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Selected AbstractsComment on ,Generalized approach for accelerated maximum likelihood based image restoration applied to three-dimensional fluorescence microscopy', by L. H. Schaefer, D. Schuster and H. Herz, J. Microsc.JOURNAL OF MICROSCOPY, Issue 1 2002107 (2001) No abstract is available for this article. [source] Protection capabilities of nanostructured shells toward cell encapsulation: A saccharomyces/paramecium modelMICROSCOPY RESEARCH AND TECHNIQUE, Issue 10 2010Raffaella Magrassi Abstract In this work we report the formation of nanostructured hybrid objects, made up of living cells encapsulated in a protective multilayer shell assembly of nanostructured polyelectrolyte. Such constructs can be hosted on nanostructured surfaces or can be installed around living organisms, at the right time. Their construction is based on the layer-by-layer (LbL) self-assembly of two oppositely alternated charged polyelectrolytes (PEs) on cell membranes as earlier done for nanocapsules or fuzzy structured nanoshells. This communication reports the optimal conditions for cell encapsulation in terms of nanoshell design and construction. Microsc. Res. Tech. 73:931,936, 2010. © 2010 Wiley-Liss, Inc. [source] AFM measurement of the stiffness of layers of agarose gel patterned with polylysineMICROSCOPY RESEARCH AND TECHNIQUE, Issue 10 2010Marco Salerno Abstract Films of agarose gel microspotted with polylysine aqueous solution have been characterized by atomic force microscopy carried out in deionized water. Thickness and surface morphology of the layers have been checked, and the effect of polylysine impregnation on the local elasticity has been investigated. An increase in contact stiffness of the organic layer at the spotted areas has been observed, correlated with the polylysine concentration. For the considered agarose layer thickness of ,0.9 ,m in dry condition, the concentration threshold at which stiffening appears is ,0.1 mg/mL. Above this threshold, the stiffening coefficient becomes approximately twofold and seems not to increase significantly with concentration in the range 0.3,0.7 mg/mL. For concentrations above the stiffening threshold, this effect is also accompanied by a locally lower film thickness. For quantitative determination of the stiffness, force,distance curves extracted from the regions of interest of spots and agarose substrate have been selected and processed. These curves were fitted to the Hertz model of purely elastic tip-surface interaction, under appropriate assumptions on both tip shape and optimum indentation depth. In this way, we could determine the Young's modulus of the agarose layer to be ,50 kPa and quantitatively confirm the stiffening due to polylysine. Microsc. Res. Tech. 73:982,990, 2010. © 2010 Wiley-Liss, Inc. [source] Multiscale observation of biological interactions of nanocarriers: From nano to macroMICROSCOPY RESEARCH AND TECHNIQUE, Issue 9 2010Su-Eon Jin Abstract Microscopic observations have played a key role in recent advancements in nanotechnology-based biomedical sciences. In particular, multiscale observation is necessary to fully understand the nano-bio interfaces where a large amount of unprecedented phenomena have been reported. This review describes how to address the physicochemical and biological interactions of nanocarriers within the biological environments using microscopic tools. The imaging techniques are categorized based on the size scale of detection. For observation of the nanoscale biological interactions of nanocarriers, we discuss atomic force microscopy (AFM), scanning electron microscopy (SEM), and transmission electron microscopy (TEM). For the micro to macro-scale (in vitro and in vivo) observation, we focus on confocal laser scanning microscopy (CLSM) as well as in vivo imaging systems such as magnetic resonance imaging (MRI), superconducting quantum interference devices, and IVIS®. Additionally, recently developed combined techniques such as AFM-CLSM, correlative light and electron microscopy (CLEM), and SEM spectroscopy are also discussed. In this review, we describe how each technique helps elucidate certain physicochemical and biological activities of nanocarriers such as dendrimers, polymers, liposomes, and polymeric/inorganic nanoparticles, thus providing a toolbox for bioengineers, pharmaceutical scientists, biologists, and research clinicians. Microsc. Res. Tech. 73:813,823, 2010. © 2010 Wiley-Liss, Inc. [source] Two and three-dimensional gene transfer from enzymatically degradable hydrogel scaffoldsMICROSCOPY RESEARCH AND TECHNIQUE, Issue 9 2010Yuguo Lei Abstract The ability to genetically modify mesenchymal stem cells (MSCs) seeded inside synthetic hydrogel scaffolds would offer an alternative approach to guide MSC differentiation. In this report, we explored gene transfer to MSCs seeded on top or inside matrix metalloproteinase (MMP) degradable hydrogels that were loaded with DNA/poly(ethylene imine) (PEI) polyplexes. DNA/PEI polyplexes were encapsulated inside poly(ethylene glycol) (PEG) hydrogels crosslinked with MMP degradable peptides via Michael Addition chemistry. Gene transfer was visualized and quantified through using a vector encoding for green fluorescent protein and luciferase. We found that gene transfer to MSCs was possible for cells seeded both in two and three dimensions. The amount of luciferase expression was similar for cells seeded in two and three dimensions even though the number of cells in three dimensions is significantly higher, indicating that gene transfer to cells seeded in two dimensions is more efficient than for cells seeded in three dimensions. The use of hydrogel scaffolds that allow cellular infiltration to deliver DNA may result in long-lasting signals in vivo, which are essential for the regeneration of functional tissues. Microsc. Res. Tech. 73:910,917, 2010. © 2010 Wiley-Liss, Inc. [source] Bioluminescent imaging of reporter gene expression in the lungs of wildtype and model mice following the administration of PEG-stabilized DNA nanoparticlesMICROSCOPY RESEARCH AND TECHNIQUE, Issue 9 2010Assem G. Ziady Abstract DNA nanoparticles (DNPs) formed by compacting DNA with polyethyleneglycolylated poly- L -lysine are a nonviral vector shown to be safe and efficacious in animals and humans. To extend our capabilities of assessing the efficacy and duration of expression achieved by DNPs, we tested the utility of bioluminescent imaging (BLI) of transgene expression in wildtype and cystic fibrosis (CF) mouse models. We tested the effect of route of administration, mouse coat color, anesthesia, dose, and promoter sequence on the level and duration of expression. Furthermore, we investigated the correlation between imaging and direct analysis of luciferase expression in lung homogenates. We found that intratracheal instillation, and the use of deep and prolonged anesthesia with avertin produced significantly higher expression compared with intranasal administration, and the use of lighter anesthesia with isoflurane. Although similar expression was observed for both dark and light coat animals, imaging signal intensity was attenuated in mice with dark fur. Furthermore, good correlation between imaging and direct homogenate analysis was observed for single dose (r = 0.96), and dose response studies in wildtype (r = 0.82) and CF mice (r = 0.87). Finally, we used imaging to track gene expression over a 56-day time course. We found that the human ubiquitin B promoter gives stable transgene expression up to 49 days following nanoparticle administration, while expression with the cytomegalovirus promoter diminished after 2 days and returned to background levels by day 14. Taken together, our results demonstrate that BLI is an effective and useful modality for measuring gene expression conferred by DNPs in the lung. Microsc. Res. Tech. 73:918,928, 2010. © 2010 Wiley-Liss, Inc. [source] Technical advances in the sectioning of dental tissue and of on-section cross-linked collagen detection in mineralized teethMICROSCOPY RESEARCH AND TECHNIQUE, Issue 8 2010Sim K. Singhrao Abstract Immunohistochemical detection of cross-linked fibrillar collagens in mineralized tissues is much desired for exploring the mechanisms of biomineralization in health and disease. Mineralized teeth are impossible to section when embedded in conventional media, thus limiting on-section characterization of matrix proteins by immunohistochemistry. We hypothesized that by using an especially formulated acrylic resin suitable for mineralized dental tissues, not only sectioning of teeth would be possible, but also our recently developed immunofluorescence labeling technique would be amenable to fully calcified tissues for characterization of dentinal fibrillar collagens, which remains elusive. The hypothesis was tested on fixed rodent teeth embedded in Technovit 9100 New®. It was possible to cut thin (1 ,m) sections of mineralized teeth, and immunofluorescence characterization of cross-linked type I fibrillar collagen was selected due to its abundance in dentine. Decalcified samples of teeth embedded in paraffin wax were also used to compare immunolabeling from either method using the same immunoreagents in equivalent concentrations. In the decalcified tissue sections, type I collagen labeling in the dentine along the tubules was "patchy" and the signal in the predentine was very weak. However, enhanced signal in mineralized samples with type I collagen was detected not only in the predentine but also at the limit between intertubular dentine, within the elements of the enamel organ and subgingival stroma. This report offers advances in sectioning mineralized dental tissues and allows the application of immunofluorescence not only for on-section protein detection but importantly for detecting cross-linked fibrous collagens in both soft and mineralized tissue sections. Microsc. Res. Tech. 73:741,745, 2010. © 2009 Wiley-Liss, Inc. [source] Physical attachment of fluorescent protein particles to atomic force microscopy probes in aqueous media: Implications for surface pH, fluorescence, and mechanical properties studiesMICROSCOPY RESEARCH AND TECHNIQUE, Issue 8 2010Susana Moreno-Flores Abstract Transfer of a fluorescently labeled protein particle from a surface to a microsized scanning probe has been induced by repetitive scanning in aqueous medium. The so-attached particle can in turn act as a probing tool to study particle,substrate and particle,particle interactions. Attachment of the fluorescent particle occurs at the apical region of an atomic force microscope (AFM) cantilever tip and it endures repetitive loading,unloading cycles against the sample surface. Fluorescence microscopy has been used to address the exact location of the attached particle in the cantilever and to identify the moment when the particle contacts the sample. Moreover, we have observed that fluorescence intensity at the contact point is lower when the probing particle contacts another fluorescent particle than when it contacts the nonfluorescent substrate. The change in fluorescence is attributed to local changes of pH and interparticle-quenching of fluorophores in the contact region. These findings are promising since they constitute a chemical-free way to attach bioparticles to AFM probes under fisiological conditions. The atomic force microscopy combined with fluorescence microscopy provides a straight forward method to study particle/particle and particle/substrate interactions, as well as to investigate mechanical properties of biocolloids. Microsc. Res. Tech. 73:746,751, 2010. © 2009 Wiley-Liss, Inc. [source] MRT Letter: Spatial distribution of vancomycin-induced damage in Staphylococcus epidermidis biofilm: An electron microscopic studyMICROSCOPY RESEARCH AND TECHNIQUE, Issue 7 2010Rachna Singh Abstract This study was planned to elucidate the efficacy of antibiotics on Staphylococcus epidermidis and Staphylococcus aureus biofilms by scanning electron microscopy (SEM). Biofilms of S. epidermidis ATCC 35984 and S. aureus ATCC 29213 were grown on black, polycarbonate membranes placed on tryptic soy agar plates for 48 h at 37°C, and then exposed to vancomycin or amikacin or ciprofloxacin at clinically achievable levels for 24 h at 37°C. The morphology of antibiotic-treated and untreated biofilms was elucidated by SEM. SEM analysis indicated a differential affection of S. epidermidis ATCC 35984 in the center and periphery of biofilm upon treatment with vancomycin. The center of biofilm revealed damaged cells with sparse distribution, smaller size, and irregular shape, whereas cells in the periphery were unaffected. This differential distribution of susceptibility within S. epidermidis ATCC 35984 biofilms was specific for vancomycin only and was not observed on exposure to amikacin or ciprofloxacin. No such response was found in S.aureus ATCC 29213 biofilms. Thus, our study suggests a spatial distribution of vancomycin-induced damage in S. epidermidis biofilms. To our knowledge, this is the first report that indicates a differential affection of S. epidermidis in the center and periphery of biofilm upon treatment with vancomycin. Studies on the factors controlling this differential distribution could provide valuable insights into the mechanisms of antimicrobial resistance in S. epidermidis biofilms. Microsc. Res. Tech., 2010. © 2010 Wiley-Liss, Inc. [source] A new bone vascular perfusion compound for the simultaneous analysis of bone and vasculatureMICROSCOPY RESEARCH AND TECHNIQUE, Issue 7 2010Krista L. Sider Abstract Bone is a highly vascular tissue, which plays an important role in bone development and healing. The ability to analyze both the bone and vasculature simultaneously can enhance the understanding of wound healing, development, and disease in bone. At present, analysis methods are limited in their ability to allow for this simultaneous analysis of bone and bone vasculature in three dimensions, without using the most recent dual-energy computed tomography (CT) techniques. In this study, we present a new barium sulfate (BaSO4) radiopaque vascular perfusion compound for performing postmortem microangiography with single-beam microcomputed tomography (microCT), which allows for such simultaneous analysis. This compound differs from currently available contrast mediums due to (1) the high weight-to-volume ratio of BaSO4 achieved, (2) small BaSO4 aggregate size (<5 ,m), (3) minimal additives, and (4) its miscibility with blood and saline. Most notably, it achieves a radiodensity of 2.4× that of cortical bone, with high perfusion of both the arterial and venous systems and the intervening capillary bed, resulting in an in vivo radiodensity that ranges from that of bone to titanium. Our results, verified using a rat femoral gap-healing model, show that the compound is uniquely suited to high-contrast imaging of the vasculature in the presence of undecalcified bone, with a versatility to be used in other tissues. Microsc. Res. Tech., 2010. © 2009 Wiley-Liss, Inc. [source] Aberrant distribution of junctional complex components in retinoic acid receptor alpha-deficient miceMICROSCOPY RESEARCH AND TECHNIQUE, Issue 6 2010Sanny S.W. Chung Abstract Retinoic acid receptor alpha (RAR,)-deficient mice are sterile, with abnormalities in the progression of spermatogenesis and spermiogenesis. In this study, we investigated whether defective retinoid signaling involved at least in part, disrupted cell,cell interactions. Hypertonic fixation approaches revealed defects in the integrity of the Sertoli-cell barrier in the tubules of RAR,-deficient testes. Dye transfer experiments further revealed that coupling between cells from the basal to adluminal compartments was aberrant. There were also differences in the expression of several known retinoic acid (RA)-responsive genes encoding structural components of tight junctions and gap junctions. Immunostaining demonstrated a delay in the incorporation of zonula occludens (ZO-1), a peripheral component protein of tight junctions, into the Sertoli cell tight junctions. Markedly reduced expression of connexin-40 in mutant pachytene spermatocytes and round spermatids was found by in situ hybridization. An ectopic distribution of vimentin and disrupted cyclic expression of vimentin, which is usually tightly regulated during spermiogenesis, was found in RAR,-deficient testes at all ages examined. Thus, the specific defects in spermiogenesis in RAR,-deficient testes may correlate with a disrupted cyclic expression of RA-responsive structural components, including vimentin, a downregulation of connexin-40 in spermatogenic cells, and delayed assembly of ZO-1 into Sertoli cell tight junctions. Interestingly, bioinformatic analysis revealed that many genes that are components of tight junctions and gap junctions contained potential retinoic acid response element binding sites. Microsc. Res. Tech., 2010. © 2009 Wiley-Liss, Inc. [source] Evaluation of the smear layer and hybrid layer in noncarious and carious dentin prepared by air abrasion system and diamond tipsMICROSCOPY RESEARCH AND TECHNIQUE, Issue 6 2010Ana Carolina Mascarenhas Oliveira Abstract Purpose: To analyze the smear layer and the hybrid layer in noncarious and carious dentin prepared by different cutting instruments and restored with composite resin. Study design: Cavities were randomly prepared in 160 specimens (noncarious and artificial carious dentin) by high-speed diamond tips (KG Sorensen 1013), air abrasion system (Prepstart, Danville Engineering), ultrasonic tip (CVDentus 8.3231-1), and ultrasonic tip associated with ultrasonic cavitation by water for 10 s. Half of the cavities in each group were conditioned with 37% phosphoric acid for 15 s. The amount of smear layer and dentinal tubules present were analyzed using scanning electron microscopy and graded from 0 to 3. Cavities were prepared in another 20 noncarious specimens and 20 carious specimens and restored with adhesive composite resin system. The restorations were hemisected longitudinally and analyzed using scanning electron microscopy to evaluate the hybrid layer and resinous prolongation characteristics, using scores ranging from 1 to 6. Results: The data were statistically analyzed using Kruskal-Wallis and Dunn tests at 5% of significance level. There was evidence that the most efficient smear layer removal was the acid etching in the noncarious dentin and the water ultrasonic cavitation in the carious dentin. The hybrid layer formed on the noncarious and carious dentin prepared by the ultrasonic tip was more regular than in the specimens prepared by high-speed diamond tip, with many resinous prolongations. Conclusion: The ultrasonic tip seems to be a promising tool for carious dentin cavity preparation. Microsc. Res. Tech., 2010. © 2009 Wiley-Liss, Inc. [source] Quantitative analysis of spatial proteoglycan content in articular cartilage with Fourier transform infrared imaging spectroscopy: Critical evaluation of analysis methods and specificity of the parametersMICROSCOPY RESEARCH AND TECHNIQUE, Issue 5 2010L. Rieppo Abstract Objective: To evaluate the specificity of the current Fourier transform infrared imaging spectroscopy (FT-IRIS) methods for the determination of depthwise proteoglycan (PG) content in articular cartilage (AC). In addition, curve fitting was applied to study whether the specificity of FT-IRIS parameters for PG determination could be improved. Methods: Two sample groups from the steer AC were prepared for the study (n = 8 samples/group). In the first group, chondroitinase ABC enzyme was used to degrade the PGs from the superficial cartilage, while the samples in the second group served as the controls. Samples were examined with FT-IRIS and analyzed using previously reported direct absorption spectrum techniques and multivariate methods and, in comparison, by curve fitting. Safranin O-stained sections were measured with digital densitometry to obtain a reference for depthwise PG distribution. Results: Carbohydrate region-based absorption spectrum methods showed a statistically weaker correlation with the PG reference distributions than the results of the curve fitting (subpeak located approximately at 1,060 cm,1). Furthermore, the shape of the depthwise profiles obtained using the curve fitting was more similar to the reference profiles than with the direct absorption spectrum analysis. Conclusions: Results suggest that the current FT-IRIS methods for PG analysis lack the specificity for quantitative measurement of PGs in AC. The curve fitting approach demonstrated that it is possible to improve the specificity of the PG analysis. However, the findings of the present study suggest that further development of the FT-IRIS analysis techniques is still needed. Microsc. Res. Tech. 2010. © 2009 Wiley-Liss, Inc. [source] Development and neuronal dependence of cutaneous sensory nerve formations: Lessons from neurotrophins,MICROSCOPY RESEARCH AND TECHNIQUE, Issue 5 2010Juan A. Montaño Abstract Null mutations of genes from the NGF family of NTs and their receptors (NTRs) lead to loss/reduction of specific neurons in sensory ganglia; conversely, cutaneous overexpression of NTs results in skin hyperinnervation and increase or no changes in the number of sensory neurons innervating the skin. These neuronal changes are paralleled with loss of specific types of sensory nerve formations in the skin. Therefore, mice carrying mutations in NT or NTR genes represent an ideal model to identify the neuronal dependence of each type of cutaneous sensory nerve ending from a concrete subtype of sensory neuron, since the development, maintenance, and structural integrity of sensory nerve formations depend upon sensory neurons. Results obtained from these mouse strains suggest that TrkA positive neurons are connected to intraepithelial nerve fibers and other sensory nerve formations depending from C and A, nerve fibers; the neurons expressing TrkB and responding to BDNF and NT-4 innervate Meissner corpuscles, a subpopulation of Merkell cells, some mechanoreceptors of the piloneural complex, and the Ruffini's corpuscles; finally, a subpopulation of neurons, which are responsive to NT-3, support postnatal survival of some intraepithelial nerve fibers and Merkel cells in addition to the muscle mechanoreceptors. On the other hand, changes in NTs and NTRs affect the structure of non-nervous structures of the skin and are at the basis of several cutaneous pathologies. This review is an update about the role of NTs and NTRs in the maintenance of normal cutaneous innervation and maintenance of skin integrity. Microsc. Res. Tech. 2010. © 2009 Wiley-Liss, Inc. [source] Surfing the wave, cycle, life history, and genes/proteins expressed by testicular germ cells.MICROSCOPY RESEARCH AND TECHNIQUE, Issue 4 2010Part 2: Changes in spermatid organelles associated with development of spermatozoa Abstract Spermiogenesis is a long process whereby haploid spermatids derived from the meiotic divisions of spermatocytes undergo metamorphosis into spermatozoa. It is subdivided into distinct steps with 19 being identified in rats, 16 in mouse and 8 in humans. Spermiogenesis extends over 22.7 days in rats and 21.6 days in humans. In this part, we review several key events that take place during the development of spermatids from a structural and functional point of view. During early spermiogenesis, the Golgi apparatus forms the acrosome, a lysosome-like membrane bound organelle involved in fertilization. The endoplasmic reticulum undergoes several topographical and structural modifications including the formation of the radial body and annulate lamellae. The chromatoid body is fully developed and undergoes structural and functional modifications at this time. It is suspected to be involved in RNA storing and processing. The shape of the spermatid head undergoes extensive structural changes that are species-specific, and the nuclear chromatin becomes compacted to accommodate the stream-lined appearance of the sperm head. Microtubules become organized to form a curtain or manchette that associates with spermatids at specific steps of their development. It is involved in maintenance of the sperm head shape and trafficking of proteins in the spermatid cytoplasm. During spermiogenesis, many genes/proteins have been implicated in the diverse dynamic events occurring at this time of development of germ cells and the absence of some of these have been shown to result in subfertility or infertility. Microsc. Res. Tech., 2010. © 2009 Wiley-Liss, Inc. [source] Surfing the wave, cycle, life history, and genes/proteins expressed by testicular germ cells.MICROSCOPY RESEARCH AND TECHNIQUE, Issue 4 2010Part 3: Developmental changes in spermatid flagellum, cytoplasmic droplet, egg plasma membrane, interaction of sperm with the zona pellucida Abstract Spermiogenesis constitutes the steps involved in the metamorphosis of spermatids into spermatozoa. It involves modification of several organelles in addition to the formation of several structures including the flagellum and cytoplasmic droplet. The flagellum is composed of a neck region and middle, principal, and end pieces. The axoneme composed of nine outer microtubular doublets circularly arranged to form a cylinder around a central pair of microtubules is present throughout the flagellum. The middle and principal pieces each contain specific components such as the mitochondrial sheath and fibrous sheath, respectively, while outer dense fibers are common to both. A plethora of proteins are constituents of each of these structures, with each playing key roles in functions related to the fertility of spermatozoa. At the end of spermiogenesis, a portion of spermatid cytoplasm remains associated with the released spermatozoa, referred to as the cytoplasmic droplet. The latter has as its main feature Golgi saccules, which appear to modify the plasma membrane of spermatozoa as they move down the epididymal duct and hence may be partly involved in male gamete maturation. The end product of spermatogenesis is highly streamlined and motile spermatozoa having a condensed nucleus equipped with an acrosome. Spermatozoa move through the female reproductive tract and eventually penetrate the zona pellucida and bind to the egg plasma membrane. Many proteins have been implicated in the process of fertilization as well as a plethora of proteins involved in the development of spermatids and sperm, and these are high lighted in this review. Microsc. Res. Tech., 2010. © 2009 Wiley-Liss, Inc. [source] Chemical and morphological features of dental composite resin: Influence of light curing units and immersion mediaMICROSCOPY RESEARCH AND TECHNIQUE, Issue 3 2010Patrícia Aleixo Dos Santos Abstract Aims: The study evaluated the influence of light curing units and immersion media on superficial morphology and chemistry of the nanofilled composite resin Supreme XT (3M) through the EDX analysis and SEM evaluation. Light curing units with different power densities and mode of application used were XL 3000 (480 mW/cm2), Jet Lite 4000 Plus (1230mW/cm2), and Ultralume Led 5 (790 mW/cm2) and immersion media were artificial saliva, Coke®, tea and coffee, totaling 12 experimental groups. Specimens (10 mm × 2 mm) were immersed in each respective solution for 5 min, three times a day, during 60 days and stored in artificial saliva at 37°C ± 1°C between immersion periods. Topography and chemical analysis was qualitative. Findings: Groups immersed in artificial saliva, showed homogeneous degradation of matrix and deposition of calcium at the material surface. Regarding coffee, there was a reasonable chemical degradation with loss of load particles and deposition of ions. For tea, superficial degradation occurred in specific areas with deposition of calcium, carbon, potassium and phosphorus. For Coke®, excessive matrix degradation and loss of load particles with deposition of calcium, sodium, and potassium. Conclusion: Light curing units did not influence the superficial morphology of composite resin tested, but the immersion beverages did. Coke® affected material's surface more than did the other tested drinks. Microsc. Res. Tech., 2010. © 2009 Wiley-Liss, Inc. [source] Methodological assessment of acid-etching for visualizing the osteocyte lacunar-canalicular networks using scanning electron microscopyMICROSCOPY RESEARCH AND TECHNIQUE, Issue 3 2010Daniel J. Kubek Abstract Osteocytes are the most abundant of the bone cells. Each osteocyte is contained within its own lacuna and connected to adjacent osteocytes via fillipodial processes, which form an intricate network of canaliculi within the matrix. Studying this intricate network of cells and their processes is difficult, because it exists embedded within a densely mineralized matrix. Scanning electron microscopy (SEM) has been shown to be a useful tool for visualizing this cellular network, yet the techniques involved for preparing specimens has not been systematically explored. The goal of this study was to investigate how variations in acid-etching, both etching media and etching duration, affect SEM-based visualization of the osteocyte lacunar,canalicular network. Bone samples were embedded in plastic and then acid etched in either 9% (10, 20, 40, and 60 s durations) or 37% (5, 10, and 15 s) phosphoric acid. Specimens were imaged using SEM, and qualitative evaluation of the lacunar,canalicular network was undertaken. Our findings show acid etchingwith a 9% phosphoric acid solution for 20 s provided the most favorable visualization of the osteocyte lacunar,canalicular network. Microsc. Res. Tech., 2010. © 2009 Wiley-Liss, Inc. [source] Parallel deconvolution of large 3D images obtained by confocal laser scanning microscopyMICROSCOPY RESEARCH AND TECHNIQUE, Issue 3 2010Piotr Pawliczek Abstract Various deconvolution algorithms are often used for restoration of digital images. Image deconvolution is especially needed for the correction of three-dimensional images obtained by confocal laser scanning microscopy. Such images suffer from distortions, particularly in the Z dimension. As a result, reliable automatic segmentation of these images may be difficult or even impossible. Effective deconvolution algorithms are memory-intensive and time-consuming. In this work, we propose a parallel version of the well-known Richardson,Lucy deconvolution algorithm developed for a system with distributed memory and implemented with the use of Message Passing Interface (MPI). It enables significantly more rapid deconvolution of two-dimensional and three-dimensional images by efficiently splitting the computation across multiple computers. The implementation of this algorithm can be used on professional clusters provided by computing centers as well as on simple networks of ordinary PC machines. Microsc. Res. Tech., 2010. © 2009 Wiley-Liss, Inc. [source] Role of MMP9 on invadopodia formation in cells from adenoid cystic carcinoma.MICROSCOPY RESEARCH AND TECHNIQUE, Issue 2 2010Study by laser scanning confocal microscopy Abstract Migration, invasion and protease activity are essential for tumor progression and metastasis. Metastatic cells rely on invadopodia to degrade and invade extracellular matrix (ECM). Invadopodia are membrane protrusions with enzymes required for ECM degradation. These protrusions contain cortactin and membrane type 1 matrix metalloproteinase (MT1-MMP) superimposed to areas of digested matrix. Here we characterized invadopodia in a cell line (CAC2) derived from human adenoid cystic carcinoma. We carried out fluorescent-substrate degradation assay to assess in situ protease activity of CAC2 cells. Digestion spots in fluorescent substrate appear as black areas in green background. Cells were cultured on Matrigel-gelatin-FITC and fixed after 1 h and 3 h. CAC2 cells were double labeled to actin and cortactin. Cells were also double stained to actin and MT1-MMP. Samples were studied by laser scanning confocal microscopy. In all time points CAC2 cells showed actin, cortactin, and MT1-MMP colocalized with digestion spots in fluorescent substrate. We searched for other proteases involved in invadopodia activity. We have previously demonstrated that MMP9 influences adenoid cystic carcinoma behavior. This prompted us to investigate role played by MMP9 on invadopodia formation. CAC2 cells had MMP9 silenced by siRNA. After 1 h in fluorescent substrate, cells with silenced MMP9 showed clear decrease in matrix digestion compared with controls. No differences were found in cells with silenced MMP9 grown for 3 h on fluorescent substrate. Our results showed that CAC2 cells exhibit functional invadopodia containing cortactin and MT1-MMP. Furthermore, MMP9 would be required in the initial steps of invadopodia formation. Microsc. Res. Tech., 2010. © 2009 Wiley-Liss, Inc. [source] An automatic integrated approach for stained neuron detection in studying neuron migrationMICROSCOPY RESEARCH AND TECHNIQUE, Issue 2 2010Yue Huang Abstract Neurons that come to populate the six-layered cerebral cortex are born deep within the developing brain in the surface of the embryonic cerebral ventricles. It is very important to detect these neurons for studying histogenesis of the brain and abnormal migration that had been linked to cognitive deficits, mental retardation, and motor disorders. The visualization of labeled cells in brain sections was performed by immunocytochemical examination and its image data were documented to microscopic pictures. Based on the fact, automatic accurate neurons labeling is prerequisite instead of time-consuming manual labeling. In this article, a fully automated image processing approach is proposed to detect all the stained neurons in microscopic images. First of all, dark stained neurons are achieved by thresholding in blue channel of image. And then a modified fuzzy c-means clustering method, called alternative fuzzy c-means is applied to achieve higher classification accuracy in extracting constraint factor. Finally, watershed based on gradient vector flow is employed to the constraint factor image to segment all the neurons, including clustered neurons. The results demonstrate that the proposed method can be a useful tool in neuron image analysis. Microsc. Res. Tech. 2010. © 2009 Wiley-Liss, Inc. [source] Color-based tumor tissue segmentation for the automated estimation of oral cancer parametersMICROSCOPY RESEARCH AND TECHNIQUE, Issue 1 2010Yung-nien Sun Abstract This article presents an automatic color-based feature extraction system for parameter estimation of oral cancer from optical microscopic images. The system first reduces image-to-image variations by means of color normalization. We then construct a database which consists of typical cancer images. The color parameters extracted from this database are then used in automated online sampling from oral cancer images. Principal component analysis is subsequently used to divide the color features into four tissue types. Each pixel in the cancer image is then classified into the corresponding tissue types based on the Mahalanobis distance. The aforementioned procedures are all fully automated; in particular, the automated sampling step greatly reduces the need for intensive labor in manual sampling and training. Experiments reveal high levels of consistency among the results achieved using the manual, semiautomatic, and fully automatic methods. Parameter comparisons between the four cancer stages are conducted, and only the mean parameters between early and late cancer stages are statistically different. In summary, the proposed system provides a useful and convenient tool for automatic segmentation and evaluation for stained biopsy samples of oral cancer. This tool can also be modified and applied to other tissue images with similar staining conditions. Microsc. Res. Tech. 2009. © 2009 Wiley-Liss, Inc. [source] Study of intussusceptive angiogenesis in inflammatory regional lymph nodes by scanning electron microscopyMICROSCOPY RESEARCH AND TECHNIQUE, Issue 1 2010Tíssiana Rachel Rossi-Schneider Abstract The aim of the present study was to verify the occurrence of intussusceptive angiogenesis in blood vessels from submandibular lymph nodes responsible for lymphatic drainage of the tongue. A surgical wound inflicted on the ventral tongue of male Wistar rats and submandibular regional lymph nodes were evaluated at different postoperative periods. Scanning electron microscopy (SEM) was used to observe 123 lymph nodes at times 2, 3, 7, 10, 14, and 21 postoperative days. During the analysis of the vascular models with SEM, intussusceptive angiogenesis was observed in all groups evaluated. This was more extensive on the second and third postoperative days (83.33% and 80%, respectively), representing in these groups the expansion of the vascular chain of lymph nodes. At 21 postoperative days, intussusceptive angiogenesis (42.85%) was suggestive of vascular remodeling. Microsc. Res. Tech. 2009. © 2009 Wiley-Liss, Inc. [source] Single photon fluorescent microlithography for live-cell imagingMICROSCOPY RESEARCH AND TECHNIQUE, Issue 1 2010Darío Kunik Abstract Using fluorescent dyes to trigger the polymerization of a commercial polyurethane resin allows a rapid fabrication of micrometer and submicrometer sized fluorescent structures by one-photon absorption. Here, we show that standard He,Ne lasers emitting at 632.8 nm can be used to start the photopolymerization and that very low laser power is required. This procedure allows the fabrication of fiduciary fluorescent references on standard glass coverslips, mica sheets, or gold-coated coverslips for laser scanning or standard fluorescent microscopy. The biocompatibility of the polymerized resin with cells in culture was tested by growing Xenopus melanophores and a standard laser scanning microscope was used to demonstrate that it is possible to use equipment readily available in several laboratories. We show that fluorescent structure with less than 10 nm in height may be used as references in fluorescence microscopy allowing a smooth environment for cell growth. Different dyes were tested and the conditions for one-photon polymerization were outlined. Microsc. Res. Tech. 2009. © 2009 Wiley-Liss, Inc. [source] Imaging of cochlear tissue with a grating interferometer and hard X-raysMICROSCOPY RESEARCH AND TECHNIQUE, Issue 12 2009Claus-Peter Richter Abstract This article addresses an important current development in medical and biological imaging: the possibility of imaging soft tissue at resolutions in the micron range using hard X-rays. Challenging environments, including the cochlea, require the imaging of soft tissue structure surrounded by bone. We demonstrate that cochlear soft tissue structures can be imaged with hard X-ray phase contrast. Furthermore, we show that only a thin slice of the tissue is required to introduce a large phase shift. It is likely that the phase contrast image of the soft tissue structures is sufficient to image the structures even if surrounded by bone. For the present set of experiments, structures with low-absorption contrast have been visualized using in-line phase contrast imaging and a grating interferometer. The experiments have been performed at the Advanced Photon Source at Argonne National Laboratories, a third generation source of synchrotron radiation. The source provides highly coherent X-ray radiation with high-photon flux (>1012 photons/s) at high-photon energies (5,70 keV). Radiographic and light microscopy images of the gerbil cochlear slice samples were compared. It has been determined that a 20-,m thick tissue slice induces a phase shift between 1/3, and 2/3,. Microsc. Res. Tech., 2009. © 2009 Wiley-Liss, Inc. [source] Gene expression analysis of immunostained endothelial cells isolated from formaldehyde-fixated paraffin embedded tumors using laser capture microdissection,A technical reportMICROSCOPY RESEARCH AND TECHNIQUE, Issue 12 2009Tomoatsu Kaneko Abstract Laser capture microdissection (LCM) allows microscopic procurement of specific cell types from tissue sections that can then be used for gene expression analysis. In conventional LCM, frozen tissues stained with hematoxylin are normally used to the molecular analysis. Recent studies suggested that it is possible to carry out gene expression analysis of formaldehyde-fixated paraffin embedded (FFPE) tissues that were stained with hematoxylin. However, it is still unclear if quantitative gene expression analyses can be performed from LCM cells from FFPE tissues that were subjected to immunostaining to enhance identification of target cells. In this proof-of-principle study, we analyzed by reverse transcription-PCR (RT-PCR) and real time PCR the expression of genes in factor VIII immunostained human endothelial cells that were dissected from FFPE tissues by LCM. We observed that immunostaining should be performed at 4°C to preserve the mRNA from the cells. The expression of Bcl-2 in the endothelial cells was evaluated by RT-PCR and by real time PCR. Glyceraldehyde-3-phosphate dehydrogenase and 18S were used as house keeping genes for RT-PCR and real time PCR, respectively. This report unveils a method for quantitative gene expression analysis in cells that were identified by immunostaining and retrieved by LCM from FFPE tissues. This method is ideally suited for the analysis of relatively rare cell types within a tissue, and should improve on our ability to perform differential diagnosis of pathologies as compared to conventional LCM. Microsc. Res. Tech., 2009. © 2009 Wiley-Liss, Inc. [source] Ontogeny of the androgen receptor expression in the fetal and postnatal testis: Its relevance on Sertoli cell maturation and the onset of adult spermatogenesisMICROSCOPY RESEARCH AND TECHNIQUE, Issue 11 2009Rodolfo A. Rey Abstract From fetal life to adulthood, the testis evolves through maturational phases showing specific morphologic and functional features in its different compartments. The seminiferous cords contain Sertoli and germ cells, surrounded by peritubular cells, and the interstitial tissue contains Leydig cells and connective tissue. Sertoli cells secrete anti-Müllerian hormone (AMH), whereas Leydig cells secrete androgens. In the fetal and early postnatal testis, Leydig cells actively secrete androgens. Sertoli cells are morphologically and functionally immature,e.g., they secrete high levels of AMH,and germ cells proliferate by mitosis but do not enter meiosis. During infancy and childhood, Leydig cells regress and testosterone secretion declines dramatically. Sertoli cells remain immature and spermatogenesis is arrested at the premeiotic stage. At puberty, Leydig cells differentiate again, and testosterone concentration increases and provokes Sertoli cell maturation,e.g., down-regulation of AMH expression,and germ cells undergo meiosis, the hallmark of adult spermatogenesis driving to sperm production. An intriguing feature of testicular development is that, although testosterone production is as active in the fetal and early postnatal periods as in puberty, Sertoli cells and spermatogenesis remain immature until pubertal onset. Here, we review the ontogeny of the androgen receptor expression in the testis and its impact on Sertoli cell maturation and the onset of pubertal spermatogenesis. We show that the absence of androgen receptor expression in Sertoli cells underlies a physiological stage of androgen insensitivity within the male gonad in the fetal and early postnatal periods. Microsc. Res. Tech., 2009. © 2009 Wiley-Liss, Inc. [source] Is there a role for thyroid hormone on spermatogenesis?MICROSCOPY RESEARCH AND TECHNIQUE, Issue 11 2009Marcia Santos Wagner Abstract Appropriate level of thyroid hormone is essential for normal development and metabolism in most vertebrate tissues and altered thyroid status impacts adversely on them. For many years the testis was regarded as a thyroid hormone unresponsive organ, but consistent evidence accumulated in the past two decades has definitively changed this classical view. Currently, the concept that thyroid hormone plays a critical role in testis development, in rats and other vertebrate species, is clearly established. Although the effects of thyroid hormone on Sertoli and Leydig cells in the immature testis are well described, its role on the adult organ remains controversial. In this review, we summarize and discuss the recent development on the thyroid hormone effects in immature and adult testes. Particularly, we have attempted to address the role of thyroid hormone in the regulation of spermatogenesis, emphasizing recent data that suggest its involvement in germ cells differentiation and survival. Microsc. Res. Tech. 2009. © 2009 Wiley-Liss, Inc. [source] Microscopic studies of the influence of main exposure time on parameters of flexographic printing plate produced by digital thermal methodMICROSCOPY RESEARCH AND TECHNIQUE, Issue 10 2009Liliya Harri Abstract The digital thermal technology of producing flexographic printing plates from photopolymer plates is one of the newest technologies. This technology allows to develop flexographic plates without the use of any solvent. The process of producing flexographic printing plates by the digital thermal method consists of several main stages: back exposure, laser exposure, main exposure, thermal development, post exposure, and light finishing. The studies carried out with the use of optical stereoscopic microscopy allowed to determine the effect of time of main exposure to ultraviolet radiation on the dot area, diameter, and edge factor of halftone dots reproduced on flexographic printing plate produced by the digital thermal method, as well as on the quality of reproducing the surface and on the profiles of free-standing printing microelements. The results of the microscopic studies performed have allowed to define the criteria of establishing optimum time of main exposure of photopolymer plates used in the digital thermal technology of producing flexographic printing plates. A precise definition of the criteria for determining the optimum time of main exposure will enable to reduce the time-consuming control tests and to eliminate errors in both the process of manufacturing flexographic printing plates and in the printing process carried out with the use of such plates. Microsc. Res. Tech., 2009. © 2009 Wiley-Liss, Inc. [source] Micro-fabrication and monitoring of three-dimensional microstructures based on laser-induced thermoplastic formationMICROSCOPY RESEARCH AND TECHNIQUE, Issue 10 2009Leyan Wang Abstract This article reports a novel laser-induced micro-fabrication method and its monitoring system for three-dimensional (3D) microstructures. The mechanism of the method is that a small zone of thermoplastic material melted by laser heating grows in liquid surrounding environment, solidifying into a convex microstructure, such as micro-dot or micro-pillar. A laser diode (808 nm) with maximum power output of 130 mW is used as power source, and a kind of paraffin mixed with stearic acid and paint serves as the thermoplastic material for 3D microstructure formation experiments. A light microscope system consisting of a charge-coupled device (CCD) and a computer is utilized to realize real-time observation of the micro-fabricating process. The distribution of local temperature rise on material surface created by laser irradiation is simulated. The effects of liquid environment on microstructure formation have been theoretically analyzed and experimentally studied. Experiments are further carried out to investigate the relationship between laser spot and fabricated microstructures. The results indicate that the widths of micro-dots or micro-pillars are mostly determined by the size of focal spot, and their heights increase with the enlargement of laser power density. With this method, a micro-dot array of Chinese characters meaning "China" has been successfully fabricated through computer programming. This method has the advantages of implementing direct, mask-less, real-time and inexpensive 3D microstructure fabrication. Therefore, it would be widely applied in the fields of micro/nano-technology for practical fabrication of different kinds of 3D microstructures. Microsc. Res. Tech., 2009. © 2009 Wiley-Liss, Inc. 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