Showing papers in "Microscopy Research and Technique in 2011"

Growth factors, CD34 positive cells, and fibrin network analysis in concentrated growth factors fraction

Abstract: An interesting clinical option for optimizing healing tissue is the use of platelet concentrate. Platelets contain high quantities of growth factors, among these TGF-β1 and VEGF, which are known to be implicated in tissue regeneration. CGF is produced by processing blood samples with a special centrifuge device; three layers are formed: top acellular plasma (PPP), middle CGF and bottom red blood cells (RBC) layers. Given that to date there are no data concerning the biological characteristic of CGF, the aim of this study was to evaluate the presence of TGF-β1 and VEGF in CGF and also in PPP and RBC layers. In addition, since circulating stem cells are recruited from blood to injured tissue for healing we also evaluated the presence of CD34 positive cells. Our data show the presence of TGF-β1 and VEGF in CGF and RBC layers. In addition, we show CD34 positive cells in CGF.

Gold nanoparticles and quantum dots for bioimaging

Abstract: KEY WORDS cancer; glia; neurons; FRET; BRET; PRET; neuroscience; in vivo imaging;inflammationABSTRACT Nanoparticles are the latest tool acquired by the science of bioimaging, servingprimarily as new contrast agents, sensors, or signal enhancing agents in established and develop-ing imaging techniques. This review focuses on the unique properties of two classes of nano-particles: gold nanoparticles (GNP) and quantum dots, and how these properties are benefitingcellular and in vivo imaging. We discuss the surface plasmon resonance of GNP and its implica-tions for various imaging techniques of biological relevance. Furthermore, the key properties ofquantum dots are reviewed, and their use alone or in combination with traditional fluorescent dyesfor biological imaging are described. The underlying principles of these techniques are provided,along with some representative examples. Microsc. Res. Tech. 00:000–000, 2010.

Multiphoton fluorescence lifetime imaging of 3D-stem cell spheroids during differentiation.

Abstract: Long-term high-resolution multiphoton imaging of nonlabeled human salivary gland stem cell spheroids has been performed with submicron spatial resolution, 10.5-nm spectral resolution, and picosecond temporal resolution. In particular, the two-photon-excited coenzyme NAD(P)H and flavins have been detected by time-correlated single photon counting (TCSPC). Stem cells increased their autofluorescence lifetimes and decreased their total fluorescence intensity during the adipogenic-differentiation process. In addition, the onset of the biosynthesis of lipid vacuoles was monitored over a period of several weeks in stem-cell spheroids. Time-resolved multiphoton autofluorescence imaging microscopes may become a promising tool for marker-free stem-cell characterization and cell sorting.

Bimodal magnetic-fluorescent probes for bioimaging.

Abstract: Fluorescent optical probes have been intensively used in the area of bio-imaging. In this review article, we describe the recent advancements in the synthesis and application of bimodal magnetic–fluorescent probes for bioimaging. The bimodal probes consist of fluorescent [semiconducting quantum dots (e.g., CdSe/ZnS) or rare-earth doped (e.g., NaYF4:Yb,Er)] nanoparticles (NPs) and magnetic (iron oxide or gadolinium based) NPs for optical and magnetic resonance (MR) imaging. Microsc. Res. Tech., 2011. © 2010 Wiley-Liss, Inc.

Transmission X-ray microscopy for full-field nano imaging of biomaterials.

Abstract: Imaging of cellular structure and extended tissue in biological materials requires nanometer resolution and good sample penetration, which can be provided by current full-field transmission X-ray microscopic techniques in the soft and hard X-ray regions. The various capabilities of full-field transmission X-ray microscopy (TXM) include 3D tomography, Zernike phase contrast, quantification of absorption, and chemical identification via X-ray fluorescence and X-ray absorption near edge structure imaging. These techniques are discussed and compared in light of results from the imaging of biological materials including microorganisms, bone and mineralized tissue, and plants, with a focus on hard X-ray TXM at ≤ 40-nm resolution.

Application of WGA lectin staining for visualization of the connective tissue in skeletal muscle, bone, and ligament/tendon studies.

Abstract: During immunostaining of specific proteins in tissue sections using monoclonal and polyclonal antibodies, visualization of general tissue staining/background or major structural features is helpful to pinpoint precise localization of the protein of interest. Often in skeletal muscle research, immunostaining with antibodies against connective tissue or plasma membrane proteins (collagen 1, laminin, and caveolin 3) are used for this purpose. Although immunostaining for these proteins works well, it is time consuming, costly, limits the number of antibodies against protein of interest that can be used on a single section, and is not applicable to some staining techniques. Lectins were frequently used in earlier publications for skeletal muscle fiber boundaries and connective tissue visualization, but are not common in the current research studies. This work investigates costaining of muscle, bone, ligament, and tendon tissue sections with fluorescently tagged wheat germ agglutinin (WGA) lectin as a tool for the visualization of connective tissue. The results of this study show that fluorescent WGA lectin costaining is a cost-effective, fast, and convenient method for connective tissue visualization, especially in the studies where extensive washes reduce staining of the structures that are the primary interest of the investigation.

Ionic liquid enables simple and rapid sample preparation of human culturing cells for scanning electron microscope analysis

Abstract: Ionic liquid is a kind of salt that stays in a molten state even at room temperature. It does not vaporize at all in vacuum and facilitates electrical conductivity to the sample surfaces for observations with a scanning electron microscope (SEM). In this study, we used an ionic liquid in SEM for the first time to observe fixed human culture cells. The condition for the cell culture using wrapping sheets and SEM settings were varied to elucidate the optimized protocol. Compared to samples prepared by the conventional way, the ionic liquid-treatment of samples gave SEM images of the cellular ultra structures in more detail, enabling observation of microvilli that made bridges between separated cells. In addition, the ionic liquid treatment is less time consuming as well as less laborious compared with the conventional way that includes dehydration, drying, and conductivity treatments. Totally, we concluded the ionic liquid is a useful reagent for SEM sample preparation.

Effects of ultramorphological changes on adhesion to lased dentin—Scanning electron microscopy and transmission electron microscopy analysis

Abstract: Dentin irradiation with erbium lasers has been reported to alter the composite resin bond to this treated surface. There is still a lack of studies reporting the effect of erbium lasers on dentin organic content and elucidating how laser treatment could interfere in the quality of the resin-dentin interface. This study aimed to evaluate the effect of erbium laser irradiation on dentin morphology and microtensile bond strength (μTBS) of an adhesive to dentin. Seventy-two dentin disks were divided into nine groups (n = 8): G1-Control (600-grit SiC paper); Er:YAG groups: G2- 250 mJ/4 Hz; G3- 200 mJ/4 Hz; G4- 180 mJ/10 Hz; G5- 160 mJ/10 Hz; Er,Cr:YSGG groups: G6- 2 W/20 Hz; G7- 2.5 W/20 Hz; G8- 3 W/20 Hz; G9- 4 W/20 Hz. Specimens were processed for cross-sectional analysis by scanning electron microscopy (SEM) (n = 3), transmission electron microscopy (TEM) (n = 2), and adhesive interface (n = 3). Forty-five dentin samples (n = 5) were restored and submitted to μTBS testing. ANOVA (α = 5%) revealed that G1 presented the highest μTBS values and irradiated groups did not differ from each other. TEM micrographs showed a superficial layer of denatured collagen fibrils. For SEM micrographs, it was possible to verify the laser effects extending to dentin subsurface presenting a rough aspect. Cross-sectional dentin micrographs of this hybridized surface revealed a pattern of modified tags with ringlike structures around it. This in vitro study showed that erbium laser irradiation interacts with the dental hard tissue resulting in a specific morphological pattern of dentin and collagen fibrils that negatively affected the bond strength to composite resin.

Determination of matrix composition based on solute-solute nearest-neighbor distances in atom probe tomography

Abstract: In this study, we propose a fast automatic method providing the matrix concentra- tion in an atom probe tomography (APT) data set containing two phases or more. The principle of this method relies on the calculation of the relative amount of isolated solute atoms (i.e., not surrounded by a similar solute atom) as a function of a distance d in the APT reconstruction. Simulated data sets have been generated to test the robustness of this new tool and demonstrate that rapid and reproducible results can be obtained without the need of any user input parameter. The method has then been successfully applied to a ternary Al-Zn-Mg alloy containing a fine dispersion of hardening precipitates. The relevance of this method for direct estimation of matrix concentration is discussed and compared with the existing methodologies. Microsc. Res. Tech. 74:257-263, 2011. V C 2010 Wiley-Liss, Inc.

Ultrastructural observations on antennal sensilla of Cnaphalocrocis medinalis (Lepidoptera: Pyralidae).

Abstract: The morphology and ultrastructure of the antennal sensilla of Cnaphalocrocis medinalis (Lepidoptera: Pyralidae) was studied by scanning and transmission electron microscopic techniques. Eight morphological types of sensillum were recorded in both sexes: sensilla trichodea (S. trichodea), sensilla basiconc (S. basiconc), sensilla coeloconica (S. coeloconica), sensilla styloconica (S. styloconica), sensilla squamous (S. squamous), sensilla auricillica (S. auricillica), Bohm bristles (B. bristles), and sensilla cavity (S. cavity). S. trichodea were the most abundant sensilla and were distributed over the entire antennal surface. Four different types of S. trichodea and S. basiconc were observed. The number of S. basiconc and S. coeloconica of males were greater than those of females of C. medinalis. S. squamous formed on the dorsal part of the antenna, as a cluster in females and as in a line in males. Higher magnification revealed that S. basiconc had an olfactory function, while the character of longer length of these sensilla suggested that they also played a rolein sensing mechanical or other chemical stimuli. Transmission electron microscopy (TEM) of S. squamous revealed nonporous walls suggesting a non-olfactory function. S. coeloconica, S. styloconica, and S. cavity may be involved in the perception of humidity, temperature, heat, and CO₂. Because of their particular location, we infer that B. bristles may function in sensing the position and movements of the antennae, while the function of S. auricillica is as yet unknown. The results obtained provide direct morphological evidence that antennae possess structures that can play a role in finding mates and locating host plants.

Ketamine effects on the urogenital system—changes in the urinary bladder and sperm motility

Abstract: Different doses of ketamine (10 mg/kg, 20 mg/kg, 30 mg/kg, 40 mg/kg, 50 mg/kg, and 60 mg/kg) were injected i.p. (I.P.), respectively, to male ICR mice to determine the optimal dosage for chronic administration. At and above 40 mg/kg I.P. injection, mice had almost no hindlimb movement during swimming test. Subsequently, 30 mg/kg was used as the dose for the study in the toxicity of long-term ketamine administration on urinary bladder and sperm motility. The treatment group were subdivided into two (n = 10 each group); one received daily ketamine treatment i.p. for 3 months and another group for 6 months. Corresponding number of mice in control groups (n = 5 each group) received saline injection instead of ketamine. Terminal dUTP nick and labeling (TUNEL) study and Sirius red staining were carried out on the sectioned slides of the urinary bladders to study the degree of apoptosis in both epithelium and muscular layers of the urinary bladder and the relative thickness of the muscular layers in this organ was also computed. Apoptosis in the bladder epithelium was observed initially in the 3-month ketamine treated mice and the number of apoptotic cells was significantly different (P < 0.05) between the 3-month and 6-month ketamine treated mice and the control. The relative thickness of muscular layers in the bladder wall also decreased significantly (P < 0.05) when the 6-month treated mice and the control were compared. Sirius red staining revealed increase of collagen in the urinary bladder of the treated mice, most evidently 6 months after ketamine treatment. In addition, the sperm motility was studied and there was a statistically significant difference between the control and ketamine treated groups in the percentages of sperms which were motile (P < 0.05). This suggested that the chronic administration of ketamine affected the genital system as well.

The effect of fluoride therapies on the morphology of bleached human dental enamel.

Abstract: The aim of this in vitro study was to evaluate qualitatively the surface morphology of enamel bleached with 35% hydrogen peroxide (HP) followed by application of fluoridated agents. Forty intact pre molars were randomly distributed into four groups (n = 10), treated as follows: Group I (control group) remained stored in artificial saliva at 37 °C, Group II - 35% HP; Group III - 35% HP + acidulated fluoride (1.23%) and Group IV - 35% HP + neutral fluoride (2%). The experimental groups received three applications of bleaching gel and after the last application all specimens were polished. This procedure was repeated after 7 and 14 days, and during the intervals of applications, the specimens were stored in artificial saliva at 37°C. Scanning electron microscopy (SEM) analysis showed superficial irregularities and porosities to varying degrees in bleached enamel compared to control group. Sample evaluation was made by attributing scores, and data were statistically analyzed using Kruskal-Wallis and Dunn tests (P < 0.05). SEM qualitative investigation demonstrated that 35% hydrogen peroxide affected human dental enamel morphology, producing porosities, depressions, and superficial irregularities at various degrees. These morphological changes were higher after the application of 1.23% acidulated fluoride gel.

Investigations on the leaf anatomy and ultrastructure of grapevine (Vitis vinifera) under heat stress

Abstract: Leaf anatomical and ultrastructural responses of "Razegui" and "Muscat Italia" grapevine cultivars to high temperatures were studied under controlled conditions (T > 36°C), based on photonic and electron microscopy. Histological studies performed on leaves from heat-stressed and control grapevines revealed thicker leaf blades under high temperature conditions. Environmental scanning electron microscopy of leaf surfaces from both cultivars allowed observing sinuate epidermal cells on the leaves of grapevines cultivated under heat stress and irregular giant oblong pores on their adaxial surface. When observed by transmission electron microscopy, leaf cross sections in grapevines cultivated under high temperature conditions exhibited folded cuticle and cell wall on the adaxial epidermis layer. Therefore, significantly greater cell wall thicknesses were measured under heat stress than control conditions in both cultivars. Regarding chloroplasts, they were more globular in shape under heat stress compared with control conditions and had disorganized thylakoids with a reduced thickness of grana stacking. The size of starch granule decreased, while the number of plastoglobules increased with heat stress, indicating a reduced carbon metabolism and a beginning of senescence within the 3-month heat stress period. This study confirms widespread adaptive properties in two grapevine cultivars in response to high temperature stress.

Abundance of antennal chemosensilla in two parasitoid wasps with different degree of host specificity may explain sexual and species differences in their response to host-related volatiles.

Abstract: The endoparasitic wasps (Hymenoptera: Braconidae), Microplitis croceipes (specialist), and Cotesia marginiventris (generalist) are parasitoids of lepidopteran larvae that differ in their degree of host specificity. Previous studies by our group have reported key differences in the response of both parasitoids to host-related volatiles. To possibly understand the basis for our results, we compared the abundance of antennal sensilla in both sexes of both parasitoid species using scanning electron microscopy and silver-staining techniques. Males of both species had relatively longer antennae than conspecific females. Five major sensilla types were recorded in both species: sensilla chaetica (nonporous), s. trichodea (nonporous), s. placodea (multiporous), s. basiconica (two types, type 1 with terminal opening and type 2 with wall pores), and s. coeloconica (nonporous). Silver staining confirmed multiple pores on the walls of s. placodea and s. basiconica type 2 and terminal openings on s. basiconica type 1, suggesting chemosensory function. In general, both putative chemosensilla types, s. placodea and s. basiconica, were more abundant in M. croceipes (specialist) than in C. marginiventris (generalist), and this was true for both sexes. Comparing the sexes, s. placodea and s. trichodea were significantly more abundant in M. croceipes males compared to females. In contrast, s. placodea was relatively more abundant in female C. marginiventris than in males. These results may explain the reported differences in the responses of both parasitoid species to host-related volatiles and are discussed in relation to the possible roles of the sensilla types in the behavioral ecology of the parasitoids. Microsc. Res. Tech. 2010. © 2010 Wiley-Liss, Inc.

The application of nanoparticles in gene therapy and magnetic resonance imaging.

Abstract: The combination of nanoparticles, gene therapy, and medical imaging has given rise to a new field known as gene theranostics, in which a nanobioconjugate is used to diagnose and treat the disease. The process generally involves binding between a vector carrying the genetic information and a nanoparticle, which provides the signal for imaging. The synthesis of this probe generates a synergic effect, enhancing the efficiency of gene transduction and imaging contrast. We discuss the latest approaches in the synthesis of nanoparticles for magnetic resonance imaging, gene therapy strategies, and their conjugation and in vivo application.

Automatic evaluation of nickel alloy secondary phases from SEM images

Abstract: Quantitative metallography is a technique to determine and correlate the microstructures of materials with their properties and behavior. Generic commercial image processing and analysis software packages have been used to quantify material phases from metallographic images. However, these all-purpose solutions also have some drawbacks, particularly when applied to segmentation of material phases. To overcome such limitations, this work presents a new solution to automatically segment and quantify material phases from SEM metallographic images. The solution is based on a neuronal network and in this work was used to identify the secondary phase precipitated in the gamma matrix of a Nickel base alloy. The results obtained by the new solution were validated by visual inspection and compared with the ones obtained by a commonly used commercial software. The conclusion is that the new solution is precise, reliable and more accurate and faster than the commercial software. Microsc. Res. Tech. 74:36-46, 2011. © 2010 Wiley-Liss, Inc.

High-resolution X-ray CT for 3D petrography of ferruginous sandstone for an investigation of building stone decay.

Abstract: Diestian ferruginous sandstone has been used as the dominant building stone for monuments in the Hageland, a natural landscape in east-central Belgium. Like all rocks, this stone type is sensitive to weathering. Case hardening was observed in combination with blackening of the exterior parts of the dressed stones. To determine the 3D petrography and to identify the structural differences between the exterior and interior parts, X-ray computed tomography was used in combi- nation with more traditional research techniques like optical microscopy and scanning electron microscopy. The 3D characterization of the ferruginous sandstone was performed with a high-resolu- tion X-ray CT scanner (www.ugct.ugent.be) in combination with the flexible 3D analysis software Morpho1, which provides the necessary petrophysical parameters of the scanned samples in 3D. Besides providing the required 3D parameters like porosity, pore-size distribution, grain size, grain orientation, and surface analysis, the results of the 3D analysis can also be visualized, which enables to understand and interpret the analysis results in a straightforward way. The complementarities between high-quality X-ray CT images and flexible 3D software and its relation with the more tradi- tional microscopical research techniques are opening up new gateways in the study of weathering processes of natural building stones. Microsc. Res. Tech. 00:000-000, 2011. V C 2011 Wiley-Liss, Inc.

Transmission electron microscopy as a tool to image bioinorganic nanohybrids: the case of phage-gold nanocomposites.

Abstract: In recent years, bioinorganic nanohybrids composed of biological macromolecules and functional inorganic nanomaterials have revealed many unique properties that show promise for the future. Transmission electron microscopy (TEM) is a popular and relatively simple tool that can offer a direct visualization of the nanomaterials with high resolutions. When TEM is applied to visualize bioinorganic nanohybrids, a treatment of negative staining is necessary due to the presence of biological molecules in the nanohybrids except for those with densely packed inorganic materials. However, the conventional negative-staining procedure for regular biological samples cannot be directly applied to such bioinorganic nanohybrids. To image a specific bioinorganic nanohybrid, negative-staining factors such as negative stain type, working pH, staining time, and drying method, should be identified. Currently, no detailed studies have been done to investigate how to adjust negative-staining factors based on specific bioinorganic nanohybrids. In this study, bacteriophage-gold nanoparticle hybrids were chosen as a model to systematically study the effects of each factor on the negative staining of the nanohybrids. The best staining conditions for gold nanoparticle-phage nanohybrids were obtained and the effects of each factor on the negative staining of general nanohybrids were discussed. This work indicates that with proper staining it is possible to use TEM to visualize directly both biological and inorganic components without introducing any artifact. Microsc. Res. Tech. 2011. © 2011 Wiley-Liss, Inc.

Effect of ultrasonic excitation on the porosity of glass ionomer cement: a scanning electron microscope evaluation

Abstract: Sao Paulo State Univ, Araraquara Dent Sch, Dept Orthodont & Pediat Dent, BR-14801903 Araraquara, SP, Brazil

Visualization of bionanostructures using transmission electron microscopical techniques.

Abstract: In the recent years, nanotechnology has rapidly evolved as promising toolbox for many applications, including sensing and drug delivery. Nanotechnology aims at forming man-designed two-dimensional and three-dimensional structures in the nanometer scale using e.g., the self-assembly properties of smaller building blocks such as DNA and RNA. The visualization and structural characterization of these nanostructures do not only provide evidence for the correct formation of the desired shapes, but can also contribute to a better understanding of their formation and functionality. Transmission electron microscopy offers the possibility to directly visualize the individual nanostructures. The vitrification of the sample by using the plunge-freezing method and subsequent electron cryomicroscopy (cryo-EM) provides in-solution snapshots of the nanostructures under cryogenic conditions and thus preserves the close-to-native structure of the particles. Here, we describe the plunge-freezing and other sample preparation protocols such as negative staining and cryo-negative staining as well as the various imaging and image processing methods, including electron crystallography, electron tomography, and single-particle EM. Typical example applications are provided together with a discussion of benefits and shortcomings of these approaches. We also discuss how deviations from an ideal symmetry and structural heterogeneity, in general, can limit the resolution. Finally, we suggest that nanotechnological approaches may not only offer new applications in the field of nanomaterial science and nanomedicine, but may also emerge as tools for structural biology and structure-related biomedical research. Microsc. Res. Tech., 2011. © 2010 Wiley-Liss, Inc.

Scanning electron microscopy with an ionic liquid reveals the loss of mitotic protrusions of cells during the epithelial–mesenchymal transition

Abstract: Epithelial-mesenchymal transition (EMT) is a key event in cancer metastasis and is characterized by increase in cell motility, increase in expression of mesenchymal cell markers, loss of proteins from cell-to-cell junction complexes, and changes in cell morphology. Here, the morphological effects of a representative EMT inducer, transforming growth factor (TGF)-β1, were investigated in human lung adenocarcinoma (A549) cells and pancreatic carcinoma (Panc-1) cells. TGF-β1 caused morphological changes characteristic of EMT, and immunostaining showed loss of E-cadherin from cell-to-cell junction complexes in addition to the upregulation of the mesenchymal marker vimentin. During scanning electron microscopy (SEM) with an ionic liquid, we observed EMT-specific morphological changes, including the formation of various cell protrusions. Interestingly, filopodia in mitotic cells were clearly observed by SEM, and the number of these filopodia in TFG-β1-treated mitotic cells was reduced significantly. We conclude that this reduction in such mitotic protrusions is a novel effect of TGF-β1 and may contribute to EMT.

Morphological and Molecular Pathology of CCl4-Induced Hepatic Fibrosis in Connexin43-Deficient Mice

Abstract: Gap junction channels, formed by connexins (Cx), are involved in the maintenance of tissue homeostasis, cell growth, differentiation, and development. Several studies have shown that Cx43 is involved in the control of wound healing in dermal tissue. However, it remains unknown whether Cx43 plays a role in the control of liver fibrogenesis. Our study investigated the roles of Cx43 heterologous deletion on carbon tetrachloride (CCl(4))-induced hepatic fibrosis in mice. We administered CCl(4) to both Cx43-deficient (Cx43(+/-)) and wild-type mice and examined hepatocellular injury and collagen deposition by histological and ultrastructural analyses. Serum biochemical analysis was performed to quantify liver injury. Hepatocyte proliferation was analyzed immunohistochemically. Protein and messenger RNA (mRNA) expression of liver connexins were evaluated using immunohistochemistry as well as immunoblotting analysis and quantitative real-time PCR. We demonstrated that Cx43(+/-) mice developed excessive liver fibrosis compared with wild-type mice after CCl(4) -induced chronic hepatic injury, with thick and irregular collagen fibers. Histopathological evaluation showed that Cx43(+/-) mice present less necroinflammatory lesions in liver parenchyma and consequent reduction of serum aminotransferase activity. Hepatocyte cell proliferation was reduced in Cx43(+/-) mice. There was no difference in Cx32 and Cx26 protein or mRNA expression in fibrotic mice. Protein expression of Cx43 increased in CCl(4)-treated mice, although with aberrant protein location on cytoplasm of perisinusoidal cells. Our results demonstrate that Cx43 plays an important role in the control and regulation of hepatic fibrogenesis.

An overview of the legislation and light microscopy for detection of processed animal proteins in feeds

Abstract: From the first cases of bovine spongiform encephalopathy (BSE) among cattle in the United Kingdom in 1986, the route of infection of BSE is generally believed by means of feeds containing low level of processed animal proteins (PAPs). Therefore, many feed bans and alternative and complementary techniques were resulted for the BSE safeguards in the world. Now the feed bans are expected to develop into a "species to species" ban, which requires the corresponding species-specific identification methods. Currently, banned PAPs can be detected by various methods as light microscopy, polymerase chain reaction, enzyme-linked immunosorbent assay, near infrared spectroscopy, and near infrared microscopy. Light microscopy as described in the recent Commission Regulation EC/152/2009 is the only official method for the detection and characterization of PAPs in feed in the European Union. It is able to detect the presence of constituents of animal origin in feed at the level of 1 g/kg with hardly any false negative. Nevertheless, light microscopy has the limitation of lack of species specificity. This article presents a review of legislations on the use of PAPs in feedstuff, the detection details of animal proteins by light microscopy, and also presents and discusses the analysis procedure and expected development of the technique.

Automated image analysis for tracking cargo transport in axons

Abstract: The dynamics of cargo movement in axons encodes crucial information about the underlying regulatory mechanisms of the axonal transport process in neurons, a central problem in understanding many neurodegenerative diseases. Quantitative analysis of cargo dynamics in axons usually includes three steps: (1) acquiring time-lapse image series, (2) localizing individual cargos at each time step, and (3) constructing dynamic trajectories for kinetic analysis. Currently, the later two steps are usually carried out with substantial human intervention. This article presents a method of automatic image analysis aiming for constructing cargo trajectories with higher data processing throughput, better spatial resolution, and minimal human intervention. The method is based on novel applications of several algorithms including 2D kymograph construction, seed points detection, trajectory curve tracing, back-projection to extract spatial information, and position refining using a 2D Gaussian fitting. This method is sufficiently robust for usage on images with low signal-to-noise ratio, such as those from single molecule experiments. The method was experimentally validated by tracking the axonal transport of quantum dot and DiI fluorophore-labeled vesicles in dorsal root ganglia neurons.

Human odontoblasts express transient receptor protein and acid-sensing ion channel mechanosensor proteins.

Abstract: Diverse proteins of the denegerin/epithelial sodium channel (DEG/ENa(+) C) superfamily, in particular those belonging to the acid-sensing ion channel (ASIC) family, as well as some members of the transient receptor protein (TRP) channel, function as mechanosensors or may be required for mechanosensation in a diverse range of species and cell types. Therefore, we investigated the putative mechanosensitive function of human odontoblasts using immunohistochemistry to detect ENa(+) C subunits (α, β, and γ) and ASIC (1, 2, 3, and 4) proteins, as well as TRPV4, in these cells. Positive and specific immunoreactivity in the odontoblast soma and/or processes was detected for all proteins studied except α-ENa(+) C. The intensity of immunostaining was high for β-ENa(+) C and ASIC2, whereas it was low for ASIC1, ASIC3, γ-ENa(+) C, and TRPV4, being absent for α-ENa(+) C and ASIC4. These results suggest that human odontoblasts in situ express proteins related to mechanosensitive channels that probably participate in the mechanisms involved in teeth sensory transmission.

Inhibitory action of neem aqueous extract (Azadirachta indica A. Juss) on the vitellogenesis of Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae) ticks.

Abstract: The present study revealed unheard of data about the action of aqueous extracts of neem leaves (Azadirachta indica) on the vitellogenesis of Rhipicephalus sanguineus ticks, proving that these extracts in 10 and 20% concentrations do not have the potential to kill the females; however, in lower concentrations (10%) provokes great morphological alterations in germinative cells such as the emergence of extended cytoplasmic vacuolization areas as well as the fragmentation of the germinal vesicle, even in those oocytes which were in initial stages of development (I-III), showing that neem is a potent agent which acts impeding one of the main metabolic stages of the ticks, i.e., the reproduction. In oocytes in final stages of development (IV-V) azadirachtin (neem's active principle) caused significant reduction in the size and quantity of proteic granules of the yolk and the inversion of their localization where the smaller granules before inside the cell (normal oocyte) were posteriorly observed in the periphery, and the bigger ones in the central region. Thus, the study showed that the alterations found both in the oocytes and in the pedicel cells indicated that azadirachtin acts on the process of tick's reproduction and signalizes that this plant can be used in the future to control ticks with the advantage of not being aggressive to nontarget organism or the environment. Furthermore, data here obtained showed that the most significant efficiency of the aqueous extract of neem is related to the concentration of 10%, proving that higher doses would not be so efficient.

Dual‐color fluorescence lifetime correlation spectroscopy to quantify protein–protein interactions in live cell

Abstract: Dual-color fluorescence correlation spectroscopy is an interesting method to quantify protein interaction in living cells. But, when performing these experiments, one must compensate for a known spectral bleed through artifact that corrupts cross-correlation data. In this article, problems with crosstalk were overcome with an approach based on fluorescence lifetime correlation spectroscopy (FLCS). We show that FLCS applied to dual-color EGFP and mCherry cross-correlation allows the determination of protein-protein interactions in living cells without the need of spectral bleed through calibration. The methodology was validated by using EGFP-mCherry tandem in comparison with coexpressed EGFP and mCherry in live cell. The dual-color FLCS experimental procedure where the different laser intensities do not have to be controlled during experiment is really very helpful to study quantitatively protein interactions in live sample.

High‐resolution imaging and nano‐manipulation of biological structures on surface

Abstract: In this mini-review we discuss our recent findings on imaging and manipulation of biological macromolecular structures by atomic force microscopy (AFM). In the first part of this review, we focus on high-resolution imaging of selected biological samples. AFM images of membrane proteins have revealed detailed conformational features related to identifiable biological functions. Different self-assembling behaviors of short peptides into supramolecular structures on various substrates under controlled environmental conditions have been systematically studied with AFM imaging. In the second part, we present a novel nano-manipulation technique for manipulating, isolating, amplifying, and sequencing of individual DNA molecules, which may find unique applications in the analysis of difficult sequence structures. Finally, we discuss how to characterize the elasticity of individual biomolecules and live cells. These results demonstrate that not only the high resolution capacity of the AFM is suited to resolve certain biological questions, but can also be applied to single molecule isolation and biomechanical analysis with its unique advantages. Microsc. Res. Tech. 74: 614-626, 2011. (C) 2010 Wiley-Liss, Inc.

Effects of fluoride or nanohydroxiapatite on roughness and gloss of bleached teeth

Abstract: Objectives: The aim of this study was to describe roughness and gloss alterations of enamel after treatment with 38% hydrogen peroxide (HP) and after polishing with 2% neutral sodium fluoride (SF) or a dental tooth paste containing nanohydroxiapatite particles (nHA) using power spectral density (PSD) description, roughness parameters (Ra, RMS, and Z range) and gloss analysis. Methods: An atomic force microscope (AFM) and a spectrophotometer were used to analyze eighteen specimens of upper incisors. After initial analyses, all specimens were bleached with 38% HP for 135 min. The specimens were analyzed after bleaching. Nine specimens were polished with SF (Group Fluor) and the other nine specimens were polished with nHA (Group nHA), then all specimens were analyzed after polishing. Roughness and gloss were analyzed with ANOVA and Tukey's t-test. Results: No statistical difference was found for Ra and RMS among initial, after bleaching and after polishing in both groups. For Z range, Group nHA showed a significant decrease after polishing. Bleaching with 38% HP did not increase the PSD in the spatial frequency of the visible light spectrum range in both groups. After polishing, nHA group showed a decrease in PSD for all morphological wavelengths. Gloss did not show statistical difference after bleaching in both groups. Gloss showed significant increase after polishing with nHA. Significance: bleaching treatment with 38% HP didn't alter enamel surface roughness or gloss. PSD analyses were very suitable to identifying the morphological changes on the surfaces. Microsc. Res. Tech., 2011. © 2011 Wiley Periodicals, Inc.

A detailed morphological study of the vomeronasal organ and the accessory olfactory bulb of cats

Abstract: The organization of the vomeronasal system (VNS) of fetal, newborn, and adult cats was investigated by microdissection and microscopic examination of sections stained conventionally or with lectins (UEA-1, LEA) or antibodies against proteins Gα i2 (associated with vomeronasal receptor type1) and Gα o (associated with receptor vomeronasal receptor type2). The feline VNS is morphologically similar to that of other mammals. Staining with lectins and anti-Gα i2 was uniform throughout the sensory epithelium of the vomeronasal organ, and throughout the nervous and glomerular layers of the accessory olfactory bulb (AOB); anti-Gα o stained no VNS tissue. This organization places the cat together with several other domestic or farm animals (dog, horse, sheep, goat, pig) in a group of mammals with just a single path of communication between the sensory epithelium of the vomeronasal organ and AOB, in contrast to the two-path model found in rodents and other mammals (in which apical and basal sensory epithelium layers project to rostral and caudal AOB areas, respectively). However, the cat differs from the sheep and pig in that the development of its VNS is still incomplete at birth. Microsc. Res. Tech. 2011. © 2011 Wiley Periodicals, Inc.