in the heat-deproteinized samples, exclusively to the hydroxyapatite crystals aggregation mode. The elemental chemical analysis documented low content of carbon and hydrogen, no detectable levels of nitrogen and significantly higher content of calcium and phosphorus in heat-deproteinized samples, as compared with dehydrated controls. In both samples, the X-ray diffraction (XRD) pattern did not show any significant difference in pattern of hydroxyapatite, with no peaks of any possible decomposition phases. Scanning electron microscopic (SEM) morphology of heat-deproteinized samples could be documented with the fracturing technique facilitated by the bone brittleness. The structure of crystal aggregates, oriented in parallel and with marks of time periods, was documented. Comparative study of deproteinized and undecalcified samples showed that the matrix inorganic phase did not undergo a coarse grain thermal conversion until it reached 500°C, maintaining the original crystals structure and orientation. Incident light stereomicroscopy, combined with SEM analysis of deproteinized bone fractured surfaces, is a new enforceable technique which can be used in morphometric studies to improve the understanding of the osteonal dynamics. Microsc. Res. Tech., 2016 . © 2016 Wiley Periodicals, Inc.

Description: ABSTRACT The influence of tuning the laser pulse energy during the analyses on the resulting microstructure in a specimen utilizing an ultra-fast laser assisted atom probe was demonstrated by a case study of a binary Al-Li alloy. The decomposition parameters, such as the size, number density, volume fraction, and composition of precipitates, were carefully monitored after each analysis. A simple model was employed to estimate the corresponding specimen temperature for each value of the laser energy. The results indicated that the corresponding temperatures for the laser pulse energy in the range of 10 to 80 pJ are located inside the miscibility gap of the binary Al-Li phase diagram and fall into the metastable equilibrium field. In addition, the corresponding temperature for a laser pulse energy of 100 pJ was in fairly good agreement with reported range of solvus temperature, suggesting a result of reversion upon heating due to laser pulsing. Microsc. Res. Tech., 2016 . © 2016 Wiley Periodicals, Inc.

Description: ABSTRACT Microscopic magnetic resonance imaging (µMRI) T2 data from canine cartilage at different tibial locations were analyzed to investigate the influences of spatial resolution and pixel position on the T2 sensitivity to osteoarthritis (OA). Five experimental factors were investigated: inaccurate pixel position, different pixel resolutions, different specimen orientations in the magnetic field, topographical variations over the tibial surface, and different OA stages. A number of significant trends were identified in this analysis, which shows the subtle but substantial influences to our abilities of detecting OA due to T2 changes. In particular, any deviation in locating the cartilage pixels may result in erratic values near the cartilage surface. Significant differences were found in T2 values between nearly any two comparison-groups under all resolutions both in the meniscus-covered and -uncovered areas, which were also showed interaction between the OA degradation stages. This multiresolution project should help to improve the detection sensitivities of MRI toward cartilage degeneration. Microsc. Res. Tech., 2016 . © 2016 Wiley Periodicals, Inc.

Description: ABSTRACT In mammals, hair cell loss is irreversible and leads to hearing loss. To develop and test the functioning of different strategies aiming at hair cell regeneration, animal models of sensorineural hearing loss are essential. Although cochleae of these animals should lack hair cells, supporting cells should be preserved forming an environment for the regenerated hair cells. In this study, we investigated how ototoxic treatment with kanamycin and furosemide changes the structure of cochlear sensory epithelium in mice. The study also compared different tissue preparation protocols for scanning electron microscopy (SEM). Cochleae were collected from deafened and nondeafened mice and further processed for plastic mid modiolar sections and SEM. For comparing SEM protocols, cochleae from nondeafened mice were processed using three protocols: osmium–thiocarbohydrazide–osmium (OTO), tannic acid–arginine–osmium, and the conventional method with gold-coating. The OTO method demonstrated optimal cochlear tissue preservation. Histological investigation of cochleae of deafened mice revealed that the supporting cells enlarged and ultimately replaced the lost hair cells forming types 1 and 2 phalangeal scars in a base towards apex gradient. The type 3 epithelial scar, flattened epithelium, has not been seen in analysed cochleae. The study concluded that mice deafened with kanamycin and furosemide formed scars containing supporting cells, which renders this mouse model suitable for testing various hair cell regeneration approaches. Microsc. Res. Tech, 2016 . © 2016 Wiley Periodicals, Inc.

Description: ABSTRACT The superhydrophobicities and the contact time of lotus leaf and reed leaf were investigated. The results indicated that both lotus leaf and reed leaf have good superhydrophobic properties, and the water contact time was 12.7 and 14.7 ms on the surface of lotus leaf and reed leaf, respectively. Surface structure plays a key role in the different contacting times. Homogeneous distribution of papillae on the surface of lotus leaf was more helpful to reduce the contact time than anisotropic groove-shape on the surface of reed leaf. Based on the bionics coupling theory, the bionics sample possessing similar lotus-leaf-like surface structure on the aluminum alloy was designed and fabricated successfully. The water contact angle was about 153 ± 2°, sliding angle less than 5°, and the water contact time was 13.4 ms on the surface of bionics sample, which presented excellent superhydrophobic property, and achieved the aim of bionic design. Microsc. Res. Tech., 2016 . © 2016 Wiley Periodicals, Inc.

Description: ABSTRACT Understanding the interaction between graphene oxide (GO) and lipid membranes is of great importance for its various applications in biotechnology. Here, we investigated the interaction between GO and charged supported lipid bilayers (SLBs) by in situ atomic force microscope (AFM) imaging. It was found that GO could peel off a single layer of positively charged SLBs and deposited on the hydrophobic part of the remaining sublayer. Then free lipid molecules would assemble on GO surface and formed 1.5 bilayers in a lipid-GO-lipid manner. For negatively charged lipid bilayers, however, GO deposited to the SLBs only when its concentration was very high. These results indicate that, in addition to electrostatic interaction, the hydrophobic interaction plays an important role when GO sheets deposit onto the charged lipid bilayers, and should be helpful to understand possible cytotoxicity and antibiosis of graphene-related nanomaterials. Microsc. Res. Tech., 2016 . © 2016 Wiley Periodicals, Inc.

Description: ABSTRACT The present study analyzed the effects of different concentrations of the hexane extract of A. oleraceae (HEAO) (Jambú) on the germ cells of semi-engorged Rhipicephalus microplus female ticks, through a morpho-histological study, evaluating the effectiveness of the extract in the genesis of the individuals. To perform this analysis, 100 semi-engorged females were divided into five groups with 20 individuals each: groups I and II, respectively constituted by distilled water control and 50% ethanol + 1% DMSO, and groups III, IV, and V constituted by treatment with HEAO in the concentrations of 12.5, 25.0 and 50.0 mg/mL, respectively. All the ticks were immersed in the different concentrations of the extract or in distilled water for 5 minutes, dried and conditioned in BOD incubator for 7 days. The individuals of the treatment groups revealed the action of this extract showing alterations in the germ cells of the females from the different groups when compared with those from the groups I and II (control groups). These alterations were mainly related to the size and shape of the oocytes; number of yolk granules; presence, number, size and location of vacuoles in the cytoplasm of all the germ cells; and the presence of nuclear alterations in these cells as well. Thus, it was demonstrated that the concentrations of HEAO affected the germ cells of R. microplus ticks. The effects of the extract are similar to those caused by renowned and efficient chemical products used to control these ticks. Microsc. Res. Tech., 2016 . © 2016 Wiley Periodicals, Inc.

Description: ABSTRACT The aim of this study was to investigate how the Surface Enlargement Factor (SEF) and the epithelial keratinization degree of sheep rumen change in response to phytomass production, and to forage fiber and water content during the pasture vegetative cycle. The study used eighteen sheep nourished with dry hay and cereals during the winter season and with fresh hay during the pasture vegetative cycle. We collected samples from rumen indicative regions for two consecutive years characterized by different rainfall and pasture productivity values. We evaluated the densities (D) of rumen papillae to estimate the rumen SEF, and the keratinization percentage of the epithelial lining; these parameters showed differentiated modifications in the four ruminal analyzed compartments in response to pasture seasonal conditions. In addition, we performed Canonical Redundancy Analysis (RDA) on the “keratinization and SEF” matrix constrained by phytomass, water, and crude fiber contents of pasture at different time in the two considered years to highlight how rumen features answer to pasture conditions. Atrium (A) and ventral sac (VS) keratinization showed a strict positive correlation to crude fiber, while SEF of VS was positively related to phytomass and forage water content. The degree of keratinization of the rumen VS epithelium proved to be a useful parameter for evaluating anatomical variations in the short term period related to pasture features; in addition, its monitoring could be carried out through biopsy, thus avoiding the killing of animals. The study also leads to the application of the 3Rs (Replacement; Reduction; and Refinement). Microsc. Res. Tech., 2016 . © 2016 Wiley Periodicals, Inc.

Description: ABSTRACT Despite the increase in the incidence of cancer, the number of women who survive cancer treatment is growing. However, one of the principal results of chemotherapy is premature ovarian failure (POF). The aim of this study was to use the in situ culture preantral follicles as an in vitro model to evaluate the toxicity of two anticancer drugs, doxorubicin (DXR) and paclitaxel (PTX), on the integrity and development of ovarian follicles. Fragments of the ovarian cortex of goats were cultured in vitro for 1 or 7 days in α-MEM + supplemented with different concentrations of DXR (0.003, 0.03, or 0.3 µg/mL) and PTX (0.001, 0.01, or 0.1 µg/mL). Analyses were performed before and after culture to evaluate tissue integrity by classical histology, apoptosis by TUNEL assay, DNA laddering kit and the detection of activated caspase 3, and DNA damage by the immune detection of phosphorylated histone H2A.x (H2AXph139). Both DXR and PTX reduced the number of morphologically normal primordial and developing follicles. Positive staining for TUNEL and active caspase 3 was detected in all the samples ( P  〈 0.05). Therefore, we propose the in situ culture of caprine preantral follicles as a useful experimental model for assessing the toxic effects of the chemotherapeutic agents on ovarian folliculogenesis. Microsc. Res. Tech, 2016 . © 2016 Wiley Periodicals, Inc.

Description: This study investigated the effect of Ca addition on the microstructure and mechanical properties of as-cast Mg–4Sm alloys. The addition of 1.0 wt% Ca led to a significant grain refinement of Mg–4.0Sm alloys owing to the formation of rod-like Mg2Ca phases that acted as active nucleates for the Mg matrix. The as-cast Mg–4.0Sm–1.0Ca alloy showed the smallest grain size at 45 μm. Furthermore, the Mg–4.0Sm–1.0Ca alloy exhibited greater hardness, higher tensile strength, and higher yield tensile strength and elongation than the other two alloys with different Ca contents. These results were attributed to the grain refinement and precipitation strengthening of the Mg2Ca and Mg41Sm5 phases. Microsc. Res. Tech., 2016 . © 2016 Wiley Periodicals, Inc.

Description: ABSTRACT We report the microemulsion synthesis of vanadium and chromium sulfide nanoparticles (NPs) and their biological application as nanoprobes for colocalization of membrane proteins. Spherical V 2 S 3 and Cr 2 S 3 NPs were prepared in reverse microemulsion droplets, as nanoreactors, obtained by the surfactant sodium bis(2-ethylhexyl) sulfosuccinate (AOT) in nonpolar organic phase (heptane). Electron microscopic data indicated that the size distribution of the nanoparticles was uniform with an average diameter between 3 ÷ 5 nm. The prepared hydrophobic nanocrystals were transferred in aqueous phase by surface cap exchange of AOT with biotin-dihydrolipoic ligands. This substitution allows the nanoparticles solubility in aqueous solutions and confer their bioactivity. In addition, we report the conjugation procedure between α- Lipoic acid (LA) and biotin (abbreviated as b iotin-LA ). The biotin-LA structure was characterized by 1D and 2D NMR spectroscopy. The biotinylated vanadium and chromium sulfide nanoparticles were tested as probes for colocalization of glutamate receptors on sodium-dodecyl-sulfate-digested replica prepared from rat hippocampus. The method suggests their high labeling efficiency for study of membrane biological macromolecules. Microsc. Res. Tech., 2016 . © 2016 Wiley Periodicals, Inc.

Description: An amino-functionalized sorbent (ACF) was prepared by grafting amine group onto cotton fiber (CF) using tetraethylenepentamine (TEPA). The resulting composite was characterized using various instrumental techniques such as scanning electron microscopy (SEM), Fourier transformed infrared (FTIR) and pHpzc analysis. FTIR spectrum and pHpzc analysis illustrated the involvement of amine groups in the X-3B sorption process. ACF sorbent was tested for its ability to remove X-3B from an aqueous solution in batch experiments. The adsorption efficiency of ACF was 100% in comparison with 5.52% of raw cotton fiber when initial concentration of active brilliant red X-3B was 30 mg/l. Results suggested that solution pH hardly affected the removal of X-3B, and ACF remained stable sorption efficiency in the pH range of 3.0–12.0. High values of correlation coefficients indicated the adsorption kinetics could best described by the pseudo-second-order kinetic equation. The sorption isotherm of ACF fitted the Langmuir equilibrium model, with the maximum adsorption capacity of 270.27 mg/g at 293K. The obtained thermodynamic parameters showed that the adsorption of X-3B onto the adsorbent was spontaneous and exothermic. The excellent regeneration performance provided strong evidence of the potential of ACF for technological applications of dye removal.

Description: The objective of study is to study the peculiarities of morphological changes in different subdivisions of the intralobular duct of the submandibular gland (SMG) in rats in case of experimental diabetes mellitus (DM). The study included sexually mature male Wistar rats. Experimental DM was induced by streptozotocin. Electron microscopic study of subdivisions of the intralobular duct of the SMG was carried out on the 14th, 28th, 42nd, 56th, and 70th days of the experiment. In early stages of experimental DM the intercalated ducts are characterized by a relatively unchanged structure, and in late stages vacuolization of the cytoplasm of their epithelial cells is observed. Since the 14th day vacuolization of mitochondria is observed in epithelial cells of the granular ducts being the most pronounced on the 28th day and not apparent over the subsequent periods. The degree of filling with granules reduces till 56th day, however, it increases sligthly on the last day of the experiment. On the 28th–70th days vacuolization of the cytoplasm is observed in epithelial cells of the striated ducts. In addition, on the 14th day the mitochondrial matrix of these cells condenses; over the next periods it becomes enlightened and mitochondrial cristae are clearly visualized and disorganized. Conclusion: In the intralobular duct of the SMG in experimental DM dystrophic changes of different intensity occur in the granular and striated ducts on the 14th day and in the intercalated ducts only since the 42nd day of the experiment.

Description: Atomic force microscopy is not only a high-resolution imaging device but also a mechanical machine, which can be used either to indent or stretch (soft) biomaterials. Due to the statistical nature of such materials (i.e., hydrogels or polymers) hundreds of force-distance curves are required to describe their mechanical properties. In this manuscript, we present an automated system for polymer unfolding detection based on continuous wavelet analysis. We have tested the automated program on elastin, which is an important protein that provides elasticity to tissues and organs. Our results show that elastin changes its mechanical behavior in the presence of electrolytes. In particular, we show that NaCl has a different effect on the contour length than CaCl 2 for similar unfolding forces. In addition, we provide the program in the supporting information for the researches facing such kind of problem.

Description: ABSTRACT In the present work, ultra-high-strength steels with multiphase microstructures containing martensite and bainite were prepared by controlling the cooling rate. A new approach was proposed for quantitatively statistical phase analysis using electron backscatter diffraction (EBSD) based on the band contrast which correlates to the quality and intensity of the diffraction patterns. This approach takes advantage of the inherently greater lattice imperfections of martensite, such as dislocations and low-angle grain boundaries, relative to that of bainite. These can reduce the intensity and quality of the EBSD patterns of martensite, which decrease the band contrast. Thus, combined with morphological observations, Gaussian two-peak fitting was employed to analyze the band contrast profile and confirm the ranges of band contrast for the two phases. The volume fractions of bainite and martensite in different samples were determined successfully. In addition, the results show that increased cooling rates improve the proportion of martensite and the ratio of martensite to bainite. Microsc. Res. Tech, 2016 . © 2016 Wiley Periodicals, Inc.

Description: ABSTRACT Light sheet microscopy is a powerful technique for rapid, three-dimensional fluorescence imaging of large specimen such as drosophila and zebrafish embryos. Yet, beam divergence results in a loss of axial resolution at the periphery of the light sheet. Here, we demonstrate how an electrically tunable lens can be utilized to maintain the minimal, diffraction-limited thickness of the light sheet over a wide field of view (〉600 µm) at high frame rates (40 fps). This mode of operation is necessary for the application of fluorescence fluctuation spectroscopy in images. Microsc. Res. Tech, 2016 . © 2016 Wiley Periodicals, Inc.

Description: Many pathogens require direct binding to mucosal cells to cause an infection. The mucosal epithelium of the digestive tract, which is covered by a mucin layer, fulfills several protective functions that are essential to maintaining the health of the digestive tract. Mucins are glycoproteins, which are found on membranes and in mucus gels and protect the underlying mucosal cells. Both membrane-associated mucins and secreted mucins are critical components of mucosal defense. The aim of this study was to determine the localization and expression of mucin profile of the abomasum via histochemistry and immunohistochemistry. The abomasums of 20 bulls and 20 rams were evaluated. Histochemical examination showed that neutral and acidic mucins were present in the mucosa and the glands of the pars cardiaca, fundus, and pars pylorica of the abomasums of both bulls and rams. However, the expression of acidic mucins was weak in the superficial glands and strong in the deep glands of the abomasum of rams. In both bulls and rams, MUC1, MUC5AC, and MUC6 were expressed in the glandular epithelial cells in all regions of the abomasum. Interestingly, while MUC2 was not expressed in the pars cardiaca and fundus, it was weakly expressed in the parietal cells of the pars pylorica in both species. In conclusion, the presence of neutral and acidic mucins and MUC1, MUC2, MUC5AC, and MUC6 proteins in luminal epithelial and glandular cells of abomasum in the bulls and rams support the hypothesis that mucins play a key role in the protection of the abomasal mucosa against infectious agents.

Description: ABSTRACT The aim of this study was to characterize the antennal morphology of Osmoderma eremita , a threatened scarab beetle inhabiting tree hollows. O. eremita males produce a sex pheromone, ( R )-(+)- γ -decalactone, responsible mainly for the attraction of females but also other males. Gross and fine morphology of microstructures including sensilla, microsculpture and pores were analyzed using Scanning Electron Microscopy. The antenna of O. eremita showed the typical lamellicorn shape of scarab beetles, with a basal scape, a pedicel, a funicle composed of five antennomeres and a club composed of three lamellae. Six different subtypes of sensilla chaetica (Ch.1 − 6), Böhm sensilla (Bo), one subtype of sensilla basiconica (Ba.1), two subtypes of sensilla coeloconica (Co.1 − 2), two subtypes of sensilla placodea (Pl.1 − 2), pores and peculiar folds were described. The two sexes did not show any significant differences in the occurrence and number of the sensilla placodea, known to be responsible for the pheromone reception. Instead, some sexual differences were found on the occurrence and topology of three different microstructures: (1) one subtype of sensillum chaeticum (Ch.2) occurring on the pedicel only in males; (2) a characteristic pore occurring on the funicle only in males; (3) a peculiar fold occurring on different antennomeres of the funicle in the two sexes, on the fourth in males and on the fifth in females. A comparison between sensilla of O. eremita and those of other Scarabaeoidea is provided. Microsc. Res. Tech., 2016 . © 2016 Wiley Periodicals, Inc.

Description: ABSTRACT Development and innovation of new technologies are necessary especially in food quality; due that most instrumental technique for measuring quality properties involves a considerable amount of manual work. Image analysis is a technique that allows to provide objective evaluations from digitalized images that can estimate quality parameters for consumer's acceptance. The aim of the present research was to study the effect of freeze drying on the microstructure and texture of legume and vegetables using scanning electron microscopy at different magnifications' combined with image analysis. Cooked and cooked freeze dried rehydrated legumes and vegetables were analyzed individually by scanning electron microscopy at different magnifications' (250, 500, and 1000×).Texture properties were analyzed by texture analyzer and image analysis. Significant differences ( P  〈 0.05) were obtained for image and instrumental texture parameters. A linear trend with a linear correlation was applied for instrumental and image features. Results showed that image features calculated from Grey level co-occurrence matrix at 1,000× had high correlations with instrumental features. In rice, homogeneity and contrast can be applied to evaluate texture parameters gumminess and adhesiviness; Lentils: contrast, correlation, energy, homogeneity, and entropy for hardness, adhesiviness, gumminess, and chewiness; Potato and carrots: contrast, energy, homogeneity and entropy for adhesiviness, chewiness, hardness, cohesiviness, and resilence. Results revealed that combing scanning electron microscopy with image analysis can be a useful tool to analyze quality parameters in legumes and vegetables. Microsc. Res. Tech., 2016 . © 2016 Wiley Periodicals, Inc.

Description: ABSTRACT Stereo light microscope (SLM) simulates stereo imaging principle of human eyes. Microscopic vision system based on SLM has become an important visual tool for micro measurement, micromanipulation, and microinjection. We develop a micromanipulation system based on SLM and present an image distortion correction method. We mainly correct two kinds of image distortions: lateral and vertical distortion. Distortion correction consists of two steps. First, a linear fitting algorithm for each row or column of target points is developed, and the fitting errors are calculated. If the fitting errors are smaller than a given threshold, the linear fitting results are kept and used. Otherwise polynomial fitting procedure will be used. Second, the parallelism of straight lines is corrected. The results show that a line in world coordinate frame (WCF) is not necessarily a straight line in image coordinate frame (ICF), or two parallel lines in WCF may be not parallel in ICF. Distortion correction can restore the parallel and linear relationship. For distorted left and right images, the magnitude of distortion exceeds 6 pixels and 4 pixels in the horizontal direction, and 1.2 pixels and 1.7 pixels in the vertical direction, respectively. After corrected, for left and right image, distortion can be reduced to 0.8 pixels and 0.7 pixels in the horizontal direction, and 0.96 pixels and 1.3 pixels in the vertical direction, respectively. The results show that distortion parameters obtained from the proposed method can effectively correct distorted images. Microsc. Res. Tech., 2016 . © 2016 Wiley Periodicals, Inc.

Description: ABSTRACT Gilthead sea bream with different age groups that collected from Seawater fisheries, Ismailia Governorate, Egypt, were examined by gross anatomy and scanning electron microscopy to assess the morphological characteristics of the oral cavity. Teeth patterns showed that the gilthead sea bream is adapted to the feeding pattern according to age development, as it modified from spiny form teeth in young fishes to obelisk-like teeth and flat dome-like teeth in growing fishes, with differentiation of teeth into three pairs of canine and conical teeth, that later differentiated to small and large flat teeth. With development, the apical pouch also showed morphological differentiation from curve-like in small fishes to Y-letter shape in medium-sized fishes, which later grow to completely covering the lower jaw in grown adult fishes. Tongue papillae, on the other hand, showed some differentiation being smoother in growing fishes than adult ones. Consistent with development differentiation, the palatine region of young fishes appeared separated from the palate by deep palatine fissure, while that the same palatine region was continuous with the palate with a remnant of palatine fissure as shallow groove was noticed in grown big fishes. Taste buds were characterized in the oral cavity of small and large fishes however in large fishes; the taste buds were more prominent especially at the palate and palatine folds. These and other morphological features of the oral cavity and the feeding habits in small and large gilthead sea bream fishes were recorded. Microsc. Res. Tech., 2016 . © 2016 Wiley Periodicals, Inc.

Description: ABSTRACT The object of the present report is to provide a method for a visualization of DNA in TEM by complementary labeling of cytosine with guanine derivative, which contains platinum as contrast-enhanced heavy element. The stretched single-chain DNA was obtained by modifying double-stranded DNA. The labeling method comprises the following steps: (i) stretching and adsorption of DNA on the support film of an electron microscope grid (the hydrophobic carbon film holding negative charged DNA); (ii) complementary labeling of the cytosine bases from the stretched single-stranded DNA pieces on the support film with platinum containing guanine derivative to form base-specific hydrogen bond; and (iii) producing a magnified image of the base-specific labeled DNA. Stretched single-stranded DNA on a support film is obtained by a rapid elongation of DNA pieces on the surface between air and aqueous buffer solution. The attached platinum-containing guanine derivative serves as a high-dense marker and it can be discriminated from the surrounding background of support carbon film and visualized by use of conventional TEM observation at 100 kV accelerated voltage. This method allows examination of specific nucleic macromolecules through atom-by-atom analysis and it is promising way toward future DNA-sequencing or molecular diagnostics of nucleic acids by electron microscopic observation. Microsc. Res. Tech., 2016 . © 2016 Wiley Periodicals, Inc.

Description: ABSTRACT The aim of the present study was to investigate immunohistochemical distribution of heat shock protein 70 (Hsp70) and proliferating cell nuclear antigen (PCNA) in the mouse placenta at different gestational stages. For this purpose a total of 18 Swiss albino female mice at 12–14 weeks of age were used. Females were sacrificed on days 3 (early), 10 (mid-), and 17 (late) of pregnancy and the implantation sites of the pregnant uterus were sampled. The sections were made transversely through the central region of the implantation site and stained with hematoxylin and eosin for histological examination. PCNA and Hsp70 was stained immunohistochemically. Since the definitive placenta was not still formed on day 3 of pregnancy, Hsp70 and PCNA positivity were evaluated in only luminal epithelium and decidual-stromal cells. On days 10 and 17 of pregnancy, Hsp70 and PCNA positivity were evaluated in labyrinth zone, junctional zone and decidual layer of placenta. Hsp70 expression was observed trophoblast cells and decidual cells and was relatively constant throughout the pregnancy. This protein was strongly labeled in the trophoblast cells; while decidual cells were displayed moderate staining. In early pregnant mouse uteri, PCNA was mainly localized in decidual-stromal cells. The trophoblast cells and decidual cells displayed highly proliferative activity at the midgestational period. However there was a significant decrease in the percentage of PCNA positive cells in late gestation. Microsc. Res. Tech., 2016 . © 2016 Wiley Periodicals, Inc.

Description: Tetrastichus howardi (Olliff) (Hymenoptera: Eulophidae), a pupal parasitoid of a great number of Lepidoptera pests, has a great potential for biological control. To investigate the olfactory system of this parasitoid, we examined the morphology and ultrastructure of the antennal sensilla of both male and female T. howardi using scanning and transmission electron microscopic techniques. Antennae of male and female T. howardi were geniculate in shape, which consisted of scape, pedicel and flagellum with 5 and 4 flagellomeres, respectively. The sexual differences were recorded in the types, structure, distribution and abundance of antennal sensilla of T. howardi . Fourteen morphologically distinct types of antennal sensilla were found on the female antennae, while seventeen on the male antennae. They were: multiporous plate sensilla (MPS1-4), chaetica sensilla (CH1-3), multiporous trichodea sensilla (MTS), aporous trichodea sensilla (ATS1-5), multiporous grooved peg sensilla (MGPS), coeloconic sensilla (COS), campaniform sensilla (CAS), terminal finger-like hairy sensilla (TFI), cuticular pore (CP), and ventral sensory plaque (VSP). MPS4, ATS (3-5), and VSP only occurred on the male antennae, while MPS2 and MPS3 only on the female antennae. The MPSs, MTS, MGPS, TFI, and CP may function as olfactory sensilla involving in detecting odor stimuli whereas the ATSs, CHs, and CAS may serve as mechanoreceptors. COS were presumed to play a role as chemo-, thermo- or hygro-receptor. The results could facilitate future studies on the biology of olfaction in T. howardi . Microsc. Res. Tech., 2016 . © 2016 Wiley Periodicals, Inc.

Description: A modified method was suggested to improve the performance of the Pluta microscope in its nonduplicated mode in the calculation of the areal craze density especially, for relatively low draw ratio (low areal craze density). This method decreases the error that is resulted from the similarity between the formed crazes and the dark fringes of the interference pattern. Furthermore, an accurate method to calculate the birefringence and the orientation function of the drawn fibers via nonduplicated Pluta polarizing interference microscope for high areal craze density (high draw ratio) was suggested. The advantage of the suggested method is to relate the optomechanical properties of the tested fiber with the areal craze density, for the same region of the fiber material. Microsc. Res. Tech., 2016 . © 2016 Wiley Periodicals, Inc.

Description: ABSTRACT A numerical description of fracture is an important step in the search of the correlation between specific micromechanisms of decohesion and material characteristics designated with the use of fracture mechanics methods. This issue is essential for the proper orientation of the search for basic relationships between chemical composition, technology, structure, and properties of materials. It often happens that fracture surfaces are well developed, which can significantly hinder or even prevent the measurement and reconstruction of the tested material surface geometry. In this article, comparative measurements of a biomaterial surface were performed using laser confocal microscopy. To this end, short working distance lenses dedicated to a focused UV laser beam and long working distance objective lenses were used. The article includes a quantitative comparative analysis and interpretation of the obtained results. Microsc. Res. Tech., 2016 . © 2016 Wiley Periodicals, Inc.

Description: Image fusion process consolidates data and information from various images of same sight into a solitary image. Each of the source images might speak to a fractional perspective of the scene, and contains both “pertinent” and “immaterial” information. In this study, a new image fusion method is proposed utilizing the Discrete Cosine Transform (DCT) to join the source image into a solitary minimized image containing more exact depiction of the sight than any of the individual source images. In addition, the fused image comes out with most ideal quality image without bending appearance or loss of data. DCT algorithm is considered efficient in image fusion. The proposed scheme is performed in five steps: (1) RGB colour image (input image) is split into three channels R, G, and B for source images. (2) DCT algorithm is applied to each channel (R, G, and B). (3) The variance values are computed for the corresponding 8 × 8 blocks of each channel. (4) Each block of R of source images is compared with each other based on the variance value and then the block with maximum variance value is selected to be the block in the new image. This process is repeated for all channels of source images. (5) Inverse discrete cosine transform is applied on each fused channel to convert coefficient values to pixel values, and then combined all the channels to generate the fused image. The proposed technique can potentially solve the problem of unwanted side effects such as blurring or blocking artifacts by reducing the quality of the subsequent image in image fusion process. The proposed approach is evaluated using three measurement units: the average of Q abf , standard deviation, and peak Signal Noise Rate. The experimental results of this proposed technique have shown good results as compared with older techniques. Microsc. Res. Tech., 2016 . © 2016 Wiley Periodicals, Inc.

Description: ABSTRACT This study evaluated the effect of dentin surface treatment on the micromorphology and shear bond strength (SBS) of a self-adhering flowable composite, Vertis Flow (VF). Flat dentin surfaces obtained from sixty extracted human molars were divided into six groups ( n  = 10) according to the following surface treatments: (G1) control, no treatment; (G2) self-etching adhesive, Optibond All-in-One; (G3) phosphoric acid etching for 15 s; (G4) polyacrylic acid for 10 s; (G5) EDTA for 60 s; and G6) sodium hypochlorite (NaOCl) for 15 s. After restoration using VF, SBS was measured in MPa. Data were analyzed using one-way ANOVA and Tamhane test ( α  = 0.05). Six additional specimens were prepared for scanning electron microscopy analysis. SBS was significantly affected by surface treatment ( P  〈 0.001). SBS of six groups from the highest to the lowest were as follows: (G3) 13.5 A ; (G5) 8.98 AB ; (G2) 8.85 AB ; (G4) 8.21 AB ; (G1) 7.53 BC ; and (G6) 4.49 C (groups with the same superscript letter were statistically similar). Morphological analysis revealed numerous long resin tags at the adhesive interface for acid-etched group, with a few short resin tags for the control group and small gap formation for NaOCl-treated group. In conclusion, dentin surface treatments tested differently affected bonding performance of VF; only acid-etching effectively improved this. Microsc. Res. Tech., 2016 . © 2016 Wiley Periodicals, Inc.

Description: ABSTRACT Dose from radiation exposure can be estimated from dicentric chromosome (DC) frequencies in metaphase cells of peripheral blood lymphocytes. We automated DC detection by extracting features in Giemsa-stained metaphase chromosome images and classifying objects by machine learning (ML). DC detection involves (i) intensity thresholded segmentation of metaphase objects, (ii) chromosome separation by watershed transformation and elimination of inseparable chromosome clusters, fragments and staining debris using a morphological decision tree filter, (iii) determination of chromosome width and centreline, (iv) derivation of centromere candidates, and (v) distinction of DCs from monocentric chromosomes (MC) by ML. Centromere candidates are inferred from 14 image features input to a Support Vector Machine (SVM). Sixteen features derived from these candidates are then supplied to a Boosting classifier and a second SVM which determines whether a chromosome is either a DC or MC. The SVM was trained with 292 DCs and 3135 MCs, and then tested with cells exposed to either low (1 Gy) or high (2-4 Gy) radiation dose. Results were then compared with those of 3 experts. True positive rates (TPR) and positive predictive values (PPV) were determined for the tuning parameter, σ . At larger σ , PPV decreases and TPR increases. At high dose, for σ  = 1.3, TPR = 0.52 and PPV = 0.83, while at σ  = 1.6, the TPR = 0.65 and PPV = 0.72. At low dose and σ  = 1.3, TPR = 0.67 and PPV = 0.26. The algorithm differentiates DCs from MCs, overlapped chromosomes and other objects with acceptable accuracy over a wide range of radiation exposures. Microsc. Res. Tech., 2016 . © 2016 Wiley Periodicals, Inc.

Description: ABSTRACT Cross-sectional TEM analysis is one of the most important techniques to characterize microstructures of films. However, the complex process, low efficiency, and low success rate of specimen preparation limit its application. This paper analyzed the main causes of low success rate and proposed an improved method for specimen preparation of films deposited on metallic substrates. This method consisting of twin-jet electropolishing and one-sided rocking ion milling is high in efficiency and success rate. Microsc. Res. Tech., 2016 . © 2016 Wiley Periodicals, Inc.

Description: ABSTRACT The introduction of scanning/transmission electron microscopes (S/TEM) with sub-Angstrom resolution as well as fast and sensitive detection solutions support direct observation of dynamic phenomena in-situ at the atomic scale. Thereby, in-situ specimen holders play a crucial role: accurate control of the applied in-situ stimulus on the nanostructure combined with the overall system stability to assure atomic resolution are paramount for a successful in-situ S/TEM experiment. For those reasons, MEMS-based TEM sample holders are becoming one of the preferred choices, also enabling a high precision in measurements of the in-situ parameter for more reproducible data. A newly developed MEMS-based microheater is presented in combination with the new NanoEx™-i/v TEM sample holder. The concept is built on a four-point probe temperature measurement approach allowing active, accurate local temperature control as well as calorimetry. In this paper, it is shown that it provides high temperature stability up to 1,300°C with a peak temperature of 1,500°C (also working accurately in gaseous environments), high temperature measurement accuracy (〈4%) and uniform temperature distribution over the heated specimen area (〈1%), enabling not only in-situ S/TEM imaging experiments, but also elemental mapping at elevated temperatures using energy-dispersive X-ray spectroscopy (EDS). Moreover, it has the unique capability to enable simultaneous heating and biasing experiments. Microsc. Res. Tech., 2016 . © 2016 Wiley Periodicals, Inc.

Description: ABSTRACT The objective of this study was to evaluate the microarchitecture and trabecular bone strength at the distal region of the femur, and its biomechanical properties with simvastatin administration with two different doses in ovariectomized (OVX) rats. Ninety rats were divided into six groups to evaluate treatment with the simvastatin drug (n = 15): SH (Sham surgery), SH-5 (5 mg simvastatin), SH-20 (20 mg simvastatin), OVX, OVX-5, and OVX-20. Euthanasia was performed at three different times, five animals per period: 7, 14, and 28 days. The effectiveness of the treatments was evaluated by mechanical testing and histomorphometric analysis of the femurs. The results of analysis by the linear model of mixed effects showed 20 mg of simvastatin results in increased trabecular bone after 14 days ( P  = 0.039) of ingestion in ovariectomized animals. However, ingestion of 5 mg of simvastatin is able to sensitize the trabecular bone only at 28 days ( P  = 0.005) of ingestion. In the mechanical tests stiffness improves within 28 days ( P  = 0.003). Regarding maximum strength, no statistical differences were observed. According to these results, it can be concluded that for a decrease in oral intake, longer treatment times are required. Microsc. Res. Tech., 2016 . © 2016 Wiley Periodicals, Inc.

Description: ABSTRACT In recent years, the surface morphology and microstructure of ladybird ( Coccinella septempunctata ) wings have been used to help design the flapping-wing micro air vehicle (FWMAV). In this study, scanning electron microscopy (SEM) was used to verify the functional roles of the ladybird forewing and hindwing. Surface morphology and the cross-sectional microstructure of the wings are presented. Detailed morphology of ladybird forewings was observed using atomic force microscopy (AFM) and the composition of the wings was characterized using Fourier transformed infrared spectroscopy (FTIR). The ladybird forewing may possess different performance characteristics than the beetle, Allomyrina dichotoma . Additionally, the circular holes in the forewing might be important for decreasing the weight of the forewing and to satisfy requirements of mechanical behavior. Microsc. Res. Tech., 2016 . © 2016 Wiley Periodicals, Inc.