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Bioorthogonal labelling of surface receptors on living lymphocytes
Paldusová, Kateřina ; Cebecauer, Marek (advisor) ; Benda, Aleš (referee)
The surface of cells displays high heterogeneity on chemical and geometrical levels. To understand the function of cells, we need to pay attention to the morphological features formed at the plasma membrane. To study cell surface with molecular specificity, there are plenty of imaging methods starting with the conventional wide-field microscopy through confocal microscopy, ending with super-resolution fluorescence microscopies and electron microscopies. Super-resolution microscopy studies conducted on the fixed cells provide detailed steady-state data about the cell surface nanoscopic organisation and distribution of molecules at the morphological structures. However, since cells are parts of living organisms and constantly change their properties in time and space, the information about dynamics of cellular structures and motility of molecules remains hidden when using this approach. Live-cell compatible methods are required to study dynamic changes of molecules at the single-molecule level. In this study we are focusing on the distribution and dynamics of molecules CD2 and CD4 expressed on the surface of non-stimulated T cells. The main aim of this thesis was to develop a novel method for live-cell imaging and single-molecule tracking of membrane-bound proteins in 3D and at nanoscale. With such a...
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Combining the light of two sources in the integration sphere
Mellová, Katarína ; Šicner, Michal (referee) ; Dostál, Zbyněk (advisor)
For the development of the new generation of the controlled holographic microscope, is necessary to design a new fluorescence module. The work discusses the basic principles and application of fluorescence microscopy, including a description of the holographic microscope. Searching for available light sources used in fluorescence microscopy is performed and evaluated. The designed source combines radiation with selected excitation wavelengths using an integrating sphere, which also has a homogenizing effect. This innovative design ensures a quick and efficient change of excitation wavelengths due to the fluorophore used and the possibility of a direct connection of the source to the microscope. In order to choose a suitable arrangement for the source, three conceptual designs of its function are proposed. The selected designs were verified by simulation and suitable optical components were selected. The resulting construction is tested.
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Emission difference in superresolution fluorescence microscopy
Havelka, Tomáš ; Kollárová, Věra (referee) ; Bouchal, Petr (advisor)
Fluorescence microscopy finds broad applications in biological sciences as well as in technical research. Due to the complete spatial incoherence of the radiation emitted from the sample, fluorescence microscopy serves as the foundation for a range of super-resolution techniques. The bachelor's thesis is focused on the method of emission difference. This method utilizes the difference between standard and vortex image to achieve super-resolution. In this work, a q-plate is used to create the vortex image. The q-plate is a liquid crystal device that replaces costly spatial light modulators used in previous implementations of the method. The method of emission difference is described in the bachelor's thesis through theoretical calculations and numerical modeling at both the point and two-dimensional imaging levels. The experimental part of the work involves the preparation of an optical setup for the practical testing of the investigated method. The main result of the thesis is the original implementation of the method of emission difference using a q-plate and the verification of its capability to achieve super-resolution in imaging fluorescence nanoparticles.
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Kalibrace fluorescenčního optického mikroskopu s pomocí elektronového obrazu
Erlich, Lukáš ; Polášek, Tomáš (referee) ; Čadík, Martin (advisor)
This master’s thesis deals with the topic of image correction in fluorescence optical microscopy using electron image as a template. The aim is to design methods to analyze observed distortions in investigated microscopic systems and propose techniques for their correction. The thesis also presents a calibration sample that is utilized within it. Additionally, the work provides an overview of some existing solutions for modeling and suppressing distortion.
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Fluorescence spectroscopy in research on the organization of biological membranes
Vítek, Ondřej ; Obruča, Stanislav (referee) ; Mravec, Filip (advisor)
This bachelor's thesis deals with the comparison and optimization of methods for studying biological membranes using fluorescence spectroscopy and microscopy, utilizing the bacteria Cupriavidus necator H16 (containing PHB granules) and PHB-4 (not containing PHB granules) as model organisms. The appropriate concentration of the membrane probe laurdan for staining biological membranes and the time required for its penetration into the cell were determined. Organisms stained in this way were further studied using steady-state spectroscopy, which helped examine the influence of temperature on the shape of the emission spectrum and the possibility of washing as one of the steps in sample preparation. Both of these measurements suggest the advantages of washing but do not provide definite results. However, a clear difference between the two bacterial strains was observed. Furthermore, the bacteria were studied by fluorescence microscopy. This method proved to be potentially useful for studying significant variations in the membrane order of the cell membrane and for imaging other larger structures. The distribution of laurdan within Cupriavidus necator H16 appears more uneven compared to PHB-4. The findings thus suggest the influence of granule presence when staining with fluorescent probes, but they cannot be interpreted definitively.
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Molecular mechanisms of the assembly and function of BBSome
Prasai, Avishek ; Huranová, Martina (advisor) ; Varga, Vladimír (referee) ; Bosáková, Michaela (referee)
Bardet Biedl syndrome is a genetic disorder caused by the dysfunction of the BBSome, an octameric cargo adaptor protein complex. The BBSome facilitates the transport of signaling receptors into and out of the primary cilium, a microtubule based sensory organelle of the cell. The first part of this thesis focuses on the elucidation of the assembly of the BBSome in living cells. We generated a library of human and mouse cells lines deficient in the individual BBSome subunits and transduced them with the other YFP tagged subunits. We employed biochemical assays, immunofluorescence and quantitative fluorescence microscopy techniques to analyze the individual steps in the BBSome assembly pathway. We revealed that the BBSome assembly occurs sequentially in spatially regulated steps. We showed that BBS4 nucleates the assembly of a pre-BBSome at the pericentriolar satellites. The translocation of the pre-BBSome to the ciliary base is facilitated by BBS1. We also revealed that in a BBS chaperonin deficient cell line, BBS12 KO cells, a small fraction of the BBSome and/or BBSome sub-complexes are still able to form and localize to the cilium. This could suggest that the BBS chaperonins might act later in the BBSome assembly pathway providing a means for quality control for the BBSome. Ciliary ectocytosis...
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Utilisation of fluorescence techniques for analysis of industrially relevant microorganisms
Müllerová, Lucie ; Kráčmar, Stanislav (referee) ; Krčma, František (referee) ; Obruča, Stanislav (advisor)
Techniky analýzy jednotlivých buněk neboli single-cell analýzy, jsou velmi důležité v kontextu studia důležitých biotechnologických mikroorganismů. V biotechnologických procesech je optimální mít rychlé, efektivní a real-time analýzy, které lze provést s minimální přípravou vzorku. Proto jsme v rámci předložené disertační práce využili autofluorescenci jako marker fyziologického stavu mikrobiální kultury, který je možné analyzovat pomocí fluorescenční mikroskopie a/nebo průtokové cytometrie. Nejdříve bylo potvrzeno, na základně emisních spekter, že zelená autofluorescence emitovaná bakteriemi Cupriavidus necator H16 a jejím polyhydroxyalkanoáty (PHA) neprodukujícím mutantem Cupriavidus necator PHB-4, pochází skutečně z flavinů. Emisní maximum bylo naměřeno v rozmezí 510–550 nm pro FAD, FMN a riboflavin – bohužel jednotlivá spektra od sebe nebylo možné rozeznat, a 515 nm pro oba bakteriální kmeny Cupriavidus necator. Byly nalezeny dvě maxima excitačních spekter, jedno v rozmezí 360–370 nm a druhé okolo 440 nm. Z těchto poznatků byl sestaven nový protokol pro fluorescenční mikroskopii – laser byl nastaven na hodnotu 467 nm a emisní filtr pro detekci byl vybrán 520/35 nm. Flaviny a jejich autofluorescence může být take použita v kombinaci s jinými fluorofory, když jsou použity multiparametrické analýzy, ale je důrazně doporučeno, aby vybrané fluorescenční sondy buď emitovaly v jiném rozmezí, než je 500–550 nm anebo byla jejich doba života excitovaného stavu jiná, než je interval 3,2–3,7 a 4,2 – 4,9 ns. Tyto doby života navíc zůstaly konstantní, i když byly buňky vystaveny kultivačním a stresovým podmínkám. To z nich dělá velmi stabilní marker v porovnání s dynamickým charakterem intenzit fluorescence. Dále byl optimalizován protokol pro značení bakterie C. necator s využitím PHA specifické sondy BODIPY 493/503. Optimální koncentrace této lipofilní sondy byla stanovena na 2,5 µg ml-1 a sondu je možné dle našich dat aplikovat společně s viabilitní sondou propidium iodidem. Tento protokol byl také využit pro studium morfologie PHA-syntetizující halofilní bakterie Halomonas halophila. Pro stanovení UV-protektivních vlastností PHA obsaženého v bakterii Cupriavidus necator, byl optimalizován další protokol, tentokrát pro ROS citlivou sondu CM-H2DCFDA, která byla stanovená jako citlivější v porovnání s její nemethylovanou formou. V této studii byly navíc potvrzeny UV-protektivní vlastnosti PHA na bakterii Cupriavidus necator H16 v porovnání s PHA nesyntetizujícím kmenem Cupriavidus necator PHB-4.
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Tracking membrane permeabilization on single lipid vesicles - method development.
Gücklhorn, David ; Šachl, Radek (advisor) ; Heřman, Petr (referee)
Protein complexes are challenging systems to study, especially when these complexes form on lipid membranes only for a short period of time. This is also the case of fibroblast growth factor 2 (FGF2), a protein that has many physiological and pathological functions in the human organism. It plays major role in the development of cancer as it promotes cell survival and angiogenesis. It also serves as a basis for development of novel treatments of nerve injuries. Despite being heavily studied for many years, it remains unclear how the protein is translocated into the extracellular space where it performs its function. To study complex systems such as FGF2 that self-assembles on the membrane into membrane penetrating pores we decided to develop a simple and efficient fluorescent microscopy method. This method is called double leakage single GUV assay (DLSGA). It utilizes giant unilamellar vesicles (GUVs) mimicking native cellular membranes. In a single experiment, up to 300 individual GUVs are imaged for the content of a leakage dye that reports on the presence of FGF2 pores. During three measurements and under different conditions, detailed information about pore-opening dynamics is gained for each GUV. Results of these measurements are then used to divide GUVs into six groups based on formation and...
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Segmentation of phase contrast images in multi epitope ligand cartography (MELC) for image quantification at the single cell level
Mívalt, Filip ; Taschner-Mandl,, Sabine (referee) ; Mehnen, Lars (advisor)
Technologie Multi-Epitope Ligand Cartography (MELC) umožňuje mikroskopickou vizualizaci více buněčných kompartmentů za pomocí více imunofluorescenčních barviv. Tato pipeline tudíž umožňuje charakterizaci fenotypu pro všechny buňky nacházející se ve vzorcích kostní dřeně u pacientů s neuroblastomem. Přiřazení protilátkového signálu k odpovídající plazmatické membráně jednotlivých buněk je založeno na segmentaci buněčného jádra a algoritmu region growing, jenomže tahle metoda skutečný tvar buňky pouze aproximuje. Tento přístup je mimořádně chybový, pokud je aplikován na překrývající se buňky kvůli nejednoznačnému přiřazení jednoho protilátkového signálu více buňkám. Následně se pak tato chyba dostává až do popisných parametrů charakterizujících každou buňku zvláště, čímž může být ovlivniněna navazující klasifikace nebo kvantifikace buněčného fenotypu. Z toho důvodu je vyžadována segmentace fázově kontrastních obrazů, které jsou pořízeny současně s každým fluorescenčním snímkem, a zobrazují celou buňku (včetně cytoplazmy a jádra). Tato segmentace poskytuje přesné segmentační masky reprezentující celou buňku. Implementovali jsme automatizovanou strategii pro segmentaci těchto fázově kontrastních obrazů využívajíce Mask R-CNN. Algoritmus dosáhl celkového F1 skóre (pro detekci objektů) 0.935 a F1 skóre (pro klasifikaci na úrovni pixelů) 0.868, přičemž byl trénován pouze s malým anotovaným datasetem. Natrénovaný model byl implementován do existující pipeline pro zpracování MELC dat. Kromě toho, poskytujeme anotovaný dataset čítající 54 fázově kontrastních obrazů Cytospin preparátů kostní dřeně obsahující celkově 1 940 buněk. Implementovaný model Mask R-CNN umožňuje studovat popisné parametry charakterizující každou buňku zvláště za použití segmentačních masek odvozených z fázově kontrastních obrazů, které reprezentující celou buňku a tímto tedy zlepšuje automatickou kvantitativní analýzu buněk nacházejících se v kostní dřeni ve výzkumu dětské rakoviny.
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Human 4E protein family in stress granules granules and their further characterization
Hrbková, Pavlína ; Frydrýšková, Klára (advisor) ; Hašek, Jiří (referee)
Eukaryotic initiation factor 4E (eIF4E) is a key part of initiation and regulation of translation in human cells. Three members of human eIF4E proteins have been characterized: eIF4E1, eIF4E2 and eIF4E3. Cellular stress causes translation initiation inhibition followed by disassembly of the polysomes, those processes are accompanied by the assembly of cytoplasmic RNA granules, called stress granules (SG). Stress granules are dynamic structures whose composition may vary depending on the cell type and the stress stimulus. In this study, human cells were subjected to the following stress conditions: high temperature (HS), sodium arsenite (AS) or hypoxia. Using fluorescence microscopy, pairs of human translational initiation factors from the 4E protein family were visualized and their localization to SG was assessed with one GFP- 4E incorporated in the stable cell line and the other one detected endogenously. Here we show eIF4E1 being a part of all the SGs, both in HS and AS conditions. Next, the eIF4E1 and eIF4E3 proteins together form more SGs than proteins eIF4E1, respectively eIF4E3, with eIF4E2. And last, that the presence of the particular 4E protein has no effect on the composition of SGs. Furthermore, selected groups of proteins were assessed for their potential to localize to the SGs under HS...
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