Dissertations / Theses on the topic 'CLSM (confocal laser scanning microscopy)'
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Amin, Anish Kiritkumar. "Chondrocyte death in injured articular cartilage : in vitro evaluation of chondroprotective strategies using confocal laser scanning microscopy." Thesis, University of Edinburgh, 2011. http://hdl.handle.net/1842/5687.
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A reproducible in vitro model of mechanically injured (scalpel cut) articular cartilage was developed in this work utilising bovine and human osteochondral tissue. Using fluorescence-mode confocal laser scanning microscopy (CLSM), the model allowed (1) spatial and temporal quantification of in situ (within the matrix) chondrocyte viability following a full thickness cartilage injury and (2) serial evaluation of three chondroprotective strategies in injured bovine and human articular cartilage: (a) medium osmolarity (b) medium calcium concentration and, (c) subchondral bone attachment to articular cartilage. Medium osmolarity significantly influenced superficial zone chondrocyte death in injured (scalpel cut) bovine and human articular cartilage. Greatest percentage cell death occurred at 0 mOsm (distilled water). Conversely, a raised medium osmolarity (600 mOsm) was chondroprotective. The majority of in situ cell death occurred within 2.5 hours of the experimental injury, with no further increase over 7 days. Exposure of articular cartilage to calcium-free media significantly decreased superficial zone chondrocyte death in injured (scalpel cut) articular cartilage compared with exposure to calcium-rich media (2-20 mM). In calcium-rich media, the extent of percentage cell death increased with increasing medium calcium concentration but remained localised to the superficial zone of injured articular cartilage over 7 days. However, in calcium-free media, there was an increase in percentage cell death within deeper zones of injured articular cartilage over 7 days. Excision of subchondral bone from injured (scalpel cut) articular cartilage resulted in an increase in chondrocyte death at 7 days that occurred in the superficial zone of injured as well as the adjacent uninjured regions of articular cartilage. However, the presence of subchondral bone in the culture medium prevented this increase in chondrocyte death within the superficial zone. Subchondral bone may have interacted with articular cartilage via soluble mediator(s) that influenced chondrocyte survival. In human articular cartilage, healthy subchondral bone also interacted with articular cartilage in explant culture and promoted in situ chondrocyte survival, while sclerotic subchondral bone was detrimental to chondrocyte viability. These findings are of translational relevance to fluid management systems used during open and arthroscopic articular surgery, clinical and experimental research into cartilage injury, repair and degeneration as well as current techniques of tissue engineering.
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Weidhase, Michael, Patrick Beckers, Christoph Bleidorn, and M. Teresa Aguado. "On the role of the proventricle region in reproduction and regeneration in Typosyllis antoni (Annelida: Syllidae)." Universitätsbibliothek Leipzig, 2016. http://nbn-resolving.de/urn:nbn:de:bsz:15-qucosa-216141.
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Background: Syllids are a species rich annelid family possessing remarkable regenerative ability, which is not only the response after traumatic injury, but also a key step during the life cycle of several syllid taxa. In these animals the posterior part of the body becomes an epitoke and is later detached as a distinct unit named stolon. Such a
sexual reproductive mode is named schizogamy or stolonization. The prostomium and the proventricle, a modified foregut structure, have been proposed to have a control function during this process, though the concrete mechanisms behind it have never been elucidated. Results: By using different experimental set-ups, histology and immunohistochemistry combined with subsequent cLSM analyzes, we investigate and document the regeneration and stolonization in specimens of Typosyllis antoni that were amputated at different levels throughout the antero-posterior body axis. The removal of the anterior end including the proventricle implies an incomplete anterior regeneration as well as severe deviations from the usual reproductive pattern, i.e. accelerated stolonization, masculinization and the occurrence of aberrant stolons. The detailed anatomy of aberrant
stolons is described. A histological study of the proventricle revealed no signs of glandular or secretory structures. The ventricle and the caeca are composed of glandular tissue but they are not involved in the reproductive and regenerative processes. Conclusions: As in other investigated syllids, the proventricle region has a significant role during stolonization and reproduction processes in Typosyllis antoni. When the proventricle region is absent, anterior and posterior regeneration are considerably deviated from the general patterns. However, proventricle ultrastructure does not show any glandular component, thereby questioning a direct involvement of this organ itself in the control of reproduction and regeneration. Our findings offer a comprehensive starting point for further studies of regeneration and reproductive control in syllids as well as annelids in general.
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Haridoss, Sujithera. "In vivo assessment of focal adhesion kinase (FAK) activity in breast cancer cells using fluorescence resonance energy transfer (FRET) sensor and confocal laser scanning microscope (CLSM)." Thesis, Högskolan i Skövde, Institutionen för hälsa och lärande, 2015. http://urn.kb.se/resolve?urn=urn:nbn:se:his:diva-15706.
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Focal Adhesion Kinase (FAK) is essential for cell migration and plays an important role in tumor metastasis. However, the complex intermolecular and intramolecular interactions that regulate FAK activity at the focal adhesion remain unresolved. We have engineered a toolbox of Fluorescence Resonance Energy Transfer (FRET) sensors for the assessment of FAK activity in human breast cancer cells (MCF-7). Major activity of cancerous cells is drastically growth of the cell in an uncontrollable manner in such cases our human anatomy system normally consists of cell growth activity. The important protein involved in cell functionality in the human body is FAK, due to FAK activity, cell motility, proliferation, survival has been managed in the human body hence, it is necessary to investigate the performance ofFAK activity on breast cancer becomes important. In our study, the differences in bleed through between zoom = 1 and for zoom >1 for donor and acceptor was evaluated. There were no significant differences in Pearson correlation coefficient and bleed through coefficient for both the zooms. With recent advances influorescent probes, instrumentation and methodologies, FRET is sure to revolutionize scientific research in the near future.
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Helm, Conrad, and María Capa. "Comparative analyses of morphological characters in Sphaerodoridae and allies (Annelida) revealed by an integrative microscopical approach." Universitätsbibliothek Leipzig, 2015. http://nbn-resolving.de/urn:nbn:de:bsz:15-qucosa-159898.
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Sphaerodoridae is a group of benthic marine worms (Annelida) characterized by the presence of spherical tubercles covering their whole surface. They are commonly considered as belonging to Phyllodocida although sistergroup relationships are still far from being understood. Primary homology assessments of their morphological features are lacking, hindering the appraisal of evolutionary relationships between taxa. Therefore, our detailed morphological investigation focuses on different Sphaerodoridae as well as on other members of Phyllodocida using an integrative approach combining scanning electron microscopy (SEM) as well as immunohistochemistry with standard neuronal (anti-5-HT) and muscular (phalloidin-rhodamine) markers and subsequent CLSM analysis of whole mounts and sections. Furthermore, we provide histological (HES) and light microscopical data to shed light on the structures and hypothetical function of sphaerodorid key morphological features. We provide fundamental details into the sphaerodorid morphology supporting a Phyllodocida ancestry of these enigmatic worms. However, the muscular arrangement and the presence of an axial muscular pharynx is similar to conditions observed in other members of the Errantia too. Furthermore, nervous system and muscle staining as well as SEM and histological observations of different types of tubercles indicate a homology of the so called microtubercles, present in the long-bodied sphaerodorids, to the dorsal cirri of other Errantia. The macrotubercles seem to represent a sphaerodorid autapomorphy based on our investigations. Therefore, our results allow comparisons concerning morphological patterns between Sphaerodoridae and other Phyllodocida and constitute a starting point for further comparative investigations to reveal the evolution of the remarkable Sphaerodoridae.
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小林, 正典. "共焦点レーザー走査顕微鏡(Confocal Laser Scanning Microscopy : CLSM)による生体関節軟骨と人工軟組織のトライボロジーに関する研究." 京都大学 (Kyoto University), 2003. http://hdl.handle.net/2433/148895.
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Chen, X. "TAGGING BIOCONTROL STREPTOMYCES TO STUDY LETTUCE COLONIZATION." Doctoral thesis, Università degli Studi di Milano, 2015. http://hdl.handle.net/2434/345187.
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The ability of the biological control agents (BCAs) to colonize plant tissues is an important feature involved in microbe-assisted plant protection. Plant-microbe interaction research increased especially in the last decade thanks to technological revolution. Molecular methods and the development of advanced microscopic techniques allow researchers to explore gene expression and localization of beneficial microorganisms within plants. The green fluorescent protein (GFP) and its modified version, enhanced GFP (EGFP), more adapt for expression in mammalian cells and GC-rich actinomycetes like Streptomyces, have been widely used as markers to study gene expression, as well as plant-microbe interactions. Aside fluorescent protein approaches, fluorescence in situ hybridization (FISH) is another frequently used technique to visualize microbial colonization patterns and community composition by application of specific fluorescent probes. Firstly, we transformed five Streptomyces strains, which showed strong inhibition activity against Sclerotinia sclerotiorum, with the EGFP construct by the conjugation method. The conjugation efficiencies varied between the strains, but were comparable to the reference strain. The fitness of transformed strains was similar to wild-type; the transformants maintained similar sporulation, mycelium growth rate, and the ability to produce important secondary metabolites and lytic enzymes. Secondly, two transformed strains, Streptomyces cyaneus ZEA17I, and Streptomyces sp. SW06W, were used to study lettuce colonization dynamics by seed coating method. Their spatio-temporal dynamics were determined in sterile substrate. The strains were consistently recovered from lettuce rhizosphere and inner root tissues up to six weeks. Finally, the colonization pattern of lettuce by Streptomyces cyaneus ZEA17I was examined by both EGFP and FISH approaches combined with confocal laser scanning microscopy (CLSM). For FISH-CLSM analysis, universal bacteria and Streptomyces genus specific probes were used to label S. cyaneus ZEA17I. The consistent presence of the labeled strain at the lettuce root one week after sowing showed that Streptomyces spores could rapidly germinate and produce filamentous mycelium on lettuce. S. cyaneus ZEA17I was detected also on two-week-old roots, indicating the long-term survival ability of this strain in lettuce rhizosphere. Altogether, the antagonistic activity, rhizosphere and root competence showed by the Streptomyces conferred their potential to act as BCA. Further studies on the complex host-pathogen-antagonist interactions will provide additional knowledge to understand the modes and mechanisms of Streptomyces-mediated plant protection.
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Murtin, Chloé Isabelle. "Traitement d’images de microscopie confocale 3D haute résolution du cerveau de la mouche Drosophile." Thesis, Lyon, 2016. http://www.theses.fr/2016LYSEI081/document.
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La profondeur possible d’imagerie en laser-scanning microscopie est limitée non seulement par la distance de travail des lentilles de objectifs mais également par la dégradation de l’image causée par une atténuation et une diffraction de la lumière passant à travers l’échantillon. Afin d’étendre cette limite, il est possible, soit de retourner le spécimen pour enregistrer les images depuis chaque côté, or couper progressivement la partie supérieure de l’échantillon au fur et à mesure de l‘acquisition. Les différentes images prises de l’une de ces manières doivent ensuite être combinées pour générer un volume unique. Cependant, des mouvements de l’échantillon durant les procédures d’acquisition engendrent un décalage non seulement sur en translation selon les axes x, y et z mais également en rotation autour de ces même axes, rendant la fusion entres ces multiples images difficile. Nous avons développé une nouvelle approche appelée 2D-SIFT-in-3D-Space utilisant les SIFT (scale Invariant Feature Transform) pour atteindre un recalage robuste en trois dimensions de deux images. Notre méthode recale les images en corrigeant séparément les translations et rotations sur les trois axes grâce à l’extraction et l’association de caractéristiques stables de leurs coupes transversales bidimensionnelles. Pour évaluer la qualité du recalage, nous avons également développé un simulateur d’images de laser-scanning microscopie qui génère une paire d’images 3D virtuelle dans laquelle le niveau de bruit et les angles de rotations entre les angles de rotation sont contrôlés avec des paramètres connus. Pour une concaténation précise et naturelle de deux images, nous avons également développé un module permettant une compensation progressive de la luminosité et du contraste en fonction de la distance à la surface de l’échantillon. Ces outils ont été utilisés avec succès pour l’obtention d’images tridimensionnelles de haute résolution du cerveau de la mouche Drosophila melanogaster, particulièrement des neurones dopaminergiques, octopaminergiques et de leurs synapses. Ces neurones monoamines sont particulièrement important pour le fonctionnement du cerveau et une étude de leur réseau et connectivité est nécessaire pour comprendre leurs interactions. Si une évolution de leur connectivité au cours du temps n’a pas pu être démontrée via l’analyse de la répartition des sites synaptiques, l’étude suggère cependant que l’inactivation de l’un de ces types de neurones entraine des changements drastiques dans le réseau neuronalAlthough laser scanning microscopy is a powerful tool for obtaining thin optical sections, the possible depth of imaging is limited by the working distance of the microscope objective but also by the image degradation caused by the attenuation of both excitation laser beam and the light emitted from the fluorescence-labeled objects. Several workaround techniques have been employed to overcome this problem, such as recording the images from both sides of the sample, or by progressively cutting off the sample surface. The different views must then be combined in a unique volume. However, a straightforward concatenation is often not possible, because the small rotations that occur during the acquisition procedure, not only in translation along x, y and z axes but also in rotation around those axis, making the fusion uneasy. To address this problem we implemented a new algorithm called 2D-SIFT-in-3D-Space using SIFT (scale Invariant Feature Transform) to achieve a robust registration of big image stacks. Our method register the images fixing separately rotations and translations around the three axes using the extraction and matching of stable features in 2D cross-sections. In order to evaluate the registration quality, we created a simulator that generates artificial images that mimic laser scanning image stacks to make a mock pair of image stacks one of which is made from the same stack with the other but is rotated arbitrarily with known angles and filtered with a known noise. For a precise and natural-looking concatenation of the two images, we also developed a module progressively correcting the sample brightness and contrast depending on the sample surface. Those tools we successfully used to generate tridimensional high resolution images of the fly Drosophila melanogaster brain, in particular, its octopaminergic and dopaminergic neurons and their synapses. Those monoamine neurons appear to be determinant in the correct operating of the central nervous system and a precise and systematic analysis of their evolution and interaction is necessary to understand its mechanisms. If an evolution over time could not be highlighted through the pre-synaptic sites analysis, our study suggests however that the inactivation of one of these neuron types triggers drastic changes in the neural network
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Uys, Charlene Ethel. "Preparation and characterisation of pheroid vesicles / Charlene Ethel Uys." Thesis, North-West University, 2006. http://hdl.handle.net/10394/1669.
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Mu, Wangzhong. "Microstructure and Inclusion Characteristics in Steels with Ti-oxide and TiN Additions." Doctoral thesis, KTH, Tillämpad processmetallurgi, 2015. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-162284.
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Non-metallic inclusions in steels are generally considered to be detrimental for mechanical properties. However, it has been recognized that certain inclusions, such as Ti-oxide and TiN, can serve as potent nucleation sites for the formation of intragranular ferrite (IGF) in low-alloy steels. The formation of IGF could improve the toughness of the coarse grained heat affected zone (CGHAZ) of weld metals. Thus, the present thesis mainly focuses on the effect of size of nucleation sites on the IGF formation. Quantitative studies on the composition, size distribution and nucleation probability for each size of the inclusions as well as the area fraction, starting temperature and morphology of an IGF have been carried out. In the present work, the Ti-oxide and TiN powders were mixed with metallic powders. The mixed powders were heated up to the liquid state and cooled with a slow cooling rate of 3.6 ºC/min. These as-cast steels with Ti-oxide and TiN additions were used to simulate the IGF formation in the CGHAZ of weld metals. Specifically, the inclusion and microstructure characteristics in as-cast steels have been investigated. The results show that the nucleant inclusion was identified as a TiOx+MnS phase in steels with Ti2O3 additions and as a TiN+Mn-Al-Si-Ti-O+MnS phase in steels with TiN additions. In addition, the TiOx and TiN phases are detected to be the effective nucleation sites for IGF formation. It is clearly shown that an increased inclusion size leads to an increased probability of IGF nucleation. This probability of IGF nucleation for each inclusion size of the TiOx+MnS inclusions is clearly higher than that of the complex TiN+Mn-Al-Si-Ti-O+MnS inclusions. In addition, the area fraction of IGF in the steels with Ti2O3 additions is larger than that of the steels with TiN additions. This result agrees with the predicted tendency of the probability of IGF nucleation for each inclusion size in the steels with Ti2O3 and TiN additions. In order to predict the effective inclusion size for IGF formation, the critical diameters of the TiO, TiN and VN inclusions, which acted as the nucleation sites of IGF formation, were also calculated based on the classical nucleation theory. The critical diameters of TiO, TiN and VN inclusions for IGF formation were found to be 0.192, 0.355 and 0.810 μm in the present steels. The calculation results were found to be in agreement with the experiment data of an effective inclusion size. Moreover, the effects of the S, Mn and C contents on the critical diameters of inclusions were also calculated. It was found that the critical diameter of the TiO, TiN and VN inclusions increases with an increased content of Mn or C. However, the S content doesn’t have a direct effect on the critical diameter of the inclusions for IGF formation. The probability of IGF nucleation for each inclusion size slightly decreases in the steel containing a higher S content. This fact is due to that an increased amount of MnS precipitation covers the nucleant inclusion surface. In the as-cast experiment, it was noted that an IGF can be formed in steels with Ti2O3 and TiN additions with a cooling rate of 3.6 ºC/min. In order to control the microstructure characteristics, such as the area fraction and the morphology of an IGF, and to investigate the starting temperature of IGF and grain boundary ferrite (GBF) formation, the dynamic transformation behavior of IGF and GBF was studied in-situ by a high temperature confocal laser scanning microscope (CLSM). Furthermore, the chemical compositions of the inclusions and the morphology of IGF after the in-situ observations were investigated by using scanning electron microscopy (SEM), electron backscatter diffraction (EBSD) and electron probe microanalysis (EPMA) which equipped wavelength dispersive spectrometer (WDS). The results show that the area fraction of IGF is larger in the steels with Ti2O3 additions compared to the steels with TiN additions, after the same thermal cycle has been imposed. This is due to that the TiOx phase provides more potent nucleation sites for IGF than the TiN phase does. Also, the area fraction of IGF in the steels is highest after at an intermediate cooling rate of 70 ºC/min, since the competing phase transformations are avoided. This fact has been detected by using a hybrid methodology in combination with CLSM and differential scanning calorimetry (DSC). In addition, it is noted that the morphology of an IGF is refined with an increased cooling rate.QC 20150325
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Kerschnitzki, Michael. "Bone material characteristics influenced by osteocytes." Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2012. http://dx.doi.org/10.18452/16479.
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In dieser Doktorarbeit wird die Hypothese geprüft, ob Osteozyten einen direkten Einfluss auf die Knocheneigenschaften in ihrer unmittelbaren Umgebung haben. Der zentrale Experimentieransatz ist dabei die Korrelation der Organisation des Osteozytennetzwerks mit den Mineraleigenschaften des Knochens auf der Submikrometerebene. Es wird gezeigt, dass bereits die anfängliche Ausrichtung der Osteoblasten entscheidend für die Synthese von hoch ausgerichtetem Knochenmaterial ist. Die dabei entstehenden Osteozytennetzwerke sind so organisiert, dass die Osteozyten und ihre Zellfortsätze jeweils einen möglichst kleinen Abstand zum Knochenmineral haben. Deshalb wird vermutet, dass genau diese Netzwerkorganisation mitentscheidend ist, wie gut die Zellen das Mineral in ih-rer Umgebung beeinflussen können. Messungen der Knochenmineraleigenschaften auf Submikrometerebene mit Röntgenkleinwinkelstreuung bestätigen diese Vermutung. Dabei wird deutlich, dass Knochenmaterial in der Nähe der Osteozyten durch andere Mineraleigenschaften geprägt ist. Um zu klären, wie Osteozyten Mineral in ihrer direkten Umgebung verändern können, werden Mechanismen der passiven Mineralherauslösung aus der mineralisierten Oberfläche des Osteozytennetzwerks untersucht. Es wird gezeigt, dass kalziumarme ionische Lösungen unter physiologischen Bedingungen große Mengen von Kalzium-Ionen aus dem Knochen lösen und diese dann durch die Osteozytennetzwerkstrukturen diffundieren können. Zum Abschluss wurde medullärer Knochen von Hühnern als ein Modellsystem für rasanten Knochenumbau untersucht. Dieser spezielle Knochentyp dient den Hennen als labiles Kalziumreservoir und ermöglicht dadurch die tägliche Eierschalenproduktion. Experimente am medullären Knochen-material zeigen insbesondere die Bedeutung von weniger stabilen Mineralstrukturen die benötigt werden um den Knochen an den schnellen, sich wiederholenden Knochenauf- sowie Abbau optimal anzupassen.This thesis aims to test the hypothesis whether osteocytes have a direct influence on bone material properties in their vicinity. In this regard, the concomitant ana-lysis of osteocyte network organization and bone ultrastructural properties on the submicron level is the central approach to answer this question. In this work, it is shown that already initial cell-cell alignment during the process of bone formation is crucial for the synthesis of highly organized bone. Furthermore it is proposed that the occurrence of highly ordered osteocyte networks visualized with confocal laser scanning microscopy (CLSM) has a strong impact on the ability of osteocytes to directly influence bone material properties. These highly organized networks are another consequence of initial cell-cell alignment and are found to be arranged such as to feature short mineral cell distances. Examination of sub-micron mineral properties with scanning small angle x-ray scattering (sSAXS) shows that bone material in the direct vicinity of osteocytes and their cell proc-esses shows different mineral properties compared to bone further away in the depth of the tissue. Moreover, mechanisms of passive mineral extraction from the mineralized surface of the osteocyte network, due to the treatment with calcium poor ionic solutions, are investigated. It is shown that this chemical process occurring under physiological conditions leads not only to the dissolution of considerable amounts of calcium, but also to efficient diffusion of these ions through the osteocyte network structures. Finally, medullary bone which is intended as a labile calcium source for daily egg shell formation in hens is used as a model system for rapid bone turnover rates. This bone type in particular indicates the importance of uniquely adapted, less stable mineral structures to fit the requirements for rapid bone resorption as well as reformation.
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Pankajakshan, Praveen. "Blind deconvolution for confocal laser scanning microscopy." Nice, 2009. http://www.theses.fr/2009NICE4057.
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La microscopie confocale à balayage laser est une technique puissante pour étudier les spécimens biologiques en trois dimensions (3D) par sectionnement optique. Bien qu’ubiquitaire, il persiste des incertitudes dans le procédé d’observations. Comme la réponse du système à l’impulsion, ou fonction de flou (PSF), est dépendante à la fois du spécimen et des conditions d’acquisition, elle devrait être estimée à partir des images observées avec l’objet. Ce problème est mal posé, sous déterminé, et comme le processus de mesure est quasi-aléatoire dans la nature, nous le traitons en utilisant l’interférence bayésienne. L’état de l’art des algorithmes concernant la déconvolution et déconvolution aveugle est exposé dans le cadre d’un travail bayésien. Dans la première partie, nous constatons que la diffraction limitée de l’objectif et le bruit intrinsèque, sont les distorsions primordiales qui affectent les images d’un spécimen fin. Une approche de minimalisation alternative (AM), restaure les fréquences manquantes au-delà de la limite de diffraction, en utilisant une régularisation de la variation totale sur l’objet, et une contrainte spatiale sur la PSF. En outre, des méthodes sont proposées pour assurer la positivité des intensités estimées, conserver le flux de l’objet, et bien manier le paramètre de la régularisation. Quand il s’agit d’imager des spécimens épais, la phase de la fonction de la pupille, due à l’aberration sphérique (SA) ne peut être ignorée. Dans la seconde partie, il est montré qu’elle dépend de la discordance de l’index de réfraction entre l’objet et le milieu d’immersion de l’objectif et de la profondeur sur la lamelle. Les paramètres d’imagerie et la distribution de l’intensité originelle de l’objet sont récupérés en modifiant les algorithmes AM. Due à l’incohérence de la microscopie à fluorescence, la phase de récupération des intensités observées est possible en contraignant la phase par l’utilisation d’optiques géométriques. Cette méthode pourrait être étendue pour restituer des spécimens affectés par la SA. Comme la PSF varie dans l’espace, un modèle de quasi-convolution est proposé, et la PSF est rendue approximative. Ainsi, en plus de l’objet, il suffit d’estimer un seul libre paramètreConfocal laser scanning microscopy is a powerful technique for studying biological specimens in three dimensions (3D) by optical sectioning. Although ubiquitous, there are uncertainties in the observation process. As the system’s impulse response or point-spread function (PSF) is dependent on both the specimen and imaging conditions, it should be estimated from the observed images along with the object. This problem is ill-posed, under-determined, and as the measurement process is quasi-random in nature, we treat the problem by using Bayesian inference. The state of the art déconvolution and blind déconvolution algorithms are reviewed within a Bayesian framework. In the first part, we recognize that the diffraction-limited nature of the objective lens and the intrinsic noise are the primary distortions that affect this specimen images. An alternative minimization (AM) approach restores the lost frequencies beyond the diffraction limit by using a total variation regularization on the objet, and a spatial constraint on the PSF. Additionally, some methods are proposed to ensure positivity of estimated intensities, conserve the object’s flux, and to handle the regularization parameter. When imaging thick specimens, the phase of the pupil function due to spherical aberration (SA) cannot be ignored; It is shown to be dependent on the refractive index mismatch between the object and the objective immersion medium, and the depth under the cover slip. The imaging parameters and the object’s original intensity distribution is recovered by modifying the AM algorithm. Due to the incoherent nature of fluorescence microscopy, phase retrieval from the observed intensities is possible by constraining the phase using geometrical optics. This method could be extended to restore specimens affected by SA. As the PSF is space varying, a quasi-convolution model is proposed, and the PSF approximated so that, apart from the object, there is only one free parameter to be estimated
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Zator, Maria Malgorzata. "Membrane fouling characterization by confocal scanning laser microscopy." Doctoral thesis, Universitat Rovira i Virgili, 2009. http://hdl.handle.net/10803/8580.
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En sectores tan diversos como la industria alimentaria, la biotecnología y el tratamiento de aguas residuales, la filtración tangencial con membranas se viene utilizando de forma creciente en la separación, purificación y clarificación de distintas corrientes de proceso que contienen gran variedad de compuestos orgánicos. La limitación principal para el empleo industrial de las técnicas de separación por membranas es el ensuciamiento de éstas. El ensuciamiento se atribuye, de forma general, a la reducción en el diámetro de los poros, a su bloqueo y/o a la formación de un depósito en la superficie de la membrana. El avance en el desarrollo de técnicas para la caracterización, el control y la prevención del ensuciamiento de las membranas ha estado limitado por la falta de técnicas adecuadas y no invasivas para la medición del ensuciamiento. El objetivo principal del presente proyecto es desarrollar estrategias apropiadas para aplicar microscopía láser confocal de barrido (CSLM) al estudio del ensuciamiento de membranas de filtración, centrándose en el ensuciamiento causado por macromoléculas biológicas. En la tesis se han llevado a cabo experimentos de microfiltración (MF) de soluciones modelo puras y de mezclas de proteínas, polisacáridos y polifenoles. Las imágenes captadas mediante CSLM de las membranas al final de diferentes experimentos de filtración, han servido para obtener información cualitativa, sobre localización de las distintas moléculas, y cuantitativa, sobre la presencia individual de cada compuesto en el interior y la superficie de la membrana. Se han realizado también intentos de aplicación de visualización en línea mediante CSLM del proceso de microfiltración.In fields such as the food and dairy industries, biotechnology, and the treatment of industrial effluents, pressure-driven membrane processes such as microfiltration are increasingly being used for the separation, purification and clarification of protein-containing solutions. A major limitation to the widespread use of membrane filtration, however, is fouling. Fouling is usually attributed to pore constriction, pore blocking or the deposition of cells and cell debris on the membrane surface and can lead to a reduction in the filtrate flux of more than an order of magnitude. Progress in developing a means for characterizing, controlling and preventing membrane fouling has been impeded by lack of suitable non-invasive fouling-measurement techniques. The main aim of this study is to develop suitable strategies for applying Confocal Scanning Laser Microscopy (CSLM) to characterise membrane fouling caused by biological macromolecules. Microfiltration experiments of single, binary and ternary model solutions of proteins, polysaccharides and polyphenols were carried out and CSLM images of the membranes at the end of the different filtration runs were obtained, in order to obtain quantitative and qualitative information about fouling patterns. Some trials of on-line monitoring of cross-flow microfiltration processes were also carried out.
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Boruah, Bosanta Ranjan. "Programmable diffractive optics for laser scanning confocal microscopy." Thesis, Imperial College London, 2007. http://hdl.handle.net/10044/1/11911.
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Yildiz, Bilge Can. "Imaging Of Metal Surfaces Using Confocal Laser Scanning Microscopy." Master's thesis, METU, 2011. http://etd.lib.metu.edu.tr/upload/12613641/index.pdf.
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Optical imaging techniques have improved much over the last fifty years since the invention of the laser. With a high brightness source many imaging applications which were once inaccessible to researchers have now become a reality. Among these techniques, the most beneficial one is the use of lasers for both wide-field and
confocal imaging systems.
The aim of this study was to design a laser imaging system based on the concept of laser scanning confocal microscopy. Specifically the optical system was based on optical fibers allowing the user to image remote areas such as the inner surface of rifled gun barrels and/or pipes with a high degree of precision (+/- 0.01 mm). In order to build such a system, initially the theoretical foundation for a confocal as well as a wide-field imaging system was analyzed. Using this basis a free-space optical confocal system was built and analyzed. The measurements support the fact that both the objective numerical aperture and pinhole size play an important role in the radial and axial resolution of the system as well as the quality of the images obtained.
To begin construction of a confocal, optical-fiber based imaging system first an all fiber wide-field imaging system was designed and tested at a working wavelength of 1550 nm. Then an all fiber confocal system was designed at a working wavelength of 808 nm. In both cases results showed that while lateral resolution was adequate, axial resolution suffered since it was found that the design of the optical system needs to take into account under-filling of the objective lens, a result common with the use of laser beams whose divergence is not at all like that of a point source.
The work done here will aid technology that will be used in the elimination process of faulty rifling fabrication in defense industry. The reason why the confocal technique is preferred to the conventional wide-field one is the need for better resolution in all directions. Theoretical concepts and mathematical background are discussed as well as the experimental results and the practical advantages of such a system.
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Luedtke, Michael A. Papazoglou Elisabeth S. "Wavelength effects on in vivo confocal scanning laser microscopy/." Philadelphia, Pa. : Drexel University, 2007. http://hdl.handle.net/1860/2518.
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Jiang, Shihong. "Non-scanning fluorescence confocal microscopy using laser speckle illumination." Thesis, University of Nottingham, 2005. http://eprints.nottingham.ac.uk/10139/.
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Confocal scanning microscopy (CSM) is a much used and advantageous form of microscopy. Although CSM is superior to conventional microscopy in many respects, a major disadvantage is the complexity of the scanning process and the sometimes long time to perform the scan. In this thesis a novel non-scanning fluorescence confocal microscopy is investigated. The method uses a random time-varying speckle pattern to illuminate the specimen, recording a large number of independent full-field frames without the need for a scanning system. The recorded frames are then processed in a suitable way to give a confocal image. The goal of this research project is to confirm the effectiveness and practicality of speckle-illumination microscopy and to develop this proposal into a functioning microscope system. The issues to be addressed include modelling of the system performance, setting up experiments, computer control and image processing. This work makes the following contributions to knowledge: * The development of criteria for system performance evaluation * The development of methods for speckle processing, whereby the number of frames required for an image of acceptable quality can be reduced * The implementation of non-scanning fluorescence confocal microscopy based upon separate recording of the speckle patterns and the fluorescence frames, demonstrating the practicality and effectiveness of this method * The realisation of real-time image processing by optically addressed spatial light modulator, showing how this new form of optical arrangement may be used in practice The thesis is organised into three main segments. Chapters 1-2 review related work and introduce the concepts of fluorescence confocal microscopy. Chapters 3-5 discuss system modelling and present results of performance evaluation. Chapters 6-8 present experimental results based upon the separate recording scheme and the spatial light modulation scheme, draw conclusions and offer some speculative suggestions for future research.
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Ghafari, H. "Confocal laser scanning microscopy of nanoparticles applied to immunosorbent assays." Thesis, Nottingham Trent University, 2011. http://irep.ntu.ac.uk/id/eprint/57/.
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The aim of this project was to demonstrate and develop a confocal readout method for fluorescent immunosorbent assays and investigate its potential advantages in comparison to traditional immunoassays. The key point of a confocal immunosorbent assay is the ability to detect the thin layer of immunoassay in the presence of unbound fluorescent reagents without washing the overlayer. Heterogeneous and homogeneous sandwich immunoassays of human IgG model were demonstrated successfully followed by the use of an empirical decomposition method for quantitative separation of the signals of the thin fluorescent assay layer from the overlayer. The detection limits for the homogeneous and heterogeneous formats of the model were 2.2 and 5.5 ng/ml, respectively. The application of confocal microscopy in kinetic analysis of the antigen-antibody reaction of the human IgG model was studied for homogeneous and heterogeneous formats and two fluorescent labels antibodies (FITC and QDs). The association rates of binding of FITC and QD605 conjugated antibodies to human IgG on prepared surfaces were 5.7×104 and 2.6×104 (M-1s-1) respectively. Confocal detection immunosorbent assay enables the detection of more than one assay along the z-axis. By replacing standard substrates with multiple 30 :m layers of substrates, a high density array of immunosorbent assays was created within a stratified medium. Stacks of up to five modified thin mica substrates of model immunoassays were detected by confocal microscopy. When applied to model assays consisting of human and mouse IgGs on different layers, the z-axis multiplexing of immunosorbant assays was demonstrated. The arrays of multiplexed immunosorbent assays were extended to 3D format by using microcontact printing and the assay density was increased twice by detecting the stack of two substrates which each contained two IgGs assays.
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Moss, Martin Christopher. "Investigations of in-vitro dental plaques using confocal laser scanning microscopy." Thesis, University of Liverpool, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.386815.
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Tefft, John. "The Study of Coating and Ink Penetration into Coating Structures Using a Confocal Laser Scanning Microscope." Fogler Library, University of Maine, 2007. http://www.library.umaine.edu/theses/pdf/TefftJ2007.pdf.
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Esposito, Elric. "Nonlinear optical frequency conversion based soures for improved confocal laser scanning microscopy." Thesis, University of Strathclyde, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.510907.
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Yio, Marcus Heo Nong. "Characterising the microstructure of cement-based materials using laser scanning confocal microscopy." Thesis, Imperial College London, 2016. http://hdl.handle.net/10044/1/55294.
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Three-dimensional (3D) pore characterisation of cement-based materials is essential for understanding the influence of topological pore parameters such as connectivity and tortuosity on transport processes. The main objective of this thesis was to develop laser scanning confocal microscopy (LSCM) for 3D imaging and quantification of pore structure of cement-based materials at submicron resolution. To enable this, a novel approach to reconstruct large volumes of cement-based materials at submicron resolution was developed by combining serial sectioning with LSCM. The method uses a series of Z-stacks with overlapping regions for stitching based on phase correlation. With this method, no information is lost and the spatial resolution is maintained with increase in image size. The effects of axial distortion in LSCM images caused by mismatch of refractive indices between immersion medium and different phases within cement-based materials on various pore attributes were examined. Results indicated that parameters including porosity, specific surface area, percolation connectivity, scalar diffusion tortuosity and formation factor are not significantly affected by axial distortion. A generic correction method was proposed based on measuring the aspect ratio of pulverised fuel ash (PFA) particles in hardened blended pastes. The representative elementary volume for 3D pore characterisation of different cementitious systems was also investigated using a statistical approach. For a given number of realisations, an image volume of 1003 μm3 was found to give comparable porosity to that measured by 2D backscattered electron (BSE) microscopy. BSE signal variation across pore-solid boundaries was simulated using a 3D Monte Carlo technique to enhance image analysis of the pore structure. It was found that a single pore of down to 1 nm can be resolved with field emitters under ideal imaging conditions. The Overflow method was also found to be able to accurately segment pores larger than 1 μm with errors of ~1% and randomly inclined pores with an average error of ~5.2%. Effects of supplementary cementitious materials including silica fume (SF), pulverised fly ash (PFA) and ground granulated blastfurnace slag (GGBS) on the 3D pore structure of cement pastes were investigated using LSCM in conjunction with BSE microscopy. Generally, results from both techniques showed that SF enhances the pore structure (i.e. decreased porosity and percolation connectivity, and increased diffusion tortuosity) from early ages whereas PFA and GGBS show improvements at later ages. The percolation connectivity decreases while diffusion tortuosity increases drastically, as porosity reduces to ~15%. Measured 3D pore characteristics were used as inputs to simple analytical equations for predicting transport properties. Predicted results agreed reasonably well with measured values, mostly within a factor of five. An exploratory study into the application of fluorescence LSCM for real-time imaging of early cement hydration is also presented. Qualitative and quantitative analyses of microstructural developments in different hydrating cementitious systems were made. The advantages and limitations of LSCM for such application are also discussed.
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Neils, Christopher Martin. "Laser scanning microscopy of broad freezing interfaces with applications to biological cells /." Full text (PDF) from UMI/Dissertation Abstracts International, 2000. http://wwwlib.umi.com/cr/utexas/fullcit?p3004349.
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Emilson, Axel. "Analysis of human epidermal Langerhans' cells and allergens with confocal laser scanning microscopy /." Stockholm, 1997. http://diss.kib.ki.se/1997/91-628-2734-0.
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Hennessy, Richard Joseph. "STUDYING MILK COAGULATION KINETICS WITH LASER SCANNING CONFOCAL MICROSCOPY, IMAGE PROCESSING, AND COMPUTATIONAL MODELING." DigitalCommons@CalPoly, 2011. https://digitalcommons.calpoly.edu/theses/587.
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The kinetics of milk coagulation are complex and still not well understood. A deeper understanding of coagulation and the impact of the relevant factors would aid in both cheese manufacturing and also in determining the nutritional benefits of dairy products. A method using confocal microscopy was developed to follow the movement of milk fat globules and the formation of a milk protein network during the enzyme-induced coagulation of milk. Image processing methods were then used to quantify the rate of coagulation. It was found that the texture of the protein network is an indicator of the current status of the milk gelation, and hence can be used to monitor the coagulation process. The imaging experiment was performed on milk gels with different concentrations of the coagulation enzyme, chymosin. Rheological measurements were taken using free oscillation rheometry to validate the imaging results. Both methods showed an inverse relationship between rennet concentration and the coagulation time.
The results from the imaging study were used to create a computational model, which created simulated images of coagulating milk. The simulated images were then analyzed using the same image analysis algorithm. The temporal protein network texture behavior in the simulated images followed the same pattern as the protein texture in the confocal imaging data. The model was developed with temperature and rennet concentration as user inputs so that it could be implemented as a predictive tool for milk coagulation.
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Maggiano, Corey. "CONFOCAL LASER SCANNING MICROSCOPY AS A TOOL FOR THE INVESTIGATION OF TETRACYCLINE FLUORESCENCE IN ARCHAEOLOGICALHUMAN BONE." Master's thesis, University of Central Florida, 2005. http://digital.library.ucf.edu/cdm/ref/collection/ETD/id/2752.
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Fluorochromes such as tetracycline have been used to label bone for histomorphometric analysis, measuring bone formation, growth, maintenance, and pathology. More recently, similar fluorescence has been observed in ancient human bone. Attributed to tetracycline (TC) exposure, this phenomenon could affect various aspects of health during life and/or preservation of remains postmortem. Standard epifluorescence microscopy is the most common tool employed in the analysis of these labels. Though valuable, this technique is limited by its inability to penetrate bone three-dimensionally and its inclusion of out-of-focus light, possibly disrupting accurate analysis. Confocal Laser Scanning Microscopy (CLSM) has been demonstrated as a valuable tool for three-dimensional histology. Its application to the study of compact bone fluorescence has been lacking, especially in archaeological and forensic sciences. In the following two papers, modern TC-controlled bone is compared to well preserved archaeological bone recovered from the Dakhleh Oasis, Egypt, using both standard wide-field and more modern confocal techniques for imaging and analysis. Spectral analysis via CLSM shows that both modern and ancient fluorescent labels in bone share the exact same fluorescence emission peak at 525 nm. Differences in the shape of the spectral curve and photobleaching characteristics are discussed. In addition, CLSM's high-resolution two- and three-dimensional imaging capabilities (in polarized light, scattered light, and fluorescence light) are found to increase the flexibility and creativity of investigations into the occurrence of tetracycline labels in archaeological bone and could have added benefits for modern medical and anatomical experimentation.M.S.
Department of Biology
Arts and Sciences
Biology
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Farber, Elliott. "A new method to achieve lithic use-wear discrimination using laser scanning confocal microscopy (LSCM)." Thesis, Florida Atlantic University, 2013. http://pqdtopen.proquest.com/#viewpdf?dispub=1524500.
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My study sought to acquire quantitative data from the surface of lithic tools and use that data to discriminate tools used on different contact materials. An experimental archaeological wear production method was conceived, whereby I and several volunteers produced wear on chert, heat-treated chert, and obsidian flakes by using those flakes on several contact materials. The flakes were then analyzed using a laser scanning confocal microscope, which recorded three-dimensional surface data from each tool.
The data was analyzed using cluster analysis to find the ideal combination of parameters which correctly discriminated the flakes based on use-wear data. After finding acceptable parameters which grouped flakes appropriately through cluster analysis, those groups were subjected to a discriminant analysis. Each analysis returned a p-value under .05, meaning that the clustering based on the parameters Sq and Sfd produced by the cluster analysis was not random, but indicative of these variables’ ability to discriminate lithic use-wear. The major advantage of the approach developed in this study is that it can quantitatively discriminate use-wear produced by different contact materials on flakes with no a priori information at all.
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Taylor, Zeike Amos. "Patient-specific models of cartilaginous tissues based on laser scanning confocal arthroscopy." University of Western Australia. School of Mechanical Engineering, 2006. http://theses.library.uwa.edu.au/adt-WU2006.0097.
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[Truncated abstract] An important field of research in orthopaedic biomechanics is the elucidation and mathematical modelling of the mechanical response of cartilaginous tissues. Such research has applications in the understanding of joint function and degenerative processes, as well as in surgical planning and simulation, and engineering of tissue replacements. In the case of surgical and tissue engineering applications especially, patient-specific mechanical properties are highly desirable. Unfortunately, obtaining such information would generally involve destructive mechanical testing of patient tissue, thus rendering the tissue functionally unusable. Development of a laser scanning confocal arthroscope (LSCA) within our School will soon allow non-invasive extraction of 3D microstructural images of cartilaginous tissues in vivo. It is also envisaged that, linked to a suitably formulated constitutive formulation, such information could allow estimation of tissue mechanical response without physical biopsy. This thesis describes the development of techniques to potentially allow non-invasive patient-specific estimation of tissue mechanical response based on confocal arthroscopy data. A microstructural constitutive model is developed which is capable of directly incorporating LSCA-derived patient-specific structural information. A fibre composite type homogenisation approach is used as the basis for the model. ... The result is a series of orientation tensors describing the 3D orientation of linear features in the image stack. The developed analysis techniques are used to estimate fibre volume fraction and orientation distribution for each of the meniscal specimens. The developed constitutive model and image-derived structural parameters are finally used to estimate the reaction force history of two meniscal cartilage specimens subjected to partially confined compression. The predictions are made on the basis of the specimens? individual structural condition as assessed by confocal microscopy and involve no tuning of material parameters. Although the model does not reproduce all features of the experimental curves, as an unfitted estimate of mechanical response the prediction is quite accurate. In light of the obtained results it is judged that more general non-invasive estimation of tissue mechanical properties is possible using the developed framework. The likely limitations and potential areas of improvement are discussed.
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Doroshenko, Mikheil [Verfasser]. "Diffusion in heterogeneous systems studied by laser scanning confocal microscopy and fluorescence correlation spectroscopy / Mikheil Doroshenko." Mainz : Universitätsbibliothek Mainz, 2014. http://d-nb.info/104870758X/34.
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Syrett, Natalie. "The nuclear localisation of human poly (ADP-ribose) polymerase-1 investigated by confocal laser scanning microscopy." Thesis, University of Essex, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.423567.
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Smith, Shea C. "LASER SCANNING CONFOCAL MICROSCOPY (LSCM): AN APPLICATION FOR THE DETECTION OF MORPHOLOGICAL ALTERATIONS IN SKIN STRUCTURE." DigitalCommons@CalPoly, 2009. https://digitalcommons.calpoly.edu/theses/198.
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Laser scanning confocal microscopy (LSCM) is an optical imaging technique that provides improved resolution and sensitivity over conventional methods of optical microscopy. However, the cost of most commercial LSCM systems exceeds the financial limitations of many smaller laboratories. The design of a custom LSCM created at a fraction of the cost of a commercial model is discussed in this paper.
The increase in the incidence rate of skin cancer in the world today is alarming, as such, it is essential to provide an early, rapid and effective method for in vivo diagnostics of human skin tissue. LSCM is capable of detecting alterations in skin morphology and configuration, as well as providing chemical composition information which may be indicative of the development of skin cancer. If developed successfully, LSCM could replace the current invasive biopsy procedures performed today with a quick, non-invasive optical scanning method that would prove beneficial for both patients and physicians alike.
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Mokwatlo, Sekgetho Charles. "Microscopic visualisation of succinate producing biofilms of Actinobacillus succinogenes." Diss., University of Pretoria, 2017. http://hdl.handle.net/2263/62782.
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Biofilms of Actinobacillus succinogenes, grown in a biofilm reactor system, were investigated for structure and cell viability, through microscopic visualisation with a confocal scanning laser microscope (CSLM) and a scanning electron microscope (SEM). Biofilms were sampled and visualised at steady state conditions with the broth containing succinic acid titres between 15 and 21 g/L. All sampled biofilm was 6 days old. Six-day-old biofilms of A. succinogenes showed a heterogeneous biofilm architecture composed of cell micro-colony pillars which varied considerably in thickness, area and shape. Microcolony pillars consisted of a densely packed entanglement of sessile cells. Quantitative analysis revealed that the pillars were mostly large, with a mean pillar diameter of 170 m and a mean thickness of 92 m, although pillar diameter and thickness were variable as they ranged from 25 – 500 m and 30 – 300 m, respectively. In the regions close to the substratum surface, pillars were characterised by having defined borders with a network of channels ranging from 40 – 200 m in width separating them. However, towards the middle of the biofilm depth some of the pillars coalesced. For this reason low cross sectional area coverage of biofilm consistently occurred at the bottom portion of the biofilm whilst the highest coverage was in the middle portion of the biofilm. Regarding cell morphology, very large differences were observed. Planktonic cells were rod-shaped, whereas sessile cells expressed an elongated rod morphology and thus were much longer and thinner compared with planktonic cells. Planktonic cells were 1 – 2 m thick and 4 – 5 m long, while sessile cells were 0.5 – 1 m thick and 5 – 100 m long. Long sessile cells resulted in extensive tangling in microcolony pillars, which may have contributed to the structural stability of the pillars. Fibre-like connections of constant diameter were observed between cells, and between the cells and surface. The diameter of these connections was approximately 20 – 30 nm. Viability stains showed that in the bottom portion (from 0 - 20 m above the substratum surface) of the biofilm, most of the cells were dead. However, the portion of covered area attributed to living cells increased past the middle of the biofilm towards the top part of the biofilm. A high percentage of living cells was thus found towards the top part of the biofilm. Overall, 65% (with 2% standard deviation) of the entire biofilm was composed of dead cells. In this way, the results show that operation at high acid conditions comes at a cost of low overall biomass productivity due to decreased active biomass.Dissertation (MEng)--University of Pretoria, 2017.
Chemical Engineering
MEng
Unrestricted
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Teo, Ying Hui. "The use of confocal laser scanning microscopy in the study of skin structure and topical dosage forms." Thesis, University of Nottingham, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.364664.
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Mohammed, Asma Hadi. "An investigation of RNR regulation in fission yeast by confocal laser scanning FRET and near-TIRF microscopy." Thesis, University of Sussex, 2011. http://sro.sussex.ac.uk/id/eprint/7401/.
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For genome integrity, adequate levels of deoxyribonucleotide (dNTPs) are essential to maintain faithful DNA replication and repair via the regulation of ribonucleotide reductase (RNR). In the fission yeast, RNR is composed of two subunits: Cdc22 and Suc22. The importance of Spd1 (RNR inhibitor) in Cdc22-Suc22 complex formation has been demonstrated by imaging of S. pombe containing fluorescent protein (FP) modified RNR subunit proteins in the presence of Spd1 and absence of Spd1 cells using confocal laser scanning microscopy. To investigate further the significant role of Spd1 in the regulation of RNR, 41 mutants created by Nestoras group. We used fluorescence resonance energy transfer (FRET) by acceptor photobleaching to investigate the RNR subunit interaction and provide evidence for a new model for the role of Spd1 in RNR regulation. Different treatments such as HU, 4NQO and heat shock have been used to investigate the effect of radical scavenging on the inhibition of RNR activity and induced DNA damage on S. pombe cell viability to elucidate further the role of Spd1 in the regulation of RNR. Finally a novel imaging technique, near-total internal reflection microscopy has been developed and applied with dual-view detection. The technique has been applied to image, simultaneously, the donor CFP and acceptor YFP channels of the FP-tagged RNR complex in the wild-type S. pombe cells and perform FRET measurements that are consistent with the confocal fluorescence results. In conclusion, a new hypothesis for the role of Spd1 has been drawn from the results, which is that the inhibitory role of Spd1 mediates the Suc22-Cdc22 (R1-R2) interaction to form a FRET competent but immature and inactive RNR complex, while with Spd1 deleted RNR is clearly active in a conformation that lacks FRET.
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Jarke, Annica. "Effect of manufacturing conditions and polymer ratio on the permeability and film morphology of ethyl cellulose and hydroxypropyl cellulose free-films produced by using a novel spray method." Thesis, Uppsala University, Department of Pharmacy, 2009. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-121842.
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This thesis considers the effect of manufacturing conditions and polymer ratio on water permeability and morphology of free-films. A novel spray method for producing ethyl cellulose (EC) and hydroxypropyl cellulose (HPC) free-films was developed where several process parameters was controlled. The process was optimised by pre-spraying solvent until the system reached a steady-state temperature. This minimised the variation of outlet air temperature to < 2.5 °C. Coating time was approximately 4 minutes excluding drying.
Free-films were produced using 94 wt% solvent (95 %-ethanol) and 6 wt% polymer. The amount of HPC in the films was varied (wt% HPC defined as HPC/(HPC+EC)*100). Films with 30-40-50-57 wt% HPC were studied. Phase diagrams was constructed to study the phase transformation of polymer mixtures. Results show that all polymer mixtures with HPC content above 30 wt% were phase separated prior to film manufacturing. Temperature had an effect on the polymer phase transformation. In the phase diagram, the 2-phase area was larger for temperatures above 40 °C.
The investigated manufacturing conditions were outlet air temperature (°C) and spray rate (g/min). Outlet air temperature was controlled by adjusting the inlet air temperature. The films were characterized by measuring water permeability (m2/s). Cross section structure of the films was analyzed with confocal laser scanning microscopy (CLSM). FITC-HPC was added for enhanced contrast between the domains.
Higher outlet air temperature gave higher water permeability of the film whereas higher spray rate gave lower water permeability. The outlet air temperature had an impact on evaporation rate. The evaporation rate together with spray rate affected the solidification and hence the structure of the film. Images show that longer solidification time smeared the domains into larger domains. Lower water permeability was caused by less connectivity between the pores.
In conclusion, experiments show that water permeability of EC/HPC free-films was highly dependent on the manufacturing conditions.
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Jones, Christopher Wynne. "Laser scanning confocal arthroscopy in orthopaedics : examination of chondrial and connective tissues, quantification of chondrocyte morphology, investigation of matirx-induced autologous chondrocyte implantation and characterisation of osteoarthritis." University of Western Australia. School of Mechanical Engineering, 2007. http://theses.library.uwa.edu.au/adt-WU2008.0061.
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[Truncated abstract] Articular cartilage (AC) covers the surface of synovial joints providing a nearly frictionless bearing surface and distributing mechanical load. Joint trauma can damage the articular surface causing pain, loss of mobility and deformation. Currently there is no uniform treatment protocol for managing focal cartilage defects, with most treatment options targeted towards symptomatic relief but not limiting the progression into osteoarthritis (OA). Autologous chondrocyte implantation (ACI) and more recently matrix-induced autologous chondrocyte implantation (MACI), have emerged as promising methods for producing hyaline or hyaline-like repair tissue, however there remains some controversy regarding the exact histological nature of the tissue formed. Histological characterisation of AC repairs requires destructive tissue biopsy potentially inducing further joint pathology thereby negating the treatment effect. OA is recognised as a major cause of pain, loss of function and disability in Western populations, however the exact aetiology is yet to be elucidated. The assessment of both OA and cartilage repair has been limited to macroscopic observation, radiography, magnetic resonance imaging (MRI) or destructive biopsy. The development of non-destructive AC assessment modalities will facilitate further development of AC repair techniques and enable early monitoring of OA changes in both experimental animal models and clinical subjects. Confocal laser scanning microscopy (CLSM) is a type of fluorescence microscopy that generates high-resolution three-dimensional images from relatively thick sections of tissue. ... Biomechanical analysis suggested that the mechanical properties of MACI tissue remain inferior for at least three months. This study showed the potential of a multi-site sheep model of articular cartilage defect repair and validated its assessment via LSCA. Finally, the LSCA was used to arthroscopically image the cartilage of an intact fresh frozen cadaveric knee from a patient with clinically diagnosed OA. Images were correlated to ICRS (Outerbridge) Grades I-IV and histology. The LSCA gave excellent visualization of cell morphology and cell density to a depth of up to 200'm. Classical OA changes including clustering chondrocytes, surface fibrillation and fissure formation were imaged. Fair to moderate agreement was demonstrated with statistically significant correlations between all modalities. This study confirmed the viability of the LSCA for non-destructive imaging of the microstructure of the OA cartilage. In conclusion, the LSCA identified histological features of orthopaedic tissues, accurately quantified chondrocyte morphology and demonstrated classical OA changes. While the development and investigation of an ovine model of cartilage repair showed the treatment benefit of MACI, some biomechanical issues remain. Ultimately, the LSCA has been demonstrated as a reliable nondestructive imaging modality capable of providing optical histology without the need for mechanical biopsy. Medical Subject Headings (MESH): articular cartilage; autologous chondrocyte implantation; matrix-induced autologous chondrocyte implantation; biomechanics; cartilage; confocal microscopy; diagnosis; histology; image analysis; immunohistochemistry; magnetic resonance imaging; microscopy; osteoarthritis
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Hottor, Bismarck Afedo. "The effect of severity of pre-eclampsia on the basal plate intervillous surface lining components : a confocal laser scanning microscopy study." Thesis, University of Leicester, 2009. http://hdl.handle.net/2381/7885.
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Renneckar, Scott Harold. "Modification of Wood Fiber with Thermoplastics by Reactive Steam-Explosion." Diss., Virginia Tech, 2004. http://hdl.handle.net/10919/11239.
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For the first time, a novel processing method of co-refining wood and polyolefin (PO) by steam-explosion was scientifically explored for wood-thermoplastic composites without a coupling agent. Traditional studies have addressed the improvement of adhesion between components of wood thermoplastic composites through the use of coupling agents such as maleated PO. The objective of this study was to increase adhesion between wood and PO through reactive processing conditions of steam-explosion. PO characteristics, such as type (polyethylene or polypropylene), form (pellet, fiber, or powder) and melt viscosity were studied along with oxygen gas content of the steam-explosion reactor vessel. Modification of co-processed wood fiber was characterized in four studies: microscopy analysis of dispersion of PO with wood fiber, sorption properties of co-processed material, chemical analysis of fractionated components, and morphological investigation of co-processed material. Two additional studies are listed in the appendices that relate to adsorption of amphiphilic polymers to the cellulose fiber surface, which is one hypothesis of fiber surface modification by co-steam-explosion.
Microscopy studies revealed that PO melt viscosity was found to influence the degree of dispersion and uniformity of the steam-exploded material. The hygroscopic nature of the co-processed fiber declined as shown by sorption isotherm data. Furthermore, a water vapor kinetics study found that all co-refined material had increased initial diffusion coefficients compared to the control fiber. Chemical changes in fractionated components were PO-type dependent. Lignin extracted from co-processed wood and polyethylene showed PO enrichment determined from an increase of methylene stretching in the Fourier Transform infrared subtraction spectra, while lignin from co-processed wood and polypropylene did not. Additionally, extracted PO showed indirect signs of oxidation as reflected by fluorescence studies. Solid state nuclear magnetic resonance spectroscopy revealed a number of differences in the co-processed materials such as increased cellulose crystallinity, new covalent linkages and an alternative distribution of components on the nanoscale reflected in the T1Ï relaxation parameter.
Steam-explosion was shown to modify wood fiber through the addition of "non-reactive" polyolefins without the need for coupling agents. In light of these findings, co-refining by steam-explosion should be viewed as a new reactive processing method for wood thermoplastic composites.Ph. D.
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Lunn, Matthew O'Brien. "A Morphological Study of the Canine Zona Pellucida: A Heterogeneous Ultrastructure and Barrier." University of Dayton / OhioLINK, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=dayton1311785290.
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Botes, Adèle. "Transdermal delivery of isoniazid and rifampicin by pheroid technology / Adèle Botes." Thesis, North-West University, 2007. http://hdl.handle.net/10394/1668.
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Limbaugh, Melissa D. "Replacement of saturated fats in a cream cheese product." The Ohio State University, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=osu1431097069.
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McVey, Alexander Ferguson. "Three-dimensional imaging of bacterial microcolonies." Thesis, University of Edinburgh, 2015. http://hdl.handle.net/1842/15774.
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Previous research into microbial colonies and biofilms shows a significant gap in our current understanding of how bacterial structures develop. Despite the huge body of research undertaken into the formation, genetic makeup, composition, and optimal growth conditions of colonies, no study has been successful in identifying all individual bacteria in a colony in three-dimensions as a function of time. This lack of bacterial cell lineage in such a simple class of organisms is conspicuous in the light of what is known about other organisms, such as Caenorhabditis elegans [1]. In this thesis I show that using laser scanning confocal microscopy in conjunction with developments in sample preparation and post acquisition image analysis, it is possible to fully reconstruct all individual bacteria within an Escherichia coli (E. coli ) microcolony grown in viscoelastic media. Additionally, I show that by further pushing the resolution of confocal microscopes, commercial systems are capable of extracting three-dimensional information on protein structures inside bacteria at early stages of growth. This thesis is in three parts. The first part shows that by pushing the resolution of a commercial laser scanning confocal microscope system it is possible to achieve single cell resolution of a bacterial colony growing in three dimensions in a viscoelastic medium (agarose) from a seed bacterium. The growth of individual bacteria is examined as the concentration of agarose in the media is altered. Results show there is a nonlinear dependence between the rate of growth of a bacterium and the concentration of the agarose in the media with a peak in growth rate at 3% (weight) concentrations of agarose in M9 media. The second part of this work presents a study of how an initially two-dimensional colony growing between a glass slide and agarose gel suddenly invades the third spatial dimension by buckling. The results show that the cells within the centre of the colony flex and buckle, due to confinement by their neighbours, creating additional layers. Indeed, flexing is not limited to the buckling event but occurs throughout the early growth cycle of a colony. The final part of this thesis shows that by further pushing the resolution of confocal microscopes, commercial systems are capable of extracting three-dimensional information about the temporal evolution of the spatial distribution of the FtsZ septation ring within the cell. As the bacterial colony grows from a seed bacterium to a microcolony, the error in placing the division accurately at the cell centre is seen to increase as the number of bacteria within the colony increases and spatial confinement occurs.
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Fúcio, Suzana Beatriz Portugal de. "Analise dos efeitos da interação entre S mutans e materiais restauradores esteticos : caracteristicas do biofilme em microscopio confocal de varredura a laser e propriedades de superficie dos materiais apos 30 dias." [s.n.], 2007. http://repositorio.unicamp.br/jspui/handle/REPOSIP/288598.
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Orientadores: Regina Maria Puppin Rontani, Renata de Oliveira Mattos-GranerDissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba
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Resumo: O desenvolvimento do biofilme de Streptococcus mutans sobre materiais restauradores e a biodegradação destes substratos em função dos metabólitos bacterianos podem ser influenciados pelas propriedades e caracterÃsticas do material. A partir de uma revisão sistemática em que se verificou a carrência de estudos a respeito dos efeitos do biofilme na superície de materiais restauradores, foi proposto investigar algumas características quantitativas e qualitativas do biofilme após 30 dias de interação com materiais restauradores, além de analisar propriedades e microestrutura da superfície dos materiais que sofreram tal interação. Para cada material testado (cerâmica - C, resina composta nanoparticulada e cimentos de inúmero de vidro modificado por resina - CIVMR e convencional - CIVC), foram confeccionados 25 discos sob condições assépticas, para distribuição em 3 grupos de estocagem: 1) 100% de umidade relativa a 37ºC (n=5); 2) meio de cultura a 37ºC (BHI + 1% sacarose) (n=5); 3) biofilme de Streptococcus mutans e meio de cultura a 37ºC (n=15). Valores de dureza do grupo 1 (valores imediatos) foram obtidos previamente à estocagem, a fim de se verificar alterações ao longo do tempo quando estocados em umidade relativa apenas. Após 30 dias de estocagem, os discos do grupo 3 foram levados para observação do biofilme corado e hidratado em microscopia de varredura confocal a laser (CLSM). As imagens obtidas auxiliaram na obtenção de valores
Abstract: Streptococcus mutans biofilm development on restorative materials and biodegradation of those materials due to bacterial acids are influenced by material properties and characteristics. Since a systematic review found a deficiency concerning studies related to effects of biofilm on the surface of restorative materials, the proposition for this investigation was to analyze some quantitative and qualitative biofilm characteristics after 30-days interaction with restorative materials. In addition, it was investigated changes on the surface properties and microstructure of materials after 30-days interaction. Twenty-five disks of each material tested (ceramic - C, nanofill composite - NC, resin-modified glass ionomer - RMGIC and conventional glass ionomer cement - CGIC) were made, at aseptic conditions, and distributed in 3 storage groups: 1) 100% relative humidity at 37ºC (n=5); 2) growth medium at 37ºC (BHI + 1% sucrose) (n=5); 3) Streptococcus mutans biofilm and growth medium at 37ºC (n=15). Vickers hardness values from group 1 were obtained previously storage, in order to observe aging by relative humidity. After 30 days storage, disks were stained, kept hydrated and observed by confocal laser scanning microscopy, whose images supported to acquire values concerning biofilm thickness, bio-volume, roughness coefficient and surface to volume ratio. Qualitative analyses related to viable / non-viable cells distribution and biofilm architecture also were realized. Subsequently, all disks were ultrasonically washed and analyzed to surface roughness, hardness and microstructure. Biofilms presented a progression more viable cells in superficial regions of the biofilm to proportionally more nonviable cells in the deeper regions of the biofilms, near the disk. Besides, cellular aggregates and fluid-filled channels were observed in biofilm developed on all materials. Concerning biofilm quantitative properties, thickness was the unique with difference statistically significant among materials. C and NC accumulated thicker biofilms than RMGIC and CGIC. There was no difference statistically significant among immediate and storage groups related to C and NC surface roughness and hardness. However, group 3 of NC showed surface biodegradation microscopically. Group 1 of RMGIC and CGIC presented higher hardness values than immediate values. Nevertheless, hardness values from RMGIC group 3 decreased compared groups 1 and 2, while surface roughness values of group 3 increased statistically. Group 3 of CGIC showed higher roughness values than other groups and no difference statistically significant among three storage groups concerning hardness values. RMGIC and CGIC micrographs also demonstrated biodegradation on the surface materials. Within this study conditions, it was concluded that there was influence of restorative materials on biofilm development and influence of biofilm on the surface properties and microstructure characteristics of materials tested, being material -dependent
Mestrado
Materiais Dentarios
Doutor em Materiais Dentários
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Oliveira, Denise Gusmão de. "Estudo in vitro da formação do biofilme de Candida albicans em resina acrílica termopolimerizável revestida por nanopartículas de dióxido de silício (revestimento cerâmico)." Universidade de São Paulo, 2013. http://www.teses.usp.br/teses/disponiveis/25/25146/tde-14102013-163201/.
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A proposta deste trabalho foi analisar um produto experimental (VIPI LTDA, Pirassununga, SP), que através da tecnologia sol-gel, modifica a superfície de resinas acrílicas para base de próteses e forma uma camada de nanopartículas de sílica (NPS) visando diminuir o acúmulo e facilitar a remoção de microrganismos. Dessa forma, inicialmente, confirmou-se a deposição de NPS e formação do revestimento cerâmico em polimetilmetacrilato (PMMA) através de espectroscopia no infravermelho por Transformada de Fourier (FTIR); e posteriormente, quantificou-se o biofilme de Candida albicans nesta superfície através da contagem de unidades formadoras de colônia (UFC/mL) e microscopia confocal (MC). Um total de 51 espécimes (10x10x3mm) de PMMA foi confeccionado e distribuído aos experimentos designados. Para a análise da composição dos espécimes em FTIR, foram avaliados 3 grupos (n=1): CN- espécime que não recebeu tratamento algum; CP- espécime que recebeu a aplicação do primer do produto; CL- espécime que passou tanto pela aplicação do primer como pelo processo sol-gel. Na etapa seguinte, foram utilizados 48 espécimes divididos em 3 grupos (n=16), de acordo com o tipo de polimento: PM3- mecanicamente polido com 3μm de rugosidade média; PM03- mecanicamente polido com 0,3μm de rugosidade; PL- polido quimicamente pelo líquido conforme instruções do fabricante. Anteriormente aos experimentos, os espécimes foram esterilizados por óxido de etileno e, então, imersos em saliva artificial por 2hs para a formação da película adquirida. Em seguida, foram secos e inoculados com 2mL de suspensão de C. albicans (1.107 cel/mL) para adesão das células fúngicas durante 90min. Após esta fase, as amostras foram lavadas em solução salina e imersas em meio estéril (RPMI) para crescimento do biofilme em estufa sob agitação (12hs a 37oC). Metade do número das amostras de cada grupo (n=8) foi destinada a contagem de UFC/mL e a outra metade dos espécimes (n=8), foi designada ao método de MC, que com o auxílio de um software (BioImageL v.2), permitiu a determinação do biovolume total (μm3), biovolume de células viáveis (μm3), biovolume de células não-viáveis (μm3) e área de cobertura do campo pelo biofilme (%). Os dados obtidos pelo FTIR foram analisados através da estatística descritiva. Os resultados obtidos pelos experimentos de quantificação após teste de normalidade Kolmogorov-Smirnov foram analisados através do teste paramétrico ANOVA, seguido do teste de Tukey para comparações entre grupos (p<0,05). Através do FTIR, observou-se a deposição satisfatória da camada de NPS, permitindo assim, o desempenho dos experimentos de quantificação. Os resultados do UFC/ml e MC demonstraram semelhança na quantificação do biofilme entre os grupos PL e PM3, e diferença quando comparados ao grupo de superfícies mais lisas, PM03. Dessa forma, observou-se que o polimento líquido experimental não foi efetivo para a diminuição da colonização de biofilme deC. albicans em superfícies de PMMA. Entretanto, maiores investigações sobre as propriedades de superfície deste revestimento devem ser realizadas, já que o processo sol-gel permite uma facilidade na modificação dessas características, podendo levar ao desenvolvimento de um material de revestimento ideal.This study investigates an experimental coating (VIPI LTDA, Pirassununga, SP) by sol-gel process that modifies acrylic resin denture base with silicon dioxide nanoparticles (SNP) to decrease C. albicans biofilm growth. Therefore, it was first investigated the presence of sol-gel ceramic coating on polymethylmethacrylate (PMMA) by Fourier Transform Infrared Spectroscopy (FTIR). Then C. albicans biofilms were quantified by colony forming units (CFU/mL) and confocal scanning laser microscopy (CSLM). Fifty-one PMMA specimens were manufactured (10x10x3mm) and assigned to the experiments. To evaluate specimens composition, it was analyzed three groups (n=1): CN- the specimen did not receive any surface treatment; CP- it was applied the coating primer on the specimen surface CL- the specimen was treated with the whole sol-gel process. In the following stage, 48 samples were divided into 3 groups (n=16) according to the polish type: PM3- 3μm of roughness mechanical polish; PM03- 0,3μm of roughness mechanical polish; PL- liquid polish. Samples of experimental group were coated according to manufacturers instructions and all the samples were sterilized with ethylene oxide. After that, they were dipped in artificial saliva for 2hs to acquire the salivary pellicle, and then, dried and inoculated with 2 mL suspension of C.albicans (1.107 cel/mL) for 90 min. Then, specimens were washed and immersed in sterile RPMI solution (37oC for 12h). Half of the samples of each group (n=8) was assigned to each quantification test (UFC/mL and CSLM). By CSLM and software (BioImageL v.2) analysis was possible to obtain the total biovolume (μm3), viable biovolume (μm3), non-viable biovolume (μm3), and covered area (%) by C. albicans biofilm. The data obtained by FTIR were analyzed by descriptive statistic. Whereas the records acquired by the quantification experiments were first analyzed by Kolmogorov-Smirnov normality test and then by one way ANOVA followed by Tukeys test to assess difference between groups (p<0,05). FTIR results showed an adequate SNP deposition, allowing the quantification tests to be performed. UFC/mL and CLSM records showed similarity between PL and PM3 groups and difference when comparing these groups to smoother surfaces group (PM03). Therefore, in this study, the experimental coating was not effective to reduce colonization by C. albicans biofilm on PMMA surfaces. Nevertheless, further investigations are required since sol-gel ceramic coating process eases the features modification of the material, possibly leading to an ideal coating development.
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Ying, Jia. "Structural Change and Its Assessment by Fluorescence Spectroscopy in Functional Polymers." 京都大学 (Kyoto University), 2014. http://hdl.handle.net/2433/192187.
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Alzahrani, Yahya A. "In vivo assessment of inflammatory cells in contact lens wearers." Thesis, Queensland University of Technology, 2016. https://eprints.qut.edu.au/93576/1/Yahya_Alzahrani_Thesis.pdf.
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Dissatisfaction with, and discontinuation from, contact lens wear is a source of major frustration and inconvenience to users, and a problem that is thought to cost the contact lens industry hundreds of millions of dollars each year. By directly and non-invasively monitoring inflammatory cells in the tissues at the front of the eye in symptomatic and asymptomatic lens wearers, the candidate has been able to demonstrate an inflammatory basis for contact lens discomfort. This finding may pave the way towards the development of strategies to make contact lenses more safe and afford greater levels of comfort.
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Souza, Joyce Gonçalves Rozário de. "Desenvolvimento do sistema reprodutivo de Echinostoma paraensei (Trematoda: Digenea) de hamsters (Mesocricetus auratus) experimentalmente infectados, analisado por microscopia de luz de campo e microscopia laser confocal." Universidade do Estado do Rio de Janeiro, 2013. http://www.bdtd.uerj.br/tde_busca/arquivo.php?codArquivo=6138.
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Coordenação de Aperfeiçoamento de Pessoal de Nível SuperiorO conhecimento da morfologia e ultraestrutura dos helmintos permite a correta classificação destes organismos, bem como fornece subsídios que poderão ser utilizados para diagnóstico e controle. A microscopia laser confocal é uma ferramenta para estudar a organização estrutural de várias espécies de helmintos, possibilitando acesso a detalhes morfológicos não evidenciados pela microscopia óptica. Echinostoma paraensei é um trematódeo, digenético, hermafrodita parasito de numerosos hospedeiros vertebrados. Neste trabalho foi investigado o desenvolvimento dos órgãos reprodutivo e a morfometria de E. paraensei, desde a fase jovem até a adulta, como contribuição ao conhecimento do desenvolvimento reprodutivo desta espécie. Os trematódeos foram recuperados aos 3, 4, 5, 6, 7, 10, 14 e 21 dias posterior à infecção (dpi) experimental em hamsters. Estes foram corados em carmim clorídrico, desidratados em série alcoólica e montados em lâmina permanente em bálsamo do Canadá, fotografados e medidos usando microscopia de luz de campo claro (MCC) e microscopia de varredura laser confocal (MVLC). Entre 3 e 4 dpi, os primórdios genitais estavam presentes e nenhuma organização do sistema reprodutivo foi visualizada por MCC e MVLC. Os primórdio do ovário, dos testículos e da bolsa do cirro foram visualizados por MCC aos 5 e 6 dpi, no entanto, MVLC dos helmintos aos 5dpi mostra que estes primórdios, o ootipo e o útero estavam presentes, como estruturas individualizadas. A bolsa do cirro apresenta metratermo e o ovário com primórdio do oótipo adjacente aos 7dpi por MVLC. A vesícula seminal, receptáculo seminal, células diferenciadas nos testículos, ducto e reservatório vitelínico e oviducto foram visualizados após 10 dias, enquanto os espermatozóides na vesícula seminal, ovos e oócitos, células vitelínicas, poro e canal de Laurer aos 14 dias. A morfometria evidencia um acelerado crescimento dos órgãos reprodutores a partir do 7 dia. Os testículos apresentam aumento significativo no comprimento do 7 ao 21 dia e o ovário durante o período de 7 à 10 dpi. Aos 21 dpi, todos os helmintos apresentaram glândulas vitelínicas, útero contendo ovos e espermatozóides no oviducto enquanto outros ovos estão sendo formados. As mudanças morfológicas acentuadas durante a gametogênese consistem no aumento do comprimento do helminto, maturação das gônadas, desenvolvimento e maturação das glândulas vitelínicas. O desenvolvimento do helminto como um todo está relacionado à maturação dos órgãos reprodutivo masculino e feminino indicando o investimento deste trematódeo em garantir a produção e eliminação dos ovos ao meio exterior.
The knowledge about morphology and ultra structure of helminthes are great importance in correct classification these organisms. The Scanning Laser Microscopy (LSM) is an important tool to study the structural organization of several helminthes species. Echinostoma paraensei is a trematode, digenetic, hermaphroditic parasite of several hosts. In this study, the development of reproductive organs and the morphometry of E. paraensei from young stage to adult worm were investigated, to contribute knowledge of the reproductive development of this specie. The trematodes were recovered on 3, 4, 5, 6, 7, 10, 14 and 21 days post infection (dpi) from experimental hamsters. It were dehydrated in alcohol series, stained with hydrochloric carmine, mounted on permanent slide using Canada balsam, photographed and measured using light microscopy (LM) and Scanning Laser Microscopy (LSM). Between 3 and 4 dpi the genital anlage were present and were not observed reproductive system organization by either LM and LSM. The anlage of ovary, testes and Cirruss sac were seen at 5 and 6dpi by LM, however LSM from 5dpi image shows theses anlage, ootype and uterus are present as individualized structure. Cirrus sac showed metraterm and ootype adjacent to primordial ovary were seen at 7 dpi by LSM. The seminal vesicle, seminal receptacle, differentiated cells in the testes, viteline ducts and oviduct were visualized after 10 days infection, while sperms in seminal vesicle, eggs, oocyte, Laurer canal and pore from 14 dpi by LSM. The morphometry shows a rapid growth of reproductive organs from 7th day. The testes have significantly increased length from 7 until 21dpi and ovary from 7 until 10dpi. All helminthes showed vitellines glands, uterus contained eggs and sperm in oviduct while another eggs were forming at 21 dpi. The marked morphologic changes during gametogenesis are increase of body length of helminthes, gonad maturation and development and maturation of vitelline glands. The development of helminthes as a whole is related to maturation of female and male organs of reproductive system showing the investment this trematode taken to ensuring the production, maintenance and delivery the eggs to external environment.
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Souza, Christiane Pezzi Gil de. "Características morfológicas de vermes adultos de Schistosoma mansoni Sambon, 1907 recuperados de camundongos alimentados com dieta hiperlípidíca na fase crônica da infecção esquistossomótica. Análise por microscopia de campo claro e confocal." Universidade do Estado do Rio de Janeiro, 2015. http://www.bdtd.uerj.br/tde_busca/arquivo.php?codArquivo=9263.
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Coordenação de Aperfeiçoamento de Pessoal de Nível SuperiorEstudos em animais experimentais evidenciaram associações significativas entre esquistossomose mansoni e hipercolesterolemia. Estudos in vitro e in vivo já demonstraram que o colesterol é essencial para Schistosoma mansoni, embora este não tenha capacidade de sintetizá-lo. A captação é realizada a partir do ambiente (cultivo ou hospedeiro) através do tegumento. O colesterol está envolvido nos mecanismos de evasão do helminto contra a resposta imunológica, além de poder participar na modulação da sinalização celular e reprodução, estimulando os órgãos reprodutores dos helmintos adultos como observado na fase aguda da infecção experimental. Este trabalho tem como objetivo avaliar se o mesmo fenômeno ocorre na fase crônica. Os helmintos foram recuperados de dez camundongos submetidos à dieta hiperlipídica ou padrão (controle) foram corados pelo carmin cloridrico e montados, individualmente, em lâmina histológica com bálsamo do Canadá. A preparação foi analisada por microscopia de campo claro nos seguintes caracteres: tegumento e o sistema reprodutor nos vermes machos (lobos testiculares, vesícula seminal, lobos testiculares supranumerários e canal ginecóforo) e, nas fêmeas (ovário, oótipo, útero, ovo, glândulas vitelínicas e espermateca). Posteriormente, algumas lâminas foram separadas para visualização pela microscopia confocal dos órgãos do sistema reprodutores acima descritos. Apesar de ter sido observado uma maior quantidade de espermatozoides, uma maior quantidade de oócitos sendo liberados no grupo da dieta, não houve diferença estatística significativa (p>0,05) entre os grupos analisados. Houve um aumento na oogênese como observado na fase aguda. Dessa forma, o colesterol pode estar relacionado com a estimulação na atividade dos órgãos reprodutores dos helmintos adultos na fase crônica da infecção.
Studies in experimental animals showed significant associations between with schistosomiasis and hypercholesterolemia. In vitro and in vivo studies have demonstrated that cholesterol is essential for Schistosoma mansoni, although this is not able to synthesize it. The capture is carried out from the environment (cultivation or host) through the tegument. The capture is carried out from the middle (cultivation or host) through the tegument. Cholesterol is involved in the helminth evasion mechanisms against the immune response, and can participate in the modulation of cell signaling and reproduction of worms by stimulating the reproductive organs of adult worms as observed in the acute phase of experimental infection. This study aims to evaluate whether the same phenomenon occurs in the chronic phase. Helminthes recovered from ten mice subjected to high fat diet or standard (control) were stained with hydrochloric carmine and mounted individually on histological slide with Canada balsam. The preparation was analyzed by bright field microscopy the following characteristics: oral sucker and ventral sucker, tubercles on tegument and the reproductive system in male worms (lobes testicular, seminal vesicles, supernumerary testicular lobes and gynaecophoric canal), and in females (ovary, ootype, uterus, egg, vitelline glands and spermatheca). Subsequently, some slides were separated for confocal microscopy for visualization of the organs of the reproductive system described above. Despite having been observed a higher amount of sperm, a larger number of oocytes are released in the diet group, there was no statistically significant difference (p> 0.05) between the groups. There was an increase in oogenesis as observed in the acute phase. Thus, cholesterol may be related to the stimulation of the activity of the reproductive organs of adult helminths in the chronic phase of infection.
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Baudin, Marine. "Couplage de rapporteurs génétiques et d’une molécule active pour l’étude de la dispersion de biofilms." Thesis, Université Paris-Saclay (ComUE), 2017. http://www.theses.fr/2017SACLC013/document.
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Les biofilms sont des communautés de microorganismes adhérant à une surface et encastrées dans une substance polymérique produite par les cellules du système, dite matrice extracellulaire. Du fait de leur nature ubiquitaire, les biofilms colonisent de nombreux environnements et causent souvent de sérieux problèmes dans les secteurs de la santé et de l’industrie. La dispersion par ajout d’agent chimique est l’une des stratégies de lutte contre les biofilms. Un acide gras, l’acide cis-2-décénoique (CDA), semble être prometteur pour ce faire, grâce à l’étendue de son action dispersante sur les espèces et règnes du vivant. L’objectif de ce travail de thèse est d’investiguer les mécanismes de dispersion des biofilms de l’espèce bactérienne Escherichia coli (E. coli) par la molécule modèle CDA. Le CDA modifie-t-il les structures du biofilm ou induit-il une réponse génétique des bactéries lors de la dispersion ? Pour répondre à ces questions, la dispersion des biofilms d’E. coli a été étudiée in situ dans des chambres microfluidiques par microscopie confocale à balayage laser (CLSM). Des souches bactériennes spécifiques ont été construites par clonage de promoteurs d’intérêt en fusion transcriptionnelle avec un gène codant pour une protéine fluorescente verte. Les résultats confirment l’activité dispersante du CDA avec une réduction significative de la biomasse, de l’épaisseur moyenne et de l’aire de recouvrement par couche du biofilm. Un outil innovant d’analyse d’images CLSM a été développé en collaboration dans le but de déterminer les propriétés structurales du biofilm et l’intensité de fluorescence in situ du rapporteur étudié. Les résultats indiquent une augmentation de l’intensité moyenne de fluorescence des biofilms après dispersion avec le CDA, au niveau global en considérant tout le biofilm et au niveau local en considérant une segmentation du biofilm en microcolonies, ainsi qu’en profondeur. Ces résultats évoquent un changement d’expression génique des bactéries en présence de CDA. Par ailleurs, les résultats montrent que le CDA ne semble pas avoir d’effet en culture planctonique, ni sur la croissance bactérienne ni sur l’activité des promoteurs sélectionnés. Ceci suggère que les effets du CDA sont biofilm-dépendantsBiofilms are microbial communities adhering to a surface and embedded in a self-produced polymeric substance, called extracellular matrix. By being ubiquitous in nature, biofilms colonize numerous environments, and they often cause serious problems for both health and industry sectors. Dispersion is one of the strategies for fighting biofilms. A fatty acid, cis-2-decenoic acid (CDA), seems to be promising for dispersing biofilms by the extent of its action on different species of microbes. The aim of this thesis work is to investigate the mechanisms of biofilm dispersion of the bacterial species Escherichia coli (E. coli) by the model molecule CDA. Does CDA modify the biofilm structures or does it induce a genetic response from bacteria during dispersion? To answer these questions, E. coli biofilm dispersal has been studied in situ in microfluidic chambers by confocal laser scanning microscopy (CLSM). Specific bacterial strains have been developed by cloning promoters of interest in transcriptional fusion with a gene encoding for a green fluorescent protein. The results confirm the dispersing activity of CDA with a significant decrease of biomass, biofilm average thickness and area over biofilm depth. A novel tool for analyzing CLSM images has been developed in collaboration in order to measure the biofilm structural properties as a function of in situ fluorescence intensity of the studied reporter. The results indicate an increase in the mean fluorescence intensity of the biofilms after dispersion with CDA, at a global level for the whole biofilm and at a local scale by considering a biofilm segmentation into microcolonies. These results evoke a change in gene expression by bacteria in the presence of CDA. Furthermore, the results show that CDA does not seem to have an effect on planktonic bacteria, neither on the bacterial growth nor on the activity of the selected promoters. This suggests that the CDA effects are biofilm-dependent
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Travascio, Francesco. "Modeling Molecular Transport and Binding Interactions in Intervertebral Disc." Scholarly Repository, 2009. http://scholarlyrepository.miami.edu/oa_dissertations/322.
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Low back pain represents a significant concern in the United States, with 70% of individuals experiencing symptoms at some point in their lifetime. Although the specific cause of low back pain remains unclear, symptoms have been strongly associated with degeneration of the intervertebral disc. Insufficient nutritional supply to the disc is believed to be a major mechanism for tissue degeneration. Understanding nutrients' transport in intervertebral disc is crucial to elucidate the mechanisms of disc degeneration, and to develop strategies for tissue repair (in vivo), and tissue engineering (in vitro). Transport in intervertebral disc is complex and involves a series of electromechanical, chemical and biological coupled events. Despite of the large amount of studies performed in the past, transport phenomena in the disc are still poorly understood. This is partly due to the limited number of available experimental techniques for investigating transport properties, and the paucity of theoretical or numerical methods for systematically predicting the mechanisms of solute transport in intervertebral disc. In this dissertation, a theoretical and experimental approach was taken in order to investigate the mechanisms of solute transport and binding interactions in intervertebral disc. New imaging techniques were developed for the experimental determination of diffusive and binding parameters in biological tissues. The techniques are based on the principle of fluorescence recovery after photobleaching, and allow the determination of the anisotropic diffusion tensor, and the rates of binding and unbinding of a solute to the extracellular matrix of a biological tissue. When applied to the characterization of transport properties of intervertebral disc, these methods allowed the establishment of a relationship between solute anisotropic and inhomogeneous diffusivity and the unique morphology of human lumbar annulus fibrosus. A mixture theory for charged hydrated soft tissues was presented as a framework for theoretical investigations on solute transport and binding interactions in cartilaginous tissues. Based on this theoretical framework and on experimental observations, a finite element model was developed to predict solute diffusive-convective-reactive transport in cartilaginous tissues. The numerical model was applied to simulate the effect of mechanical loading on solute transport and binding interactions in cartilage explants and intervertebral disc.
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Dantas, Talita Souza. "Influência da conicidade do pilar e tipo de agente cimentante na retentividade de coroas metálicas cimentadas sobre pilares personalizáveis de implantes." Universidade Federal de Uberlândia, 2011. https://repositorio.ufu.br/handle/123456789/16942.
Full textAbstract:
Several factors must be considered in the selection of angled abutments. In these situations, the clinician should find a practical way to overcome the lack of retention. The purpose of this study was to evaluate the bond strength of metallic crowns cemented to straight and angled customizable abutments with different definitive luting agents. Ninety one regular external hex analogs and abutments were divided in Control group (C): customizable straight abutment cemented with zinc phosphate (n = 10); three groups (n = 10) with 17° angled abutment cemented with zinc phosphate (A17ZP), Panavia F (A17RM) and RelyX U100 (A17R) cements and more three groups (n = 10) with 30° angled abutment also cemented with zinc phosphate (A30ZP), Panavia F (A30RM) and RelyX U100 (A30R) cements. The metal copings were cemented onto their corresponding metal dies and crowns cemented with Panavia F were pre-treated with an alloy primer containing an acid-phosphated monomer (MDP). Data from the all groups were compared to control group with a 1-way ANOVA (α=.05) and Dunnet s test, and comparison between tested groups were done with 2-way ANOVA (α=.05) and Tukey s test. SEM and CLSM evaluation were performed (n = 3) aiming to investigate microscopic features of the abutment-cement-crown interfaces. The mean force (SD) required to dislodge the crowns in the C, A17ZP, A17RM, A17R, A30ZP, A30RM and A30R groups was 357,26 (62,21) N; 251,50 (20,13) N; 397,05 (88,48) N; 328,71 (79,87) N; 276,70 (17,96) N; 377,81 (90,61) N and 335,42 (88,34) N respectively. Panavia F presented the higher tensile bond strength results and zinc phosphate the lower between the tested groups. Only A17ZP group was different from control group (p=.007) and the abutment taper has no influence in retentive values. Zinc phosphate showed an inhomogeneous cement line in SEM and CLSM analysis. The presence of MDP primer could be perceived in CLSM images of Panavia F group. Within the limitations of this in vitro study, the Panavia F presented the higher bonding strength between tested groups, however, all 3 cements tested were similar to control group except A17ZP group, showing that in the conditions of the study they all can be successfully used.Vários fatores devem ser considerados na seleção dos pilares angulados, assim, muitas vezes, o clínico deve encontrar uma forma prática de superar a falta de retenção resultante das características desse tipo de pilar. O objetivo deste estudo foi avaliar a resistência de união de coroas metálicas cimentadas sobre pilares personalizáveis retos e angulados, cimentados com diferentes agentes de cimentação definitivos. Noventa e um análogos de implantes regulares do tipo hexágono externo foram divididos em Grupo controle (C): pilar reto personalizável cimentado com fosfato de zinco (n = 10); três grupos (n = 10) com pilares angulados de 17° cimentados com fosfato de zinco (A17F), Panavia F (A17RM) e RelyX U100(A17R) e ainda mais três grupos (n = 10) com pilares angulados de 30° também cimentados com cimento de fosfato de zinco (A30F), Panavia F (A30RM) e RelyX U100 (A30R). Coroas de Níquel-Cromo foram confeccionadas e cimentadas em seus pilares correspondentes sendo que as coroas cimentadas com Panavia F foram pré-tratadas com um primer para metal contendo monômero ácido fosfatado (MDP). Dados de todos os grupos foram comparados ao grupo controle por meio da análise de variância ANOVA - One way (α =0,05) e teste de Dunnet, e a comparação entre os grupos testados foi realizada por meio de análise de variância ANOVA Two way (α =0,05) e teste de Tukey. Ainda as avaliações em MEV e MVCL foram realizadas com 3 amostras representativas de cada grupo com o objetivo de investigar as características microscópicas das interfaces pilar-coroa-cimento. A média de força (DP) necessária para deslocar as coroas nos grupos C, A17F, A17RM, A17R, A30F, A30RM e grupos A30R foram, respectivamente, 357,26 (62,21) N; 251,50 (20,13) N; 397, 05 (88,48) N; 328,71 (79,87) N; 276,70 (17,96) N; 377,81 (90,61) N e 335,42 (88,34) N. O cimento Panavia F apresentou os resultados mais elevados de resistência a tração (RT) e o fosfato de zinco apresentou os menores valores de RT entre os grupos testados. Apenas o grupo A17F foi estatisticamente diferente do grupo controle (p = 0,007) e a inclinação das paredes axiais do pilar não influenciou os valores de retenção obtidos. O fosfato de zinco apresentou uma linha de cimentação não homogênea na análise em MEV e MVCL. A presença de primer contendo MDP pode ser percebida nas imagens de MVCL do grupo Panavia F. Dentro das limitações deste estudo in vitro, o Panavia F apresentou a maior resistência de união entre os grupos testados, no entanto, todos os três cimentos testados foram semelhantes ao grupo controle, exceto o grupo A17F grupo, mostrando que nas condições do estudo todos eles podem ser usados com sucesso.
Mestre em Odontologia