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Behaviour of hydrophilic fluorescent probe Oregon Green 488 in hydrogel environment
Lacko, Kata ; Venerová, Tereza (referee) ; Mravec, Filip (advisor)
The aim of this bachelor’s thesis is to study the behavior of a hydrophilic fluorescent probe named Oregon Green 488 in hydrogel environment. The hydrogel was created using sodium hyaluronan and CTAB (cetyl trimethylammonium bromide) as a cationic surfactant. Solution of 0.15 M NaCl was used as a solvent, which provides physiologic conditions in the sample. The behavior of the fluorescent probe was characterized by steady-state and time-resolved fluorescent measurements. To obtain the emission spectra of the probe and to find the emission maximum the method named TRES (time-resolved emission spectra) was used. Fluorescence intensity decay measurements as a function of wavelengths allowed to create deconvolution of the emission spectra, which provided information about the fluorescent lifetime and the probe’s states’ relative representation in the solution. Hydrogels were examined 5 days and 1 month after the preparation and the results were compared. Oregon Green 488 demonstrated one fluorescent lifetime in the hydrogel environment in the range, 4,14,45 nm in the fresh hydrogel and 4,14,6 nm in the stable hydrogel. The emission maximum was obtained at 525 nm in both cases. The fluorescent probe’s behavior in the hydrogel environment is stable, presumably there are no interactions occurring between the hydrogel and the probe. Furthermore, no interactions were recorded between the probe and hyaluronan.
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Multiparametric fluorescence spectroscopy
Lacko, Kata ; Venerová, Tereza (referee) ; Mravec, Filip (advisor)
This diploma thesis deals with the possibilities of multiparametric fluorescence spectroscopy, since the main objective of this experiment was to evaluate the possibilities of multiparametric measurements in the fluorescence spectroscopy laboratory. A suitable fluorescence probe was proposed for this type of experiment that shows high sensitivity for pH changes in the environment, SNARF-4F AM, based on a literature research. The fluorophore was dissolved in solutions of different pH and this system was examined using a time-resolved spectrofluorimeter. The method named TRES (time-resolved emission spectra) was used to obtain the emission spectra of the probe and to find the emission maximum. Fluorescence intensity decay measurements as a function of wavelengths allowed to create deconvolution of the emission spectra, which provided information about the fluorescent lifetime and the relative representation of the states of probes in the solution. Later, the probe was dissolved in solutions of different density and pH - this system served for anisotropic measurements, during which the individual correlation-rotational times of the fluorophore were obtained. The obtained results were then used as the basis for multiparametric analysis, which was performed by using a fluorescence correlation microscope and a spectrograph. This combination allows to measure the necessary fluorescence parameters in one step. A standard operating procedure was created for the spectrograph’s control. On the basis of the obtained information the suitability, accuracy and sensitivity of the multiparametric analysis were qualified.
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Multiparametric fluorescence spectroscopy
Lacko, Kata ; Venerová, Tereza (referee) ; Mravec, Filip (advisor)
This diploma thesis deals with the possibilities of multiparametric fluorescence spectroscopy, since the main objective of this experiment was to evaluate the possibilities of multiparametric measurements in the fluorescence spectroscopy laboratory. A suitable fluorescence probe was proposed for this type of experiment that shows high sensitivity for pH changes in the environment, SNARF-4F AM, based on a literature research. The fluorophore was dissolved in solutions of different pH and this system was examined using a time-resolved spectrofluorimeter. The method named TRES (time-resolved emission spectra) was used to obtain the emission spectra of the probe and to find the emission maximum. Fluorescence intensity decay measurements as a function of wavelengths allowed to create deconvolution of the emission spectra, which provided information about the fluorescent lifetime and the relative representation of the states of probes in the solution. Later, the probe was dissolved in solutions of different density and pH - this system served for anisotropic measurements, during which the individual correlation-rotational times of the fluorophore were obtained. The obtained results were then used as the basis for multiparametric analysis, which was performed by using a fluorescence correlation microscope and a spectrograph. This combination allows to measure the necessary fluorescence parameters in one step. A standard operating procedure was created for the spectrograph’s control. On the basis of the obtained information the suitability, accuracy and sensitivity of the multiparametric analysis were qualified.
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Behaviour of hydrophilic fluorescent probe Oregon Green 488 in hydrogel environment
Lacko, Kata ; Venerová, Tereza (referee) ; Mravec, Filip (advisor)
The aim of this bachelor’s thesis is to study the behavior of a hydrophilic fluorescent probe named Oregon Green 488 in hydrogel environment. The hydrogel was created using sodium hyaluronan and CTAB (cetyl trimethylammonium bromide) as a cationic surfactant. Solution of 0.15 M NaCl was used as a solvent, which provides physiologic conditions in the sample. The behavior of the fluorescent probe was characterized by steady-state and time-resolved fluorescent measurements. To obtain the emission spectra of the probe and to find the emission maximum the method named TRES (time-resolved emission spectra) was used. Fluorescence intensity decay measurements as a function of wavelengths allowed to create deconvolution of the emission spectra, which provided information about the fluorescent lifetime and the probe’s states’ relative representation in the solution. Hydrogels were examined 5 days and 1 month after the preparation and the results were compared. Oregon Green 488 demonstrated one fluorescent lifetime in the hydrogel environment in the range, 4,14,45 nm in the fresh hydrogel and 4,14,6 nm in the stable hydrogel. The emission maximum was obtained at 525 nm in both cases. The fluorescent probe’s behavior in the hydrogel environment is stable, presumably there are no interactions occurring between the hydrogel and the probe. Furthermore, no interactions were recorded between the probe and hyaluronan.
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