Showing papers in "Microscopy Research and Technique in 2017"

A beginner's guide to atomic force microscopy probing for cell mechanics

Abstract: Atomic Force microscopy (AFM) is becoming a prevalent tool in cell biology and biomedical studies, especially those focusing on the mechanical properties of cells and tissues. The newest generation of bio-AFMs combine ease of use and seamless integration with live-cell epifluorescence or more advanced optical microscopies. As a unique feature with respect to other bionanotools, AFM provides nanometer-resolution maps for cell topography, stiffness, viscoelasticity, and adhesion, often overlaid with matching optical images of the probed cells. This review is intended for those about to embark in the use of bio-AFMs, and aims to assist them in designing an experiment to measure the mechanical properties of adherent cells. In addition to describing the main steps in a typical cell mechanics protocol and explaining how data is analysed, this review will also discuss some of the relevant contact mechanics models available and how they have been used to characterize specific features of cellular and biological samples. Microsc. Res. Tech. 80:75-84, 2017. © 2016 Wiley Periodicals, Inc.

Modified cuckoo search algorithm in microscopic image segmentation of hippocampus

Abstract: Microscopic image analysis is one of the challenging tasks due to the presence of weak correlation and different segments of interest that may lead to ambiguity It is also valuable in foremost meadows of technology and medicine Identification and counting of cells play a vital role in features extraction to diagnose particular diseases precisely Different segments should be identified accurately in order to identify and to count cells in a microscope image Consequently, in the current work, a novel method for cell segmentation and identification has been proposed that incorporated marking cells Thus, a novel method based on cuckoo search after pre-processing step is employed The method is developed and evaluated on light microscope images of rats' hippocampus which used as a sample for the brain cells The proposed method can be applied on the color images directly The proposed approach incorporates the McCulloch's method for levy flight production in cuckoo search (CS) algorithm Several objective functions, namely Otsu's method, Kapur entropy and Tsallis entropy are used for segmentation In the cuckoo search process, the Otsu's between class variance, Kapur's entropy and Tsallis entropy are employed as the objective functions to be optimized Experimental results are validated by different metrics, namely the peak signal to noise ratio (PSNR), mean square error, feature similarity index and CPU running time for all the test cases The experimental results established that the Kapur's entropy segmentation method based on the modified CS required the least computational time compared to Otsu's between-class variance segmentation method and the Tsallis entropy segmentation method Nevertheless, Tsallis entropy method with optimized multi-threshold levels achieved superior performance compared to the other two segmentation methods in terms of the PSNR

Influence of scanning rate on quality of AFM image: Study of surface statistical metrics

Abstract: The purpose of this work is to study the dependence of AFM-data reliability on scanning rate. The three-dimensional (3D) surface topography of the samples with different micro-motifs is investigated. The analysis of surface metrics for estimation of artifacts from inappropriate scanning rate is presented. Fractal analysis was done by cube counting method and evaluation of statistical metrics was carrying out on the basis of AFM-data. Combination of quantitate parameters is also presented in graphs for every measurement. The results indicate that the sensitivity to scanning rate growths with fractal dimension of the sample. This approach allows describing the distortion of the images against scanning rate and could be applied for dependences on the other measurement parameters. The article explains the relevance and comparison of fractal and statistical surface parameters for characterization of data distortion caused by inappropriate choice of scanning rate.

Retinal imaging analysis based on vessel detection.

Abstract: With an increase in the advancement of digital imaging and computing power, computationally intelligent technologies are in high demand to be used in ophthalmology cure and treatment. In current research, Retina Image Analysis (RIA) is developed for optometrist at Eye Care Center in Management and Science University. This research aims to analyze the retina through vessel detection. The RIA assists in the analysis of the retinal images and specialists are served with various options like saving, processing and analyzing retinal images through its advanced interface layout. Additionally, RIA assists in the selection process of vessel segment; processing these vessels by calculating its diameter, standard deviation, length, and displaying detected vessel on the retina. The Agile Unified Process is adopted as the methodology in developing this research. To conclude, Retina Image Analysis might help the optometrist to get better understanding in analyzing the patient's retina. Finally, the Retina Image Analysis procedure is developed using MATLAB (R2011b). Promising results are attained that are comparable in the state of art.

Silk fibroin for vascular regeneration

Abstract: Cardiovascular disease is the primary cause of morbidity and mortality in today's world. Due to the lack of healthy autologous vessels, more tissue-engineered blood vessels are needed to repair or replace the damaged arteries. Biomaterials play an indispensable role in creating a living neovessel with biological responses. Silk fibroin produced by silkworms possesses good cytocompatibility, tailorable biodegradability, suitable mechanical properties, and minimal inflammatory reactions. In addition, regenerated silk fibroin solutions can be processed into various forms of scaffolds such as films, fibers, tubes, and porous sponges. These features make silk fibroin a promising biomaterial for small-diameter vascular grafts. The present article focuses on the applications of silk fibroin for vascular regeneration. A brief overview of the properties of silk fibroin is provided, following which the current research status and future directions of the main types of silk fibroin scaffolds for vascular regeneration are reviewed and discussed. Microsc. Res. Tech. 80:280-290, 2017. © 2015 Wiley Periodicals, Inc.

Resolution and super-resolution

Abstract: Many papers have claimed the attainment of super-resolution, i.e. resolution beyond that achieved classically, by measurement of the profile of a feature in the image. We argue that measurement of the contrast of the image of a dark bar on a bright background does not give a measure of resolution, but of detection sensitivity. The width of a bar that gives an intensity at the center of the bar of 0.735 that in the bright region (the same ratio as in the Rayleigh resolution criterion) is λ/(13.9×numerical aperture) for the coherent case with central illumination. This figure, which compares with λ/(numerical aperture) for the Abbe resolution limit with central illumination, holds for the classical case, and so is no indication of super-resolution. Theoretical images for two points, two lines, arrays of lines, arrays of bars, and grating objects are compared. These results can be used a reference for experimental results, to determine if super-resolution has indeed been attained. The history of the development of the theory of microscope resolution is outlined.

Atomic force microscopy combined with optical microscopy for cells investigation

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. 80:109-123, 2017. © 2016 Wiley Periodicals, Inc.

Antimicrobial effect, frictional resistance, and surface roughness of stainless steel orthodontic brackets coated with nanofilms of silver and titanium oxide: a preliminary study.

Abstract: Nano-silver and nano-titanium oxide films can be coated over brackets in order to reduce bacterial aggregation and friction. However, their antimicrobial efficacy, surface roughness, and frictional resistance are not assessed before. Fifty-five stainless-steel brackets were divided into 5 groups of 11 brackets each: uncoated brackets, brackets coated with 60 µm silver, 100 µm silver, 60 µm titanium, and 100 µm titanium. Coating was performed using physical vapor deposition method. For friction test, three brackets from each group were randomly selected and tested. For scanning electron microscopy and atomic-force microscopy assessments, one and one brackets were selected from each group. For antibacterial assessment, six brackets were selected from each group. Of them, three were immediately subjected to direct contact with S. mutans. Colonies were counted 3, 6, 24, and 48 h of contact. The other three were stored in water for 3 months. Then were subjected to a similar direct contact test. Results pertaining to both subgroups were combined. Groups were compared statistically. Mean (SD) friction values of the groups 'control, silver-60, silver-100, titanium-60, and titanium-100' were 0.55 ± 0.14, 0.77 ± 0.08, 0.82 ± 0.11, 1.52 ± 0.24, and 1.57 ± 0.41 N, respectively (p = .0004, Kruskal–Wallis). Titanium frictions were significantly greater than control (p .05, Dunn). In the uncoated group, colony count increased exponentially within 48 h. The coated groups showed significant reductions in colony count (p < .05, two-way-repeated-measures ANOVA). In conclusions, all four explained coatings reduce surface roughness and bacterial growth. Nano-titanium films are not suitable for friction reduction. Nano-silver results were not conclusive and need future larger studies.

Preparation of silk fibroin carriers for controlled release.

Abstract: Silk fibroin provides a new option for controlled release systems as a result of its excellent biodegradability, biocompatibility, and mechanical properties. As the core material, silk fibroin can be designed and widely used in drug/gene delivery, regenerative medicine, and other biomedical fields. This review focuses on the preparation methods, loading molecules, and applications of silk fibroin-based controlled release systems including microspheres, microcapsules, films, microparticles, microneedles, liposomes, and hydrogels. These systems provide numerous advantages such as high encapsulation efficiency, avoiding loss of bioactivity and maintaining desirable range without peaks and valleys in comparison to the traditional administration approaches. Microsc. Res. Tech. 80:312-320, 2017. © 2015 Wiley Periodicals, Inc.

Antibacterial activity of glass ionomer cement modified by zinc oxide nanoparticles.

Abstract: This study evaluated the antibacterial activity of zinc oxide nanoparticles incorporated into self-cured glass ionomer cement (GIC) and light-cured resin-reinforced GIC on Streptococcus mutans biofilm. The GICs, Fuji II (GC America) and Fuji II LC (GC America), were incorporated with nanoparticles at concentrations of 0%, 1%, and 2% by weight, and the biofilm maturation time was one and seven days. Circular specimens of each GIC type were prepared. The antibacterial activity was evaluated by determining the number of colony forming units of S. mutans strain per milliliter. Morphology of the biofilm was analyzed by scanning electron microscopy (SEM). The data obtained for each GIC were analyzed by two-way ANOVA (α = 5%). For chemically activated GIC, no significant difference was observed in relation to the time of biofilm maturation (p = 0.744), concentration of nanoparticles (p = 0.966), and their interaction (p = 0.800). The results from analysis of GIC modified by light-polymerized resin showed that only of the maturing time significantly affected the number of adhered cells on the biofilm (p = 0.034, F = 4.778). The more mature the biofilm, higher the number of cells. SEM analysis showed no change in cell morphology in relation to the type of GIC, maturation time, and nanoparticles concentration. We conclude that the inclusion of zinc oxide nanoparticles at concentrations of 1% and 2% by weight into the GICs evaluated here, did not promote their antimicrobial activity against S. mutans.

Elastic properties of hydrogels and decellularized tissue sections used in mechanobiology studies probed by atomic force microscopy

Abstract: 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. Microsc. Res. Tech. 80:85-96, 2017. © 2016 Wiley Periodicals, Inc.

Effects of metals cadmium and chromium alone and in combination on the liver and kidney tissue of male Spraque‐Dawley rats: An ultrastructural and electron‐energy‐loss spectroscopy investigation

Abstract: Heavy metal pollution has increased in the last decades. Water sources are contaminated and human exposure is often long term exposure to variable amounts of different metals. In this study, male Sprague-Dawley rats were exposed via oral gavage for 28 days to cadmium (Cd) and chromium (Cr), alone and in combination at concentrations 1000 times the human World Health Organization's acceptable water limits. Rat equivalent dosages were used. Blood markers of liver and kidney function were measured, changes to cellular morphology was determined with transmission electron microscopy and the intracellular metal localisation was determined with the electron energy-loss spectroscopy and energy filtered transmission electron microscopy analysis. Both Cd and Cr caused changes to the nuclear and mitochondrial membranes and irregular chromatin condensation of hepatocytes. Cr exposure caused dilation of the rough endoplasmic reticulum (rER). The combination caused nuclear and mitochondrial membrane damage as well as irregular chromatin condensation. In the kidney tissue, Cd caused irregular chromatin condensation in the cells of the proximal convoluted tubule (PCT). Cr caused changes to the outer nuclear and mitochondrial membrane and chromatin structure. The combination group caused membrane damage, irregular chromatin condensation and rER changes in the PCT. All the metal groups showed damage to the endothelial cells and pedicles, but not to the mesangial cells. Cd and Cr bio-accumulation was observed in the nucleus, mitochondria and rER of the liver and kidney and therefore are responsible for the cellular observed damage that can cause functional changes to the tissues and organs.

Scanning electron and light microscopy of foliar epidermal characters: A tool for plant taxonomists in the identification of grasses.

Abstract: SEM and light microscopy are of special interest for biologist to observe various features of the living bodies. In the current study we observed the foliar epidermal micro-morphological characters of 44 grass species using SEM and Light microscopy to assess their taxonomic utility for taxonomists in the identification process. The aim of this study is to use the foliar epidermal structural variations in both upper and lower surfaces for identification of grasses. Significant diversity was observed in both qualitative and quantitative characters using SEM and Light microscopy. Variations were observed in stomatal number, size, guard cells shape, silica bodies, macro-hairs, micro-hairs, epidermal cell number, subsidiary cells, prickles, hooks, papillae, and short and long cells. A taxonomic key is prepared using these variations for the identification of grass species. Based on these findings, SEM and Light microscopy of foliar epidermal features can be of special interest for taxonomists in the identification of complex grass taxa.

Tip in-light on: Advantages, challenges, and applications of combining AFM and Raman microscopy on biological samples.

Abstract: Scanning probe microscopies and spectroscopies, especially AFM and Confocal Raman microscopy are powerful tools to characterize biological materials. They are both non-destructive methods and reveal mechanical and chemical properties on the micro and nano-scale. In the last years the interest for increasing the lateral resolution of optical and spectral images has driven the development of new technologies that overcome the diffraction limit of light. The combination of AFM and Raman reaches resolutions of about 50-150 nm in near-field Raman and 1.7-50 nm in tip enhanced Raman spectroscopy (TERS) and both give a molecular information of the sample and the topography of the scanned surface. In this review, the mentioned approaches are introduced, the main advantages and problems for application on biological samples discussed and some examples for successful experiments given. Finally the potential of colocated AFM and Raman measurements is shown on a case study of cellulose-lignin films: the topography structures revealed by AFM can be related to a certain chemistry by the colocated Raman scan and additionally the mechanical properties be revealed by using the digital pulsed force mode. Microsc. Res. Tech. 80:30-40, 2017. © 2016 Wiley Periodicals, Inc.

Effects of different desensitizers and lasers on dentine tubules: An in-vitro analysis.

Abstract: Background Dentin hypersensitivity is an important clinical problem affecting a large percentage of the population, and various therapies have been suggested for its treatment—among them desensitizing agents and lasers. The aim of this study was to evaluate the in vitro effects of different in-office desensitizing agents and different type lasers, alone or in combination, on human dentinal tubules. Materials and Methods Eighty-four dentinal specimens obtained from freshly extracted impacted third molars were included and subsequently divided into seven groups: Group 1 (Control), Group 2 (Gluma desensitizer), Group 3 (Teethmate desensitizer), Group 4 (Nd:YAG laser), Group 5 (Er:YAG laser), Group 6 (Gluma desensitizer + Er:YAG laser), and Group 7 (Teethmate desensitizer + Er:YAG laser). The dentinal specimens of all groups were evaluated using SEM. A total of 12 images per sample were taken, and the numbers of open, partially occluded, and totally visible (open + partially occluded) dentinal tubules were counted. AFM was used to further substantiate the blocking mechanism. The data were analyzed by one-way ANOVA and Dunnett-C tests. Results Comparison of the number of occluded dentinal tubules per 100 µm2 revealed that the laser and laser combined-treated groups showed the most occluded dentinal tubules. Gluma desensitizer + Er:YAG laser combination demonstrated significantly more tubule occlusion than all the other groups (p < .05). Conclusions The results of this study show that combined treatment methods were effective in the occlusion of dentinal tubules.

Identification of “Huoshan shihu” Fengdou: Comparative authentication of the Daodi herb Dendrobium huoshanense and its related species by macroscopic and microscopic features

Abstract: “Huoshan shihu,” derived from Dendrobium huoshanense, is a well-known, valuable, and rare Daodi herb. Because of its higher price and scarce resources, its related species D. officinale and D. moniliforme are usually presented as “Huoshan shihu” for sale. Fengdou, the processed form of D. huoshanense, its identification is particularly important. To effectively identify Fengdou of D. huoshanense and protect the Daodi herb, the morphological and microscopic characteristics of the stems of three Dendrobium species were examined. The results showed that macroscopic and microscopic features helpful for identification of the three species were the total lengths and internode numbers of stems, diameter and length of each internode, number of vascular bundles, and presence or absence of silica masses. The key features useful for distinguishing between D. huoshanense and its related species, and also between their Fengdou, were the stem lengths and the trends of change in diameter in different internodes. The results of the study indicated that a combination of macroscopic and microscopic identification techniques might conveniently and effectively be applied for identification of Dendrobium species. “Longtou Fengwei” is the main feature of the Daodi herb “Huoshan shihu,” (D. huoshanense Fengdou) and reflects the wisdom behind the protection of Daodi herbs in ancient times.

Investigating cell-substrate and cell-cell interactions by means of single-cell-probe force spectroscopy.

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. 80:124-130, 2017. © 2016 Wiley Periodicals, Inc.

Evaluation of smear layer removal and marginal adaptation of root canal sealer after final irrigation using ethylenediaminetetraacetic, peracetic, and etidronic acids with different concentrations

Abstract: The purpose of this study was to investigate the effects of different irrigation solutions on the smear layer removal and marginal adaptation of a resin-based sealer to root canal dentine. A total of 152 instrumented roots were irrigated with the following irrigants: 9,18% etidronic acid (HEBP), 0.5, 1,2% peracetic acid (PAA), 17% ethylenediaminetetraacetic acid (EDTA), saline. The amount of smear layer was evaluated using scanning electron microscope (SEM) in seventy root samples. Eighty-two roots were filled with AH Plus and gutta-percha. Slices obtained from apical third of each specimen were viewed with SEM to assess marginal adaptation. Use of 9% and 18% HEBP resulted in more efficient smear layer removal in the apical third than the other chelators (p < 0.05). Higher smear layer scores in the coronal and middle thirds were obtained from 0.5%, 1% PAA groups. Regarding marginal adaptation, 18% HEBP group showed the lowest gap size values (p < 0.05), and better marginal adaptation. Etidronic acid is a promising candidate for final irrigation of root canals.

The effects of neem oil (Azadirachta indica A. JUSS) enriched with different concentrations of azadirachtin on the integument of semi‐engorged Rhipicephalus sanguineus sensu lato (Acari: Ixodidae) females

Abstract: Several studies searching for methods to control Rhipicephalus sanguineus s.l., (dog tick) infestations have been developed aiming to minimize the damages caused by these ectoparasites to the hosts and the environment, which is harmed by the indiscriminate use of toxic acaricide products. In this scenario, neem oil has been used as a natural alternative against ticks, once this chemical has repellent properties and interferes in the growth regulation of these ectoparasites, inhibiting ecdysis. The present study evaluated the effects of azadirachtin-enriched neem oil on the integument of semi-engorged R.sanguineus s.l., females through morphohistological techniques. The results showed the occurrence of significant morphological and histochemical alterations, mainly in the females exposed to higher concentrations, which demonstrates the dose-dependent action of the chemical. A decrease in the cuticle thickness was observed, as well as a modification in the distribution of the epithelial cells, which displayed pyknotic and fragmented nuclei, and intensely vacuolated cytoplasm, indicating that these cells would be undergoing death processes. These morphological alterations observed in the integument of the females exposed to the azadirachtin-enriched neem oil encourage the use of this chemical as a strategy to control these ectoparasites.

Melatonin prevents mitochondrial dysfunctions and death in differentiated skeletal muscle cells

Abstract: Oxidative stress increase induces cellular damage and apoptosis activation, a mechanism believed to represent a final common pathway correlated to sarcopenia and many skeletal muscle disorders. The goal of this study is to evaluate if melatonin, a ROS scavenger molecule, is able to counteract or modulate myotube death. Here, differentiated C2C12 skeletal muscle cells have been treated with melatonin before chemicals known to induce apoptotic death and oxidative stress, and its effect has been investigated by means of morpho-functional analyses. Ultrastructural observations show melatonin protection against triggers by the reducing of membrane blebbing, chromatin condensation, myonuclei loss and in situ DNA cleavage. Moreover, melatonin is able to prevent mitochondrial dysfunctions which occur in myotubes exposed to the trigger alone. These findings demonstrate melatonin ability in preventing apoptotic cell death in skeletal muscle fibers in vitro, suggesting for this molecule a potential therapeutic role in the treatment of various muscle disorders.

Nonparametric de‐noising filter optimization using structure‐based microscopic image classification

Abstract: The Local Polynomial Approximation (LPA) is a nonparametric filter that performs pixel-wise polynomial fit on a certain neighborhood. This filter can be supported by the Intersection of Confidence Interval rule (ICI) as an adaptation algorithm to identify the most suited neighborhood at which the polynomial assumptions provide superior fit for the observations. However, the LPA-ICI is considered to be a near-optimal de-noising filter. Moreover, the ICI rule has several parameters that affect its performance. The current study applied an optimization algorithm, namely the Particle swarm optimization (PSO) to determine the optimal ICI parameter values for microscopic images de-noising. As the ICI parameters are image's structure based, bag-of-features classifier is used to classify the images based on their structure into different classes. Afterward, a generated optimal ICI parameters' table was created using the LPA-ICI-PSO for further direct use without optimization. This table included the optimal ICI parameters based on the image structure. Based on the image category, the generated table can be used to attain the suitable ICI optimal parameters without using PSO. This guarantees less computational time along with the optimal de-noising compared to the LPA-ICI as established by the performance metrics. The experimental results established the superiority of the proposed LPA-ICI-PSO over the classical LPA-ICI filter.

Morphostructural and immunohistochemical study on the role of metallothionein in the detoxification of heavy metals in Apis mellifera L., 1758

Abstract: Honey is a valuable food produced by bees from sugary substances that they gather in nature. The transformation the nectar into honey, by bees, is long and complex. Except for honey, where heavy metals are absent or are found only in traces, the bees and their products have always been considered excellent biomarkers of such contaminants. We have assumed that the absence of heavy metals in honey is due to the presence of a detoxification system in the digestive system of bees, which involves metallothioneins, proteins that have a role in the homeostatic control of essential and non-essential metals. We have placed the beehives in three different zones: industrial, urban and rural. Investigations were carried out with ICP-MS method for the detection of heavy metals in the guts of honey bees and honey. The metallothioneins have been identified by Immunohistochemical and Western-blotting analisys. The investigations have shown the presence of heavy metals only in bees guts but not in honey, while the presence of metallothionein has been highlighted only in epithelium of the honey sac, demonstrating the existence of an efficient system of detoxification of heavy metals.

Comparison of cleaning methods for delicate insect specimens for scanning electron microscopy

Abstract: The objective of the present study was to compare cleaning methods for delicate insect specimens for investigations with scanning electron microscopy (SEM). As typical specimens we used aquatic larvae of mosquitoes, springtails, larvae of mayflies and caterpillars because they are very fragile and large parts of their body consist of soft tissue. Additionally their cuticle is very often covered with dirt, soil particles or other materials. Cleaning with ultrasonic sound, as the most common cleaning method used for SEM, will destroy fragile insects. Therefore we tested different procedures to remove the dirt particles. In a first approach we compared cleaning with Potassium hydroxide (KOH), Proteinase K, and Triton X in aquatic larvae of flies, which were available in numbers and kept under the same conditions. As our results showed that the treatment with KOH gives the best results we treated in a second approach springtails, larvae of mayflies and caterpillars only with KOH. The springtails and caterpillars were largely free of particles after treatment with KOH; however, the larvae of mayflies were still covered with remnants of diatoms and precipitates of calcium carbonate of the algae. KOH dissolves organic impurities, on the other hand silicon dioxide and lime crusts are not solved. With this limitation, treatment with KOH is a simple technique for routine use as cleaning method for fragile insect specimens for SEM.

Label-free identification of antibiotic resistant isolates of living Escherichia coli: Pilot study

Abstract: We introduce a label-free spectroscopic method to classify subtypes of quinolone-nonsusceptible Escherichia coli (E. coli) isolates obtained from human blood cultures. Raman spectroscopy with a 30-nm gold-deposited, surface-enhanced Raman scattering (SERS) substrate was used to evaluate three multilocus sequencing typing (MLST)-predefined groups including E. coli ATCC25922, E. coli ST131:O75, and E. coli ST1193:O25b. Although there was a coffee-ring effect, the ring zone was selected at the ideal position to screen E. coli isolates. Strong Raman peaks were present at 1001-1004 cm-1 (CC aromatic ring breathing stretching vibrational mode of phenylalanine), 1447-1448 cm-1 (CH2 scissoring deformation vibrational mode), and 1667 cm-1 (amide I α-helix). Although the three MLST-predefined E. coli isolates had similar Raman spectral patterns, a support vector machine (SVM) learning algorithm-assisted principal component analysis (PCA) analysis had superior performance in detecting the presence of quinolone-nonsusceptible E. coli isolates as well as classifying similar microbes, such as quinolone-nonsusceptible E. coli ST131:O75 and E. coli ST1193:O25b isolates. Therefore, this label-free and nondestructive technique is likely to be useful for clinically diagnosing quinolone-nonsusceptible E. coli isolates with the MLST method.

Immunohistochemical colocalization of G protein alpha subunits and 5-HT in the rectal gland of the cartilaginous fish Scyliorhinus canicula.

Abstract: Serotonin [5-hydroxytryptamine (5-HT)] is an important neuromodulator involved in a wide range of physiological functions The effects of serotonin are mediated by an extended family of receptors coupled to multiple heterotrimeric G-proteins, associated with cellular membrane G proteins connect receptors to effectors and thus trigger intracellular signaling pathways These cellular processes several regulate systemic functions such as embryonic development, gonadal development, learning and memory, and organismal homeostasis Generally, elasmobranch fish dwell a hypersaline environment and utilize a specialized extrarenal salt secreting organ, the rectal gland, to face ionic homeostasis In this study in addition to the morphological, histochemical and immunohistochemical description of the Scyliorhinus canicula rectal gland, for the first time, the presence of serotonin (5-HT), and distribution of different types of G protein alpha subunits (Gα o, Gα q/11, and Gα s/olf) has been investigated in the rectal gland epithelium by confocal immunofluorescence techniques Colocalization G proteins and 5-HT in the secretory epithelium of the gland suggests serotonin acts as a hormone and involves G proteins in an autocrine-paracrine control of rectal gland homeostasis

Silk fibroin scaffold as a potential choice for female pelvic reconstruction: A study on the biocompatibility in abdominal wall, pelvic, and vagina

Abstract: The aim of this study was to compare the tissue reactions to silk fibroin scaffolds in the abdominal wall, vagina, and pelvic vesico-uterine of rats. Silk fibroin scaffolds were implanted subcutaneously in the abdominal, pelvic vesico-uterine space, and under the vaginal mucosa of 16 rats. The animals were euthanized at 2, 4, 8, and 12 weeks postoperatively. Hematoxylin and eosin staining was performed to evaluate cellular infiltration, the percentage of macrophages and granulocytes inside and around the scaffolds. The amounts of M1/M2 macrophages at the interface of scaffolds and host tissue were evaluated through an immunofluorescence assay. The degree of acute inflammation was similar among the three groups, and lasted no more than 4 weeks. A faster ingrowth of fibroblasts was found in the abdominally implanted silk fibroin scaffolds, while vaginal implanted scaffolds committed a slower tissue ingrowth rate and much more macrophages infiltration than the pelvic and abdominal group. However, a significantly higher amount of M2 cells were seen in the three groups. In general, silk fibroin has nice biocompatibility in the abdominal, vagina, and pelvic tissue, eliciting healthy tissue formation, and might be a potential choice for female pelvic reconstruction. Microsc. Res. Tech. 80:291-297, 2017. © 2016 Wiley Periodicals, Inc.

Zinc oxide 3D microstructures as an antimicrobial filler content for composite resins.

Abstract: The aim of this study was to evaluate the antibacterial activity of a composite resin modified by 3D zinc oxide (ZnO) microstructures and to verify possible alterations on its mechanical properties. ZnO was synthesized by hydrothermal approach and characterized by X-ray diffraction (XRD), surface area by Brunauer, Emmett and Teller (BET), Fourier transform infrared spectroscopy (FTIR) and Field emission scanning electron microscopy (FESEM). The minimum inhibitory concentrations of ZnO against Streptococcus mutans, Escherichia coli, Staphylococcus aureus, and Candida albicans were determinated. The composite resin FiltekTM Z350XT (3M of Brazil) was blended with 0.2%, 0.5%, and 1% in weight of ZnO and submitted to antibacterial assay by direct contact test against S. mutans, the leading cause of dental caries and the most cariogenic oral streptococci. Additionally, it was performed compressive and diametral tensile strength tests of the modified composite resin. Microrods and hollow microrods of ZnO were obtained and its MIC values were found to be 125 μg/mL for S. mutans, 500 μg/mL for C. albicans and 62.5 μg/mL for S. aureus. For the tested concentrations, it was not found MIC against E. coli. The direct contact test showed a significant antibacterial capacity of modified composite resin (p > 0.05 for all concentrations). The compressive and diametral tensile strength remains no changed after inclusion of microparticles (p > 0.05 for all concentrations). The modification of the composite resin with small amounts of ZnO microparticles significantly inhibited the S. mutans growth on resin surface without significant alterations of its mechanical strength.

Texture analysis of parasitological liver fibrosis images.

Abstract: Liver fibrosis accurate staging is vital to define the state of the Schistosomiasis disease for further treatment. The present work analyzed the microscopic liver images to identify and to differentiate between healthy, cellular, fibrocellular, and fibrous liver pathologies by proposing a fast, robust, and highly discriminative method based on texture analysis. The multiclass classification based on the "one-versus- all" method that built a voting rule approach to classify the liver images based on the liver state. Specifically, quantitative parameters, such as the anisotropy and laminarity are proposed based on the relative orientation of the pixel pairs in a global and local coherence of gradient vectors approach. Analysis of the tissue texture data using both gradient vector and gradient angle co-occurrence matrix approaches facilitated more definitive identification of the abnormal tissue. The experimental results established that the local anisotropy based texture measures are appropriated for the microtexture analysis in order to discriminate between pathologies. Macrotexture description using the global features provided only integral anisotropy coefficient that has a confidence level similar to those provided by the local feature.

Influence of passive ultrasonic irrigation on the efficiency of various irrigation solutions in removing smear layer: a scanning electron microscope study.

Abstract: To evaluate the influence of passive ultrasonic irrigation (PUI) to remove the smear layer with different irrigation solutions. Seventy-eight single rooted mandibular premolars were used. The working lengths were measured and the root canals were prepared by using nickel titanium rotary files. The specimens were subjected to six different irrigation regimens: sodium hypochloride (NaOCl) with classic irrigation (CI); NaOCl with PUI; EDTA with CI; EDTA with PUI; QMix with CI; and QMix with PUI. The specimens were split longitudinally. Scanning electron microscopy images were taken to evaluate the amount of residual smear layer and scored. Statistical analysis was performed with the Kruskal-Wallis test. The Dunn's test was used for post hoc testing after the Kruskal-Wallis test. The smear layer scores of apical thirds were significantly higher than coronal and middle thirds except for NaOCl + CI group (p < 0.001). The irrigation with PUI improved the efficacy of all irrigation solutions on removal of smear layer in both coronal and middle thirds (p < 0.001). The PUI activation increased the activity of NaOCl, EDTA, and QMix, respectively without any significance. All irrigation regimens were significantly more effective in coronal and middle thirds compared to apical third. PUI significantly increased the efficacy of smear layer removal regardless of irrigation solution.

Effect of the long‐term administration of Cyclosporine A on bone healing around osseointegrated titanium implants: A histomorphometric study in the rabbit tibia

Abstract: The aim of this study was to investigate the effect of cyclosporine A (CsA) therapy on bone healing around osseointegrated implants in the rabbit by means of descriptive histologic and histomorphometric analyses. Eighteen rabbits received one implant each in the right tibia proximal metaphysis. After the bone-healing period of 3 months, 6 randomly selected animals were sacrificed (Group CTL-12) before the immunosuppression therapy initiation to obtain a standard of implant integration. Another six randomly selected animals were submitted to a daily subcutaneous (sc) injection of 10 mg/kg CsA (Group CsA) whereas the six remaining animals received daily sc injections of saline solution (Group CTL-24). Groups CTL-24 and CsA were sacrificed after another 12 weeks. Bone-to-implant contact (BIC), and bone area fraction occupancy (BAFO) within the implant threads were measured. Bone density (BD) and descriptive histological analyses were also accomplished. Rabbits under CsA therapy presented statistically lower percentage of BIC (33.28 ± 6.19) compared to the animals of CLT-24 (55.6 ± 17.73). No differences were found between CsA and CTL-12 (38.7 ± 7.87). Significant decreased percentage of BAFO between the implant threads in the CsA (48.43 ± 9.48) group compared to the CTL-24 (72.26 ± 11.72) was observed. No differences were found between CsA and CTL-12 (61.99 ± 13.94) groups. BD analysis showed significant lower BD in the CsA (48.56 ± 9.32) group compared to the CTL-12 (68.74 ± 10.89) and CTL-24 (77.96 ± 4.96) groups. Collectively, our findings demonstrated that CsA therapy negatively affect the bone healing around osseointegrated implants due to the significant lower values for BIC, BAFO, and BD.