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Multiphoton and non-linear Raman microscopy through a multimode fibre
Pikálek, Tomáš ; Marte, Monika Ritsch- (referee) ; Zemánek, Pavel (referee) ; Čižmár, Tomáš (advisor)
Multimodová vlákna je možné využít jako miniaturní endoskopy pro zobrazování hluboko uvnitř mozku myších zvířecích modelů s minimálním poškozením okolní tkáně. S využitím metod tvarování vlnoplochy je možné svazek vystupující z vlákna fokusovat do jediného bodu, a vytvořit tak miniaturní skenovací mikroskop. Různé mikroskopické zobrazovací metody již byly tímto způsobem implementovány, a to včetně několika in-vivo demonstrací. Tato práce se zabývá implementací zobrazování pomocí koherentního anti-Stokesova Ramanova rozptylu (CARS), nelineární metody umožňující zobrazování s chemickým kontrastem bez značení vzorku. Tato metoda má potenciální využití pro klinické aplikace například při diagnóze nádorů. Práce je zaměřena zejména na optimalizaci endoskopu a vláknových sond pro fokusaci světla z pulzních laserů včetně kontroly disperze s cílem umožnit nelineární zobrazování. Takto optimalizovaný systém je následně využit pro demonstraci zobrazování metodou CARS uvnitř tkáně (například lipidové dvojvrstvy buněčných membrán kolem axonů) a chemického kontrastu na polymerových kuličkách. Systém je též použit k demonstraci multimodálního zobrazování fixované myší tkáně kombinací CARS a dvoufotonové mikroskopie.
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Study of optical properties of organic materials for nonlinear optical applications
Hrbková, Silvie ; Hrabal, Michal (referee) ; Vala, Martin (advisor)
This work deals with a development of a method for determination of two-photon parametres and its application. The theoretical part includes a description of two-photon absorption processes, most frequently used methods to determine two-photon cross sections and a summary of molecules researched to date in the field. The molecules studied in this work are Rhodamin B and Rhodamin 6G in ethanol, which are applied as standards for the implementation of the method. After that, Diketopyrrolopyrroles U24, U37, U49 and U50 dissolved in DMSO, toluene, acetone and chloroform, are researched. Basic optical characteristics - absoption and emission spectra were experimentally acquired for all the materials. Two-photon characteristics were obtained using a picosecond laser and a Two-Photon Excited Fluorescence (TPEF) method. TPA cross-sections and TPA spectra of Rhodamin B and Rhodamin 6G were measured and discussed. Subsequently, this work focuses on absorption and emission solvatation processes of the selected Diketopyrrolopyrroles. These processes indicate their two-photon efficiency. Finally, TPA spectra of the substances are discussed. Contribution of this work is a developed method for measuring the two-photon characteristics suitable for further use. The outcomes achived, enable extensive research of the Diketopyrrolopyrrols characteristics with the use of the measured standards of the Rhodamin B and Rhodamin 6G.
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Modern optical in vivo methods in neurophysiological research
Tomáška, Filip ; Novák, Ondřej (advisor) ; Elsnicová, Barbara (referee)
Accurate visualization of structures and events at subcellular level is one of the major challenges of current neuroscience. Optical methods based on fluorescence imaging were optimized to record and control neural activity, thus presenting a powerful approach complementary to historically dominant electrophysiological techniques. The employment of two-photon excitation enabled in vivo imaging of neurons up to 1 mm from the sample surface without causing significant photodamage. The application of methods of molecular biology has yielded protein-based genetically targetable indicators of neural activity, possessing performance comparable to the traditional organic dyes. Moreover, heterologous expression of microbial opsins proved capable of light-induced neural excitation or silencing in a single-component manner. The combination of these optogenetic tools offers two-way control over neuronal populations with single cell resolution. If coupled with calcium or voltage fluorescent indicators and transgenic animal models, such systems represent a non- invasive, all-optical tool for simultaneous control and imaging of specific neuronal subtypes. Its application supported by electrical recordings may finally provide the data necessary for the uncovering of fundamental principles of neural functioning.
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Actuoecology of testate amoebae in fresh water and soil environment in enteraction with fungi and their analysis with new microscopic techniques
Burdíková, Zuzana ; Holcová, Katarína (advisor) ; Jansa, Jan (referee) ; Hudáčková-Hlavatá, Natália (referee)
4 Abstract The present thesis focuses on testate amoebae (TA) and their relationship to their natural environment, as well as on relevant microscopic imaging methods. The bulk of the data has been published in original scientific papers and is compiled into three separate chapters (Pt I, Pt II and Pt III), each annotated by a brief introduction. (Pt I) The methods section is devoted to specialized microscopic techniques employed to broaden the scope of the ecological analyses. In particular, precise discrimination between live and dead individuals, biomass determination inside individual tests and a multi-modal visualization of the cytoplasm and organelles enhance the data. Laser scanning confocal microscopy and two-photon microscopy are the main imaging modalities employed to study TA morphology in detail. The data have implications for taxonomy and ecophysiology, including the use of TA as bioindicators of pollution. (Pt II) An actuoecological analysis focuses on the seasonal variability of TA species composition in a freshwater ecosystem, namely the Komo any ponds in Prague, during the course of the year. The species composition variation is correlated to simultaneously recorded limnological parameters such as temperature, pH, contamination by (heavy) metals (As, Cd, Mn, Ni, Fe, Pb), polycyclic aromatic...
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Modern optical in vivo methods in neurophysiological research
Tomáška, Filip ; Novák, Ondřej (advisor) ; Elsnicová, Barbara (referee)
Accurate visualization of structures and events at subcellular level is one of the major challenges of current neuroscience. Optical methods based on fluorescence imaging were optimized to record and control neural activity, thus presenting a powerful approach complementary to historically dominant electrophysiological techniques. The employment of two-photon excitation enabled in vivo imaging of neurons up to 1 mm from the sample surface without causing significant photodamage. The application of methods of molecular biology has yielded protein-based genetically targetable indicators of neural activity, possessing performance comparable to the traditional organic dyes. Moreover, heterologous expression of microbial opsins proved capable of light-induced neural excitation or silencing in a single-component manner. The combination of these optogenetic tools offers two-way control over neuronal populations with single cell resolution. If coupled with calcium or voltage fluorescent indicators and transgenic animal models, such systems represent a non- invasive, all-optical tool for simultaneous control and imaging of specific neuronal subtypes. Its application supported by electrical recordings may finally provide the data necessary for the uncovering of fundamental principles of neural functioning.
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Actuoecology of testate amoebae in fresh water and soil environment in enteraction with fungi and their analysis with new microscopic techniques
Burdíková, Zuzana ; Holcová, Katarína (advisor) ; Jansa, Jan (referee) ; Hudáčková-Hlavatá, Natália (referee)
4 Abstract The present thesis focuses on testate amoebae (TA) and their relationship to their natural environment, as well as on relevant microscopic imaging methods. The bulk of the data has been published in original scientific papers and is compiled into three separate chapters (Pt I, Pt II and Pt III), each annotated by a brief introduction. (Pt I) The methods section is devoted to specialized microscopic techniques employed to broaden the scope of the ecological analyses. In particular, precise discrimination between live and dead individuals, biomass determination inside individual tests and a multi-modal visualization of the cytoplasm and organelles enhance the data. Laser scanning confocal microscopy and two-photon microscopy are the main imaging modalities employed to study TA morphology in detail. The data have implications for taxonomy and ecophysiology, including the use of TA as bioindicators of pollution. (Pt II) An actuoecological analysis focuses on the seasonal variability of TA species composition in a freshwater ecosystem, namely the Komo any ponds in Prague, during the course of the year. The species composition variation is correlated to simultaneously recorded limnological parameters such as temperature, pH, contamination by (heavy) metals (As, Cd, Mn, Ni, Fe, Pb), polycyclic aromatic...
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Study of optical properties of organic materials for nonlinear optical applications
Hrbková, Silvie ; Hrabal, Michal (referee) ; Vala, Martin (advisor)
This work deals with a development of a method for determination of two-photon parametres and its application. The theoretical part includes a description of two-photon absorption processes, most frequently used methods to determine two-photon cross sections and a summary of molecules researched to date in the field. The molecules studied in this work are Rhodamin B and Rhodamin 6G in ethanol, which are applied as standards for the implementation of the method. After that, Diketopyrrolopyrroles U24, U37, U49 and U50 dissolved in DMSO, toluene, acetone and chloroform, are researched. Basic optical characteristics - absoption and emission spectra were experimentally acquired for all the materials. Two-photon characteristics were obtained using a picosecond laser and a Two-Photon Excited Fluorescence (TPEF) method. TPA cross-sections and TPA spectra of Rhodamin B and Rhodamin 6G were measured and discussed. Subsequently, this work focuses on absorption and emission solvatation processes of the selected Diketopyrrolopyrroles. These processes indicate their two-photon efficiency. Finally, TPA spectra of the substances are discussed. Contribution of this work is a developed method for measuring the two-photon characteristics suitable for further use. The outcomes achived, enable extensive research of the Diketopyrrolopyrrols characteristics with the use of the measured standards of the Rhodamin B and Rhodamin 6G.
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