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Optical Micromanipulation Techniques Combined with Microspectroscopic Methods
Pilát, Zdeněk ; Prášil,, Ondřej (referee) ; Mojzeš, Peter (referee) ; Zemánek, Pavel (advisor)
Předložená dizertační práce se zabývá kombinací optických mikromanipulací s mikrospektroskopickými metodami. Využili jsme laserovou pinzetu pro transport a třídění živých mikroorganismů, například jednobuněčných řas, či kvasinek. Ramanovskou spektroskopií jsme analyzovali chemické složení jednotlivých buněk a tyto informace jsme využili k automatické selekci buněk s vybranými vlastnostmi. Zkombinovali jsme pulsní amplitudově modulovanou fluorescenční mikrospektroskopii, optické mikromanipulace a jiné techniky ke zmapování stresové odpovědi opticky zachycených buněk při různých časech působení, vlnových délkách a intenzitách chytacího laseru. Vyrobili jsme různé typy mikrofluidních čipů a zkonstruovali jsme Ramanovu pinzetu pro třídění mikro-objektů, především živých buněk, v mikrofluidním prostředí.
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Cell inclusions in microscopic eukaryotes
Pilátová, Jana
This thesis reports, for the first time, a systematic study identifying various cell inclusions across the broad diversity of all major eukaryotic supergroups. Raman microspectroscopy technique was employed as a powerful technique, which is becoming the method of the first choice for such studies. In Chapter 1, the spatial reconstruction of chloroplasts of the unicellular zygnematophyte Cylindrocystis sp. (Streptophyta) revealed "empty spaces" inside. Subsequent analysis showed massive accumulations of polyphosphate, which is not located in the cytoplasm as in other species, but inside the chloroplasts. This newly discovered ability of the zygnematophytes, a sister group of terrestrial plants, to accumulate polyphosphate leads us to speculate about a possible adaptation to extreme Arctic conditions or even a preadaptation leading to plant terrestrialization. Chapter 2 summarizes the serendipitous discovery of purine crystalline inclusions in dinoflagellates and other microalgae. Investigated dinoflagellate species comprised zooxanthellae, the endosymbionts of reef-building corals, and the species causing toxic algal blooms. Purine crystals were further shown to act as dynamic high-capacity nitrogen storage. To show how widespread purine inclusions are, a revision of crystalline inclusions in all...
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Cell inclusions in microscopic eukaryotes
Pilátová, Jana ; Schwarzerová, Kateřina (advisor) ; Gierlinger, Notburga (referee) ; Prášil, Ondřej (referee)
This thesis reports, for the first time, a systematic study identifying various cell inclusions across the broad diversity of all major eukaryotic supergroups. Raman microspectroscopy technique was employed as a powerful technique, which is becoming the method of the first choice for such studies. In Chapter 1, the spatial reconstruction of chloroplasts of the unicellular zygnematophyte Cylindrocystis sp. (Streptophyta) revealed "empty spaces" inside. Subsequent analysis showed massive accumulations of polyphosphate, which is not located in the cytoplasm as in other species, but inside the chloroplasts. This newly discovered ability of the zygnematophytes, a sister group of terrestrial plants, to accumulate polyphosphate leads us to speculate about a possible adaptation to extreme Arctic conditions or even a preadaptation leading to plant terrestrialization. Chapter 2 summarizes the serendipitous discovery of purine crystalline inclusions in dinoflagellates and other microalgae. Investigated dinoflagellate species comprised zooxanthellae, the endosymbionts of reef-building corals, and the species causing toxic algal blooms. Purine crystals were further shown to act as dynamic high-capacity nitrogen storage. To show how widespread purine inclusions are, a revision of crystalline inclusions in all...
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Surface-enhanced Raman microspectroscopy of biomolecules and biological systems
Šimáková, Petra ; Procházka, Marek (advisor) ; Michl, Martin (referee) ; Šloufová, Ivana (referee)
The aim of this thesis was using surface-enhanced Raman scattering (SERS) microspectroscopy for the study of biomolecules and biological systems. The main probe molecule was cationic porphyrin H2TMPyP, however, other porphyrins, tryptophan and two lipids were also used. The sensitivity and reproducibility of several solid SERS substrates: (i) Au and Ag nanoparticles (NPs) immobilized via a bifunctional linker, (ii) AgNPs immobilized by drying, (iii) highly ordered Au and Ag film-over-nanosphere (FON) and (iv) Ag-coated insect wings were compared. On most of the solid substrates, the lowest detected H2TMPyP concentration was ~10-8 M. The highest sensitivity was provided by the dried drops of AgNPs/analyte mixture, where concentrations 1×10-10 M TMPyP, 1×10-5 M tryptophan, 2×10-7 M DSPC and 3×10-7 M DMTAP were detected. Nevertheless, the spectral reproducibility was decreased due to porphyrin metallation and perturbation of the lipid spectra in comparison to their Raman spectra from solution. The highest reproducibility was achieved by AuFON and Ag-coated insect wings. Finally, the AgNPs modified by PEG polymers were tested for intracellular application using HeLa cancer cells. Metallation of H2TMPyP served to probe the accessibility of PEG- AgNPs surface. The results proved that the accessibility...
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Raman Microspectroscopy in Microfluidic Devices
Peksa, Vlastimil ; Mojzeš, Peter (advisor) ; Šloufová, Ivana (referee)
Miniaturization of devices to study chemical interactions and processes in liquid samples has led to the emergence of microfluidics and construction of lab-on-a-chip systems. Present work was devoted to implementation, development and testing of microfluidic systems with detection by confocal Raman microscopy and surface enhanced Raman scattering under the conditions of training department. Several options of performing standard macroscopic measurements in microscopic scales were explored. A method for measuring thermal stability of biopolymers in microsystems with contactless detection of temperature has been designed and tested. Furthermore, possibilites for studying the SERS effect within microfluidic channels were explored. It was demonstrated that the microfluidic chips provide promising opportunity to study hydrodynamics of liquids at microscopic level and chemical reactions and kinetics.
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Surface-enhanced Raman microspectroscopy of biomolecules and biological systems
Šimáková, Petra ; Procházka, Marek (advisor) ; Michl, Martin (referee) ; Šloufová, Ivana (referee)
The aim of this thesis was using surface-enhanced Raman scattering (SERS) microspectroscopy for the study of biomolecules and biological systems. The main probe molecule was cationic porphyrin H2TMPyP, however, other porphyrins, tryptophan and two lipids were also used. The sensitivity and reproducibility of several solid SERS substrates: (i) Au and Ag nanoparticles (NPs) immobilized via a bifunctional linker, (ii) AgNPs immobilized by drying, (iii) highly ordered Au and Ag film-over-nanosphere (FON) and (iv) Ag-coated insect wings were compared. On most of the solid substrates, the lowest detected H2TMPyP concentration was ~10-8 M. The highest sensitivity was provided by the dried drops of AgNPs/analyte mixture, where concentrations 1×10-10 M TMPyP, 1×10-5 M tryptophan, 2×10-7 M DSPC and 3×10-7 M DMTAP were detected. Nevertheless, the spectral reproducibility was decreased due to porphyrin metallation and perturbation of the lipid spectra in comparison to their Raman spectra from solution. The highest reproducibility was achieved by AuFON and Ag-coated insect wings. Finally, the AgNPs modified by PEG polymers were tested for intracellular application using HeLa cancer cells. Metallation of H2TMPyP served to probe the accessibility of PEG- AgNPs surface. The results proved that the accessibility...
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Raman Microspectroscopy and Mapping of Single Cells
Gregorová, Šárka ; Mojzeš, Peter (advisor) ; Plášek, Jaromír (referee)
Raman microspectroscopy enables one to acquire spectra of Raman scattering with a spatial resolution in the order of a few μm3 and thus to study the natural composition of biological objects such as tissues, single cells and cellular organelles in a non-invasive way. In this work, we used Raman microspectroscopy to investigate vacuoles of the opportunistic human yeast pathogen Candida albicans. Large sets of Raman spectra of vacuoles were collected based on different cultivation protocols. The sets of the spectra were evaluated using the multivariate statistical method of singular value decomposition. Based on the spectral analysis, we characterized the chemical composition of the vacuoles. We found out that the vacuoles of cells cultured differently or in different media vary particularly in the concentration of polyphosphate, represented in the spectra by the peak near 1155 cm-1 . Interestingly, the wavenumber position of the polyphosphate peak may also be shifted by several cm-1 . We studied these shifts in vitro with sodium hexametaphosphate as a model of vacuolar polyphosphate. Based on these experiments, we suggest that the peak position is significantly influenced by the concentration of divalent cations.
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Vibrational spectroscopy in pharmaceutical analysis
Průchová, Kristýna ; Němec, Ivan (advisor) ; Šmejkal, Petr (referee)
The aim of this diploma thesis is the application of vibrational spectroscopy in pharmaceutical analysis in studying solid pharmaceutical forms. The surface of tablet samples containing the active substance from the group of statins has been studied especially by the methods infrared microscopy. Spectral maps of samples were collected thanks to the techniques of specular reflection, attenuated total reflection (ATR) and "inverse" ATR after determining optimal conditions for measurements. In order to evaluate these measured maps, one-dimensional analysis and principal component analysis were used. As the same samples of tablets were also measured by Raman microscopy, the comparison has been provided. The measured distribution maps enable both a determination of substances in the sample and conclusion concerned a method of tablets' preparation. The method in this case is a granulation, which has been found out from a comparison of maps of generic and original medicament. The specular reflection method was selected to be the most appropriate technique for obtaining the maps of the surface of a tablet, via confrontation of particular methods consequently with consideration of their advantages and disadvantages in the measurement and data processing.
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Raman Microspectroscopy in Microfluidic Devices
Peksa, Vlastimil ; Mojzeš, Peter (advisor) ; Šloufová, Ivana (referee)
Miniaturization of devices to study chemical interactions and processes in liquid samples has led to the emergence of microfluidics and construction of lab-on-a-chip systems. Present work was devoted to implementation, development and testing of microfluidic systems with detection by confocal Raman microscopy and surface enhanced Raman scattering under the conditions of training department. Several options of performing standard macroscopic measurements in microscopic scales were explored. A method for measuring thermal stability of biopolymers in microsystems with contactless detection of temperature has been designed and tested. Furthermore, possibilites for studying the SERS effect within microfluidic channels were explored. It was demonstrated that the microfluidic chips provide promising opportunity to study hydrodynamics of liquids at microscopic level and chemical reactions and kinetics.
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Optical Micromanipulation Techniques Combined with Microspectroscopic Methods
Pilát, Zdeněk ; Prášil,, Ondřej (referee) ; Mojzeš, Peter (referee) ; Zemánek, Pavel (advisor)
Předložená dizertační práce se zabývá kombinací optických mikromanipulací s mikrospektroskopickými metodami. Využili jsme laserovou pinzetu pro transport a třídění živých mikroorganismů, například jednobuněčných řas, či kvasinek. Ramanovskou spektroskopií jsme analyzovali chemické složení jednotlivých buněk a tyto informace jsme využili k automatické selekci buněk s vybranými vlastnostmi. Zkombinovali jsme pulsní amplitudově modulovanou fluorescenční mikrospektroskopii, optické mikromanipulace a jiné techniky ke zmapování stresové odpovědi opticky zachycených buněk při různých časech působení, vlnových délkách a intenzitách chytacího laseru. Vyrobili jsme různé typy mikrofluidních čipů a zkonstruovali jsme Ramanovu pinzetu pro třídění mikro-objektů, především živých buněk, v mikrofluidním prostředí.
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