The Diploma thesis deals with object surface reconstruction by means of confocal microscope. It includes part of mathematical theory which is associated with this problem. The mathematical algorithm of the object surface reconstruction is illustrated. This Diploma thesis result is the application development for this reconstruction. The thesis also include the results of specific object.

The diploma thesis deals with a design of illuminating system for tandem-scanning confocal microscope using a high-power LEDs.

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O mecanismo de formação e origem das alterações nucleares ainda é pouco conhecido, sendo o micronúcleo a mais estudada. As células tumorais geralmente apresentam vários tipos de alterações nucleares que estariam associadas à instabilidade genética. O objetivo deste trabalho foi analisar as possíveis associações entre alterações nucleares e citoesqueleto em células HK2 e A549, derivadas de carcinoma de pulmão humano. Otimizamos a metodologia de uso do MCVL para redefinir as alterações nucleares e caracterizar os principais filamentos do citoesqueleto em preparações coradas por Feulgen ou imunofluorescência. As células da linhagem HK2 apresentaram fibras de actina dispostas concentricamente e em "clusters" e os filamentos de tubulina apareceram de forma radial, enquanto que o padrão de distribuição em A549 foi mais semelhante ao das células normais (BRL3A). Os filamentos de lamina B foram os mais importantes para evidenciar as alterações nucleares, porém essas alterações não puderam ser relacionadas com alterações do citoesqueleto. / The origin and mechanism of formation of the nuclear alterations is largely unknown, with the micronucleus being the most well studied alteration. Tumor cells generally present various types of nuclear alterations witch can be associated with genetic instability. The propose of this study was to analyze the possible association between nuclear alterations and the cytoskeleton in the human lung carcinoma cells HK2 and 549. The method of LSM was optimized to redefine the nuclear alterations and to characterize the principal cytoskeletal filaments in preparations stained with Feulgen’s reagent or submitted to imunofluorescent methods. The HK2 cells presented actin fibres arranged either concentrically or in clusters and tubulin filaments arranged radially, while in the A549 cells the distribution pattern was similar to that of normal cells (BRL3A). The lamin B filaments were the most important to identify nuclear alterations, as these alterations could not be related to cytoskeletal alterations.

O mecanismo de formação e origem das alterações nucleares ainda é pouco conhecido, sendo o micronúcleo a mais estudada. As células tumorais geralmente apresentam vários tipos de alterações nucleares que estariam associadas à instabilidade genética. O objetivo deste trabalho foi analisar as possíveis associações entre alterações nucleares e citoesqueleto em células HK2 e A549, derivadas de carcinoma de pulmão humano. Otimizamos a metodologia de uso do MCVL para redefinir as alterações nucleares e caracterizar os principais filamentos do citoesqueleto em preparações coradas por Feulgen ou imunofluorescência. As células da linhagem HK2 apresentaram fibras de actina dispostas concentricamente e em "clusters" e os filamentos de tubulina apareceram de forma radial, enquanto que o padrão de distribuição em A549 foi mais semelhante ao das células normais (BRL3A). Os filamentos de lamina B foram os mais importantes para evidenciar as alterações nucleares, porém essas alterações não puderam ser relacionadas com alterações do citoesqueleto. / The origin and mechanism of formation of the nuclear alterations is largely unknown, with the micronucleus being the most well studied alteration. Tumor cells generally present various types of nuclear alterations witch can be associated with genetic instability. The propose of this study was to analyze the possible association between nuclear alterations and the cytoskeleton in the human lung carcinoma cells HK2 and 549. The method of LSM was optimized to redefine the nuclear alterations and to characterize the principal cytoskeletal filaments in preparations stained with Feulgen’s reagent or submitted to imunofluorescent methods. The HK2 cells presented actin fibres arranged either concentrically or in clusters and tubulin filaments arranged radially, while in the A549 cells the distribution pattern was similar to that of normal cells (BRL3A). The lamin B filaments were the most important to identify nuclear alterations, as these alterations could not be related to cytoskeletal alterations.

This thesis focuses on advanced methods of detecting contours of the cardiac cells and measuring their contraction. The theoretical section describes the types of confocal microscopes, which are used for capturing biological samples. The following chapter is devoted to the methods of cardiac cells segmentation, where we are introduced to the generally applied approaches. The most widely spread methods of segmentation are active contours and mathematical morphology, which are the crucial topics of this thesis. Thanks to the those methods we are able in the visual data to accurately detect required elements and measure their surface chnage in time. Acquired theoretical knowledge leads us to the practical realization of the methods in MATLAB.

Les opérations de hernies abdominales sont l’une des chirurgies les plus répandues dans le monde. Pourtant, malgré des progrès considérables en particulier dans le développement des textiles prosthétiques pour consolider la paroi abdominale, le taux de récurrence reste très élevé. Il apparaît donc nécessaire de développer des modèles numériques patient-spécifiques de la paroi abdominale afin de mesurer puis améliorer l’impact des solutions de soins. Les tissus assurant la cohésion et la stabilité de la paroi abdominale sont les gaines rectusiennes antérieure et postérieure et la ligne blanche, qui sont des tissus conjonctifs. Leur structure particulière, composée d’un entremêlement de fibres de collagène et d’élastine sont au cœur de cette étude.Ce mémoire a permis de mettre en évidence la relation entre architecture microscopique et comportement macroscopique des tissus fibreux. Grâce à des campagnes expérimentales combinant essais mécaniques et observations microscopiques, la structure des tissus a pu être identifiée et corrélée aux paramètres mécaniques. Des outils d’analyse d’images tridimensionnelles ont été développés afin d’estimer automatiquement l’anisotropie d’une texture. Les informations recueillies ont alors menées au développement d’un modèle constitutif anisotrope hypo-paramétré. Basé sur une description tridimensionnelle du réseau fibrillaire, le modèle a été écrit de manière à décorréler les paramètres liés à la structure et ceux relatifs à la nature même du matériau. En considérant les paramètres matériau similaires pour tous les individus, le modèle a démontré sa capacité à prédire le comportement mécanique à partir d’informations texturales / Abdominal hernia operations are one of the most common surgeries in the world. However, despite considerable progress, particularly in the development of prosthetic textiles to strengthen the abdominal wall, the recurrence rate remains very high. It therefore appears necessary to develop patient-specific numerical models of the abdominal wall in order to measure and improve the impact of care solutions. The tissues that ensure the cohesion and stability of the abdominal wall are the anterior and posterior rectus sheaths and the linea alba, which are connective tissues. Their particular structure, composed of an intertwining of collagen and elastin fibers, is at the heart of this study. This thesis highlighted the relationship between microscopic architecture and macroscopic behaviour of fibrous tissues. Thanks to experimental campaigns combining mechanical tests and microscopic observations, the structure of tissues has been identified and correlated to mechanical parameters. Three-dimensional image analysis tools have been developed to automatically estimate the anisotropy of a texture. The collected information then led to the development of a hypo-parameterized anisotropic constitutive model. Based on a three-dimensional description of the fibrillary network, the model was written in such a way as to uncorrelate the parameters related to the structure and those related to the nature of the material. By considering similar material parameters for every individual, the model demonstrated its ability to predict mechanical behaviour based on textural information

Visualization is used for stainless steel container wall and lid cross section characterization. Two specific types of containers are examined: 3013 and SAVY. The container wall examined is from a sample of the inner container of a 3013 container. The inner lid cross section examined is from a SAVY container. Laser confocal microscope data and photographic data are used to determine features of the surfaces. The surface features are then characterized by various feature statistics, such as, maximum depth, area, eccentricity, and others. The purpose of this pilot study is to demonstrate the effectiveness of using the methodology to detect potential corrosion events on the inner container surfaces. The features are used to quantify these corrosion events. An automatic image analysis system uses this methodology to classify images for possible further human analysis by flagging possible corrosion events. A manual image analysis methodology is used to determine the amount of MnS on the SAVY container lid cross section. Visualization is an integral component of the analysis methodology.

Indiana University-Purdue University Indianapolis (IUPUI) / Plasma membranes are complex supramolecular assemblies comprised of lipids and membrane proteins. Both types of membrane constituents are organized in highly dynamic patches with profound impact on membrane functionality, illustrating the functional importance of plasma membrane fluidity. Exemplary, dynamic processes of membrane protein oligomerization and distribution are of physiological and pathological importance. However, due to the complexity of the plasma membrane, the underlying regulatory mechanisms of membrane protein organization and distribution remain elusive. To address this shortcoming, in this thesis work, different mechanisms of dynamic membrane protein assembly and distribution are examined in a polymer-tethered lipid bilayer system using comple-mentary confocal optical detection techniques, including 2D confocal imaging and single molecule-sensitive confocal fluorescence intensity analysis methods [fluorescence correlation spectroscopy (FCS) autocorrelation analysis and photon counting histogram (PCH) method]. Specifically, this complementary methodology was applied to investigate mechanisms of membrane protein assembly and distribution, which are of significance in the areas of membrane biophysics and cellular mechanics. From the membrane biophysics perspective, the role of lipid heterogeneities in the distribution and function of membrane proteins in the plasma membrane has been a long-standing problem. One of the most well-known membrane heterogeneities are known as lipid rafts, which are domains enriched in sphingolipids and cholesterol (CHOL). A hallmark of lipid rafts is that they are important regulators of membrane protein distribution and function in the plasma membrane. Unfortunately, progress in deciphering the mechanisms of raft-mediated regulation of membrane protein distribution has been sluggish, largely due to the small size and transient nature of raft domains in cellular membranes. To overcome this challenge, the current thesis explored the distribution and oligomerization of membrane proteins in raft-mimicking lipid mixtures, which form stable coexisting CHOL-enriched and CHOL-deficient lipid domains of micron-size, which can easily be visualized using optical microscopy techniques. In particular, model membrane experiments were designed, which provided insight into the role of membrane CHOL level versus binding of native ligands on the oligomerization state and distribution of GPI-anchored urokinase plasminogen activator receptor (uPAR) and the transmembrane protein αvβ3 integrin. Experiments on uPAR showed that receptor oligomerization and raft sequestration are predominantly influenced by the binding of natural ligands, but are largely independent of CHOL level changes. In contrast, through a presumably different mechanism, the sequestration of αvβ3 integrin in raft-mimicking lipid mixtures is dependent on both ligand binding and CHOL content changes without altering protein oligomerization state. In addition, the significance of membrane-embedded ligands as regulators of integrin sequestration in raft-mimicking lipid mixtures was explored. One set of experiments showed that ganglioside GM3 induces dimerization of α5β1 integrins in a CHOL-free lipid bilayer, while addition of CHOL suppresses such a dimerization process. Furthermore, GM3 was found to recruit α5β1 integrin into CHOL-enriched domains, illustrating the potential sig-nificance of GM3 as a membrane-associated ligand of α5β1 integrin. Similarly, uPAR was observed to form complexes with αvβ3 integrin in a CHOL dependent manner, thereby causing the translocation of the complex into CHOL-enriched domains. Moreover, using a newly developed dual color FCS and PCH assay, the composition of uPAR and integrin within complexes was determined for the first time. From the perspective of cell mechanics, the characterization of the dynamic assembly of membrane proteins during formation of cell adhesions represents an important scientific problem. Cell adhesions play an important role as force transducers of cellular contractile forces. They may be formed between cell and extracellular matrix, through integrin-based focal adhesions, as well as between different cells, through cadherin-based adherens junctions (AJs). Importantly, both types of cell adhesions act as sensitive force sensors, which change their size and shape in response to external mechanical signals. Traditionally, the correlation between adhesion linker assembly and external mechanical cues was investigated by employing polymeric substrates of adjustable substrate stiffness containing covalently attached linkers. Such systems are well suited to mimic the mechanosensitive assembly of focal adhesions (FAs), but fail to replicate the rich dynamics of cell-cell linkages, such as treadmilling of adherens junctions, during cellular force sensing. To overcome this limitation, the 2D confocal imaging methodology was applied to investigate the dynamic assembly of N-cadherin-chimera on the surface of a polymer-tethered lipid multi-bilayer in the presence of plated cells. Here, the N-cadherin chimera-functionalized polymer-tethered lipid bilayer acts as a cell surface-mimicking cell substrate, which: (i) allows the adjustment of substrate stiffness by changing the degree of bilayer stacking and (ii) enables the free assembly of N-cadherin chimera linkers into clusters underneath migrating cells, thereby forming highly dynamic cell-substrate linkages with remarkable parallels to adherens junctions. By applying the confocal methodology, the dynamic assembly of dye-labeled N-cadherin chimera into clusters was monitored underneath adhered cells. Moreover, the long-range mobility of N-cadherin chimera clusters was analyzed by tracking the cluster positions over time using a MATLAB-based multiple-particle tracking method. Disruption of the cytoskeleton organization of plated cells confirmed the disassembly of N-cadherin chimera clusters, emphasizing the important role of the cytoskeleton of migrating cells during formation of cadherin-based cell-substrate linkages. Size and dynamics of N-cadherin chimera clusters were also analyzed as a function of substrate stiffness.