This diploma thesis deals with analysing of modern methods for cell detection, visualization and quantification in 3D space. The first section deals with optical methods for cells detection. There is detailed discussion about cell labeling and detection on confocal microscopy. There is also description about developed algorithm for whole cell volume quantification from microscopy images. This could made a comparsion of fluorescence signal according to time of cell labeling and according to cell shapes. There was also optimalization of handmade tissue phantoms visualization. It could be compared a possibilities of cell detections in these phantoms by confocal microscopy and OCT. It was also implemented algorithm for quantification of cells from OCT images. Besides confocal microscopy and OCT cells are also analyzed by other methods. The last part is the Conclusion of results and comparison of used methods.

The mechanisms of invazivity and regulation of transcription of cancer cells Cancer originates in cells that overcome the control mechanisms of the organism. Cancer cells can be eventually released from the site of origin and spread through tissues. Cancer cells can acquire certain mechanisms that enable them to more effectively invade surrounding tissue or layers of other cells. The research on the migration of cancer cells is important for the understanding of the origin and spreading of metastases and consequently for anticancer therapy. In my Ph.D. work, I participated in the research of the properties of invasive metastatic cells. We compared non-invasive rat sarcoma cell line with a higly metastatic cell line derived from it. We showed that cells of the invasive cell line use amoeboid mode of migration, have upregulated Rho/ROCK signaling, and have accumulated actin and myosin at the leading edge. It is at the leading edge where the cells generate their traction forces. Cells of non-invasive cell line use mesenchymal mode of migration and generate forces mainly at their retracting end. We also compared two breast cancer cell lines derived from a single carcinoma. We showed that the more invasive cell line, derived from its parental line by neoplastic transformation, displayed elevated cytoskeletal...

Zuzana Kubínová: Study of Quantitative Parameters of Norway Spruce Needle Structure under the Effect of Elevated CO2 Concentration and Different Irradiance, Doctoral Thesis, Prague 2019 Abstract Atmospheric concentration of CO2 is increasing, while its influence on plants is still not fully elucidated. Norway spruce (Picea abies L. Karst.) is an abundant conifer tree in European temperate and boreal forests, which behave as carbon sink in the global carbon cycle. The physiological response to elevated CO2 concentration may be interconnected with changes in leaf anatomy and morphology. Needle structure is also determined by other factors in addition to CO2 concentration, irradiance being the most important one. Thus, effect of irradiance was also included in our studies. The effects of elevated CO2 concentration and irradiance on Norway spruce needle structure were studied using new applications of well-established quantitative methods and novel methods enabling effective and unbiased analysis of needle structural traits. The General Procrustes analysis showed to be effective for needle shape on cross section comparison and the disector method proved to be suitable for chloroplast number estimates. The influence of elevated CO2 concentration and different irradiance on needle structure was studied at two...

Ce travail a été réalisé dans l’I. R. P. H. E. (Institut de Recherche sur les Phénomènes Hors Équilibre), unité de recherche de l’Université d’Aix-Marseille en collaboration avec l’Université de la Sarre, la Faculté de Physique Expérimentale. Cette étude est consacrée à une meilleure compréhension de la microcirculation du sang in vitro, ainsi que des phénomènes collectifs qui prennent place dans la microcirculation. Il se concentre principalement sur la margination en fonction du contrast de rigidité dans une suspension de globules rouges. L’expérience modale a été développée pour étudier la margination, causée exclusivement par le contraste de la déformabilité entre les deux sous-populations de globules rouges: les saines et les rigidifiées / This work was carried out in collaboration between I.R.P.H.E. (Institut de Recherche sur les Phénomènes Hors Équilibre), research unit of Aix-Marseille University and University of Saarland, Faculty of Experimental Physics (Naturwissenschaftlich-Technische Fakultät der Universität des Saarlandes) and aims to investigate microcirculatory hydrodynamics of blood in vitro. The study is dedicated to better understanding of complex collective phenomena that take place in microcirculation of blood through microfluidic in vitro experiments. It mainly focuses rigidity based margination in suspension of RBCs. For this purpose, model experiment was developed to examine margination caused exclusively by contrast of deformability between two sub-populations of RBCs

A stimuli-responsive microgel is a three-dimensional polymer network that is able to absorb and expel a solvent (commonly water). These materials are unique in the fact that their sponge-like behavior can be actuated by environmental cues, like temperature, ion concentration, pH, and light. Because of the dynamic properties of these materials they have found applications in drug-delivery systems, micro-assays, selective filtration, artificial muscle, and non-fouling surfaces. The most well-known stimuli-responsive polymer is Poly(N-isopropylacrylamide) or PNIPAAm and it experiences a switchable swelling or deswelling over a critical temperature ( Tc=~32°C). Below the critical temperature, the gel begins mixing with the surrounding solvent and swells; above this temperature, the opposite is true. The unconstrained hydrogel will continue to swell in all directions until equilibrium is established between its propensity for mixing with the surrounding solvent and the elastic restoring forces of the gel matrix. The strength of the elastic restoring forces is dependent on the interconnectedness of the polymer network and is therefore a function of crosslink density. An increase in crosslink density results in a decreased swelling and vice versa. If the hydrogel is mechanically constrained to a surface, it can experience various wrinkling and buckling conformations upon swelling, as the stresses associated with its confinement are relieved. These conformation characteristics are a strong function of geometry (aspect ratio) and extent of swelling (i.e. crosslink density). In order to capitalize on the utility of this material, it is imperative that its volume transition is well characterized and understood. Toward this end, pNIPAAm gels have been created with 1x10-7 to 2x10-³ mol/cm³ crosslink density and characterized. This was done by first examining its bulk, unattached swelling ability and then by evaluating its microscale properties as a surfaceconfined monolithe. The latter was achieved through the use of confocal microscopy and copolymerization with a fluorescent monomer. This method allows for a detail analysis of the deformations experienced (bulk-structural bending and surface undulating) and will ultimately lend itself to the correlation between crosslink density and the onset of mechanical phenomena.

Sucrose is the major transported photoassimilate in plants and is degraded concurrently by two enzymes: invertases and sucrose synthase. Sucrose synthase catalyzes the reversible conversion of UDP and sucrose to form fructose and UDP-glucose, the latter being the activated substrate for many metabolic processes including cellulose biosynthesis. There is evidence that sucrose synthase is phosphorylated as a regulatory mechanism of carbon allocation at a conserved N-terminal serine residue. The phosphorylation or dephosphorylation at this specific site has also been found to shift the protein localization in a tissue and species specific manner. A literature study of the functional regulation of sucrose synthase in plants has highlighted several scientific questions: Is sucrose synthase cellular localization regulated by phosphorylation of an N-terminal conserved serine residue? What are the regulatory mechanisms underlying within and between species variation in sucrose synthase localization? Does sucrose synthase associate with the cellulose synthase enzyme complex? Can cellulose biosynthesis be increased by over-expression of the membrane-associated form of sucrose synthase? The aim of this M.Sc study was to determine the subcellular localization of Eucalyptus grandis sucrose synthase 1 (EgSUSY1) fusion proteins expressed in Arabidopsis thaliana plants. This was investigated through modifying the 11th serine residue of EgSUSY1 into either a non-polar alanine residue that cannot be phosphorylated (S11A), or into a negatively charged glutamic acid residue which may mimic phosphorylation at this site (S11E). The modified proteins were translationally fused to green fluorescent protein (GFP) and expressed in transgenic Arabidopsis thaliana. The proteins’ subcellular localization were analysed in planta using laser scanning confocal microscopy (LSCM). Findings in this study point to the peripheral localization of modified and unmodified GFPEgSUSY1 proteins with a prominent cytoplasmic component. No evidence was found for the localization of modified or unmodified GFP-EgSUSY1 proteins within the extracellular matrix. The current study did not establish nor negate plasma membrane association of any of the GFP-EgSUSY1 fusion proteins. It was concluded that alternative methodologies need to be explored to further address issues surrounding subcellular localization of sucrose synthase. These studies will not only aid in defining the role of this enzyme in carbon allocation, but also add to our expanding knowledge of cellulose biosynthesis and cell wall formation. Copyright 2011, University of Pretoria. All rights reserved. The copyright in this work vests in the University of Pretoria. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of the University of Pretoria. Please cite as follows: Sauer, J 2011, The subcellular localization of eucalyptus grandis sucrose synthase 1 (EgSUSY1) fusion proteins expressed in Arabidopsis thaliana, MSc dissertation, University of Pretoria, Pretoria, viewed yymmdd < http://upetd.up.ac.za/thesis/available/etd-02102012-102209 / > C12/4/111/gm / Dissertation (MSc)--University of Pretoria, 2011. / Genetics / unrestricted

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To understand the role of nonmuscle myosin II in cardiac and skeletal muscle, we used a number of polyclonal antibodies, three detecting nonmuscle myosin heavy chain II-B (NMHC II-B) and two detecting NMHC II-A, to examine the localization of these two proteins in fresh-frozen, acetone-fixed sections of normal human and mouse hearts and human skeletal muscles. Results were similar in both species and were confirmed by examination of fresh- frozen sections of human hearts subjected to no fixation or to treatment with either 4% p-formaldehyde or 50% glycerol. NMHC II-B was diffusely distributed in the cytoplasm of cardiac myocytes during development, but after birth it was localized to the Z-lines and intercalated discs. Dual labeling showed almost complete colocalization of NMHC II-B with α-actinin. Whereas endothelial cells, smooth muscle cells and fibroblasts showed strong immunoreactivity for NMHC II-A and NMHC II-B, cardiac myocytes only showed reactivity for the latter. The Z-lines of human skeletal muscle cells, in contrast to those of cardiac myocytes, gave positive reactions for both NMHC II-A and NMHC II-B. The presence of a motor protein in the Z-lines and intercalated discs raises the possibility that these structures may play a more dynamic role in the contraction/relaxation mechanism of cardiac and skeletal muscle than has been previously suspected.

Corrosion-casted capillary systems of the kidney glomerulus were imaged with confocal microscopy because of the fluorescence properties of the casting plastic. Acquisition of a z-series through the glomerular capillaries provided three-dimensional data sets from which surface-rendered models were generated. These models could be rotated and viewed from any angle and also contained quantitative information allowing cast surface area and volume measurements to be calculated. The computer-generated models were also skeletonized to form a one-voxel-thick skeleton of the original model. The skeleton exhibited the three-dimensional topology and network of the capillary bed, and interior capillary relations could also be viewed. Quantitative information such as the total capillary length and number of capillary intersects was calculated from the skeletonized model. Extending this method to noncorroded kidney specimens revealed not only the casted vessels but also cellular features of the adjacent tissues surrounding the capillaries.

Breaking Rayleigh’s "curse" and resolving infinitely small spatial separations is one motivation for developing super-resolution in imaging systems. It has been shown that an arbitrarily small distance between two incoherent point sources can be resolved through the use of a spatial mode sorter, by treating it as a parameter estimation problem. However, when extending this method to general objects with many point sources, the added complexity of multi-parameter estimation problems makes resolution of general objects quite challenging. In the first part of this thesis, we propose a new approach to deal with this problem by generalizing the Richardson-Lucy (RL) deconvolution algorithm to accept the outputs from a mode sorter. We simulate the application of this algorithm to an incoherent confocal microscope using a Zernike spatial mode sorter rather than the conventional pinhole. Our method can resolve general scenes with arbitrary geometry. For such spatially incoherent objects, we find that the resolution enhancement of the sorter-based microscopy using the generalized RL algorithm is over 30% higher than the conventional confocal approach using the standard RL algorithm. This method is quite simple and potentially can be used for various applications including fluorescence microscopy. It could also be combined with other super-resolution techniques for enhanced results. The second part of this thesis explores the potential for the Herriott cell to be used as an image-preserving delay line. In quantum imaging, entangled photons are often utilized to take advantage of their spatial and temporal correlations. One photon (“the signal”) interacts with the system to be measured and the other (“the herald”) is used to trigger the detection of the signal. However, for a typical high-sensitivity camera, there is a delay on the order of 20 ns between the trigger and the sensor becoming active allowing for the signal to be recorded. An image-preserving delay line (IPDL) serves to store a photon without distorting the spatial structure and losing the spatial and temporal correlations. It is commonly made with a series of 4f systems to repeatedly image the light field. We propose to use the Herriott cell as an image-preserving delay line. We tested 10 of the lower-order HG modes and found it was able to preserve almost all of them with high fidelities (>90%), with the only exceptions being the largest modes (HG03 and HG30) at the longest delay (7.9 m) where the fidelity was still >86%. In addition to these modes, it was also able to store general images. This application of the Herriott cell affords insights into miniaturizing IPDLs, which tend to occupy a significant amount of space. Overall, these two projects offer novel insight and application to the world of quantum imaging.