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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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Intensity Normalization of Raman Spectra by Means of Various Internal Standards
Sláma, Vladislav ; Mojzeš, Peter (advisor) ; Procházka, Marek (referee)
The work deals with the testing of selected inorganic anions as internal standards for intensity normalization of Raman spectra. Using the concentration and temperature series of aqueous solutions of PO4 3- , (CH3)2AsO2 - , NO3 - a SO4 2- , impact of the spectral changes of the standard bands for reliability and accuracy of the intensity calibration was studied. Temperature and concentration series of the anion Raman spectra are then used to test normalization by means of the bands of stretching and deformation vibrations of water molecules. In addition, reliability and robustness of the calibration procedures exploiting different spectral parameters present in the Raman spectra of standards was tested. Results of the study reveal possible sources of artifacts and misinterpretations arising from insufficient understanding of the standard behavior due to changes of the physical and chemical parameters, or due to improper calibration procedure applied.
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Complexes of Cationic Porphyrins with Nucleic Acids Studied by Surface Enhanced Resonance Raman Spectroscopy
Lásková, Barbora ; Mojzeš, Peter (advisor) ; Michl, Martin (referee)
Complexes of cationic porphyrins with nucleic acids are interesting from the point of view of delivery of therapeutic oligonucleotides into cells as well as for photodynamic therapy of cancer. Diploma thesis deals with study of the complexes of cationic metalloporphyrin CuTMPyP4 with poly(dG-dC)2, where intercalation of porphyrin within polynucleotide's base pairs is supposed and with poly(dA-dT)2, poly(dA)·poly(dT), in which external groove-binding of CuTMPyP4 on the helix is assumed. The measurements were made by SERRS, RRS and absorption spectroscopies. From the time evolution of the SERRS spectra for each complex it was found, that intensity of SERRS spectra and rate of SERRS kinetics for complexes fall short of intuitive supposition about low SERRS signal and its slow increasing in the case of intercalation of porphyrin, because for complex of CuTMPyP4 with poly(dG-dC)2, the SERRS kinetic are very fast and final SERRS signal is more intense than for poly(dA-dT)2, poly(dA)·poly(dT). Comparing the SERRS and RRS spectra of the complexes it was found that SERRS spectra correspond to the porphyrin molecules released from the complexes rather that to the entire complexes. Study of various colloidal systems used for SERRS measurements revealed that SERRS signal evolves with time of exposition, namely for...
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Construction and Testing of Microfluidic Device for Raman Microscopy
Peksa, Vlastimil ; Procházka, Marek (referee) ; Mojzeš, Peter (advisor)
Miniaturization of devices to study chemical interactions of liquids has led to the emergence of microfluidics and construction of lab-on-a-chip. Present work is devoted to explore the possibility of microfluidics with detection by confocal Raman microscopy and the surface enhanced Raman scattering. The microfluidic device was assembled from commercially available components and tested. Methodology of its operation has been developed and some of its operational limits were set. Kinetics of the SERS-active particles formation was studied by its mean. It was demonstrated that the microfluidic chips provide promising opportunity to study chemical reactions, hydrodynamics of liquids at microscopic level and construction of the SERS-active surfaces.
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In situ Luminescence Detection of Singlet Oxygen
Vyklický, Vojtěch ; Dědic, Roman (advisor) ; Mojzeš, Peter (referee)
The thesis summarises already published results of singlet oxygen luminiscence detection in relation to photodynamic therapy treatment. The spectroscopic and photosensitising properties of the photosensitizer TMPyP and their comparison with already published properties of TPPS4 are presented. Also the inuence of phosphatidylcholine on photosensitising properties of TPP and singlet oxygen lifetime was investigated. The thesis is mainly focused on adaptation and optimisation of experimental setup for singlet oxygen luminescence deteciton from the surface of solid samples with time and spectral resolution using optical fibres with luminescence probe. The usability was tested on polymer matrix (PMMA or polystyrene) containing protoporphyrine IX. Finally, the kinetics of singlet oxygen phosphorescence measured from surface of thin layer of 3T3 murine fibroblasts containing TPPS4 or TMPyP are presented. Thereby the feasibility of singlet oxygen detection in situ was demonstrated using this setup.
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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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