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Segmentation of Multi-Dimensional Multi-Parametric Microscopic Data of Biological Samples Using Convolutional Neural Networks
Backová, Lenka ; Benda, Aleš (advisor) ; Schätz, Martin (referee)
Multi-parametric highly dimensional images have become a standard way of imaging biological samples. To quantify results from these images, segmentation must be often applied first. However, due to the underlying shortcomings of the fluorescence microscopy of biological samples, i.e. low signal-to-noise ratio, convolutional neural networks have become widely used for automatization of the segmentation. Convolution neural networks showed to be versatile in their potential uses and able to segment complex images. In this work, we utilise neural network U-Net for segmentation of images, which contain not only intensity information, but fluorescence excited state lifetime information as well. We try different representations of the data to assess, whether the added information of the pixel values leads to improved performance. We present an application of the segmentation results with phasor analysis to study the fertility of mice sperm. 1
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Real-time monitoring of cellular processes - current approaches
Švecová, Iva ; Hodný, Zdeněk (advisor) ; Groušl, Tomáš (referee)
This thesis aims to provide an overview of real-time live-cell imaging methods with a focus on the signalling pathways. The first, most thorough section is about fluorescence methods and is followed by sections about bioluminescence and label-free methods. In the fluorescence section, we will at first introduce the types of fluorophores and respective labelling approaches. Subsequently, we will go through the individual techniques, starting with single-fluorophore and FRET biosensors, continuing with kinetic modelling approaches, a FLIM method used to detect changes in the cellular environment, and ending with two methods used to improve the resolution. With each technique, we will shortly explain the working principle and look at the examples at which this method was used. Finally, we will look at the example of live-cell imaging of one signalling cascade.
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Study of UV-generated fluorescent zinc complexes by fluorescence spectroscopy
Havlíková, Martina ; Vaculovičová, Markéta (referee) ; Mravec, Filip (advisor)
This thesis focuses on the study of UV light-generated zinc complexes with cadmium and organic molecules SAM, SAH, CYS, HCYS and GSSG, specifically at 375 nm. Furthemore, the aim of the work is to characterize the precursors spectrally and temporally before and after irradiation in the transilluminator at 250 nm. Study of genesis these complexes was performed by FLIM. Thanks to this method, it was found that the formation of complexes occurs only with Zn:SAH, Zn:GSSG and Zn:Cd. The formation of complexes is influenced by the method of preparation. The spectral characteristic was performed on a fluorimeter where the increase in fluorescence intensity of the irradiated solution with the precursors was expected. These were turbid solutions where sedimentation of the particles was observed and the intensity of fluorescence was changed. In the Zn:SAM and Zn:CYS sample, the sedimentation increased in intensity, while in Zn:SAH and Zn:HCYS decreased. The Zn:Cd precursor solution was clear and there was no change in intensity. Zn:Cd showed the best spectral properties, while the Zn:SAM sample, whose excitation and emission maxima are very close to each other, appeared to be the worst. A sample with Zn:CYS and Zn:HCYS showed almost the same spectra and respective peak results. Based on lifetime characteristics by TCSPC, the sample with Zn:CYS, Zn:HCYS and Zn:GSSG, which showed 3 lifetimes, was best treated. Lifetime could not be unambiguously determined for SAM and SAH samples. Zn:Cd had 4 lifetimes
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Correlation between steady-state fluorescence anisotropy measurement on fluorimeter and fluorescence microscope
Moslerová, Lenka ; Venerová, Tereza (referee) ; Mravec, Filip (advisor)
This Work concentrates on detection fluorescent anisotropy on a model system of fluorescent probe and glycerol. In this thesis, the ATTO 488 was used as a fluorescent substance and glycerol solutions of different concentrations were used to simulate different viscous environments. A fluorescence spectrofluorometer and confocal fluorescence microscope were used for the measurements. A linear encrease anisotropy with increasing viscosity of the environment was observed on fluorometer. The same trend was detected on fluorescence microscope. The values were compared and correlation factors were determined. The accuracy of the measurement was verified by calculations using the Perrin´s equation.
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Intrinsic fluorescence of bacteria Cupriavidus necator
Marková, Kateřina ; Obruča, Stanislav (referee) ; Mravec, Filip (advisor)
This thesis focuses on autofluorescence of flavins in gram-negative bacteria Cupriavidus necator H16 and its mutant strain PHB-4. The main methods used were fluorescence microscopy and flow cytometry. To confirm the presence of flavins, excitation and emission spectra of the bacterial suspension were measured, which were compared with flavin standards. In the part of testing cells without stress response, the autofluorescence of bacteria in PBS buffer and cell suspensions stained with fluorescence probe BODIPY 493/503 was measured. The ratio of short fluorescence lifetime to long autofluorescence lifetime, and its dependence on fluorescence probe was compared with previous conditions. Autofluorescence of the supernatant was measured; it was found that the relative amplitude of long lifetime was multiple times higher than in the cell. In the part devoted to the stress response, this thesis was focused on the amount of dissolved oxygen in the production medium and the effect on bacterial autofluorescence. Then differently concentrated hydrogen peroxide was used, the best results were obtained from the concentration of 100 mM in media. For comparison a combination of hydrogen peroxide with ferro-ammonium sulphate was used, but there was no big difference. Sodium azide and antimycin A were selected as substances that directly influence on bacterial respiratory chain. Both compounds affected change in the ratio of the relative amplitudes, but the distribution of these lifetimes and the autofluorescence change over time was affected only by sodium azide.
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Influence of lipid composition and model peptides on lateral organization of lipid layers
Veľas, Lukáš ; Heřman, Petr (advisor) ; Malínský, Jan (referee)
Oxidized phospholipids (OxPLs) are known to be present in living organisms due to oxidative stress. However, the physiological function of OxPLs is still not fully understood. They have been shown to be present in many inflammatory diseases such as atherosclerosis and neurodegenerative diseases like Parkinson's and Alzheimer's disease. In this work we present the influence of two truncated OxPLs on the lateral heterogeneity of a model lipid membrane. Specifically, we studied the effect of 1-palmitoyl-2-(5'-oxo-valeroyl)-sn-glycero-3- phosphocholine (POVPC) and 1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine (PGPC) on the formation of nanodomains present in giant unilamellar vesicles containing 1,2- dioleoyl-sn-glycero-3-phosphocholine (DOPC), cholesterol and sphingomyelin. Only few techniques are capable of detecting nanometer-sized domains in the membrane with high resolution. Time resolved Förster resonance energy transfer (TR-FRET) combined with Monte Carlo (MC) simulations provide a strong tool not only to detect lateral heterogeneities but also characterize them with the resolution of 2 nm. Profound effects on the nanodomain size were observed in the presence of both studied OxPLs and differences were detected, as PGPC with a carboxylic group drives formation of larger nanodomains than POVPC...
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Organization and mobility of G protein-coupled receptors in plasma membrane
Merta, Ladislav ; Svoboda, Petr (advisor) ; Sýkora, Jan (referee)
This diploma thesis deals with the analysis of structural and dynamic organization of thyrotropin releasing hormone receptor (TRH-R) and δ-opioid receptor (DOR) within plasma membrane (PM) in relation to the specific sub-compartments of PM denominated as domains or membrane rafts. Modern fluorescence microscopy techniques FLIM, FRAP and RICS were used for this purpose. The experiments were performed on the live cells derived from HEK293 cell line. To reach the main goal of this work, the integrity of PM structure was altered by depletion of cholesterol which was performed by incubation of cells with β cyclodextrin. Results clearly support our previously suggested idea that the vast majority of TRH-R is localized in non-raft regions of plasma membrane. This work also compared different modes of performance of FRAP and results obtained by FRAP and RICS because these methods are to some extent analogous. This is one of the first works that used the RICS approach to characterize the G protein-coupled receptors. In the second part of this work, the setup of transient transfection of the HEK293 cells with DOR-ECFP and DOR EYFP constructs was established. Simultaneously, the functionality of these constructs, i.e. the ability of DOR to activate the cognate G protein was determined. Powered by TCPDF (www.tcpdf.org)
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Software FLIM system in confocal microscopy
Petrula, Jakub ; Odstrčilík, Jan (referee) ; Čmiel, Vratislav (advisor)
This bachelor thesis deals with the FLIM system utilization in confocal microscopy. It describes basic concepts which are closely related to the topic, such as principles of fluorescence, fluorescent dyes and principles of both fluorescence and confocal microscopy. Practical part involves work with the confocal microscope and set of intensity images acquisition of both the animal and plant cells. Final product of pratical part is fluorescence lifetime image. The thesis also describes an algorithm for generating a pseudocolor frames and created user interface.
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pH sensitive fluorescence probes
Marková, Kateřina ; Obruča, Stanislav (referee) ; Mravec, Filip (advisor)
The goal of this thesis was to suggest the suitable method for measuring bacterial cytosolic pH in bacteria strain Cupriavidus necator. Fluorescent microscopy was chosen to obtain scan of bacteria and time-resolved fluorescence was chosen to obtain a calibration curve. BCECF-AM was used as a pH-sensitive fluorescent probe. It was suggested that the external calibration is more suitable than internal one for the prokaryotic type of cells. Bacteria H16 shows a long fluorescence lifetime only on the granules containing PHB, while in PHB-4 the longest fluorescence lifetime occurs randomly throughout the cell.
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