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Anisotropy techniques in study of cytoplasm
Sýkorová, Kateřina ; Obruča, Stanislav (referee) ; Mravec, Filip (advisor)
The main goal of this thesis was to compare experiments using time-resolved anisotropy and steady-state anisotropy for measuring in bacteria strain Cupriavidus necator. Fluorescent probe for anisotropy imaging was chosen BCECF_AM, which is derivate of fluorescein. Using experiment in system glycerol/water with fluorescein, anisotropy has been verified and calculated molecular hydrodynamic volume of a single fluorescein molecule, which approximately corresponded with real value. By using fluorescence imaging anisotropy microscopy, images and values of average anisotropy in cells were taken. Images of living cells (bacteria) of CN H16 and mutant CN PHB-4 showed differences, mainly in the uniformity of the inside environment.
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Advanced Fluorescence Techniques in Research on Micellar Systems and Their Interactions with Biopolymers
Holínková, Petra ; Burgert, Ladislav (referee) ; Táborský, Petr (referee) ; Pekař, Miloslav (advisor)
The dissertation thesis deals with study of advanced steady-state and time-resolved fluorescence techniques, which can be used for study of micellar systems properties. Selected fluorescence techniques were used for characterization of Septonex and CTAB cationic micellar systems and theirs interactions with hyaluronan. Fluorescent probe pyrene was used for determination of critical micelle concentration (CMC) and micellar aggregation number of these surfactants. The changes of fluorescence behaviour of fluorescein and prodan were studied in wide concentration range of Septonex. Next chapter of thesis deals with study of Förster resonance energy transfer between perylene and fluorescein in Septonex and CTAB micellar solutions and the effect of hyaluronan addition to these systems. Also steady-state and time-resolved fluorescence anisotropy studies were used for research of the effect of hyaluronan addition to micellar solutions. The last chapter of this thesis is focused on photophysical behaviour of Prodan in different solutions (water, Septonex solutions below CMC, hyaluronan solution, Septonex micellar solution and Septonex micellar solution with hyaluronan), which was discussed on the basis of time-resolved emission spectra.
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Inverse FCS in colloidal systems research
Richterová, Veronika ; Venerová, Tereza (referee) ; Mravec, Filip (advisor)
This diploma thesis is focused on the study of inverse fluorescence correlation spectroscopy, especially with the regard for the usage of different fluorescent probes and different sized analysed particles. At first, the proper concentration of fluorescent probes was determined. In this concentration is the probe considered as a medium surrounding the analysed particles. Based on this concentration, which was determined as 400 M, several sets of samples were prepared. This samples contained different concentration of polystyrene particles of 100 and 500 nm diameter and multilamellar liposomes. Then, the FCS curves of samples with different fluorescent probes were measured. Fluorescein, rhodamine 6G and Atto 488 were used as fluorescent probes. As a result from experiments, it was found, that particles with 100 nm diameter cannot be analysed with none of the fluorescent probes. Inverse FCS method can be applied to systems, that contains particles with 500 nm diameter and fluorescein. Systems with rhodamine 6G have the same behaviour as typical FCS measurement. It is caused by dimerization of this probe and it cannot be used for 500 nm particles. Liposome samples can be established with iFCS method, but the results are biased by random distribution of liposomes size.
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Microviscosity probes in study of aggregation in a biopolymer-surfactant system.
Vašíčková, Kamila ; Vala, Martin (referee) ; Mravec, Filip (advisor)
The effect of cationic surfactant concentration and ionic strength on anisotropy of fluorescence of probes diphenylhexatrien and fluorescein has been investigated in the system of cationic surfactant and in the system of cationic surfactant and hyaluronan. The investigation has been done by fluorescence emission spectroscopy. Obtained anisotropy gives information about microviscosity of investigated systems. Subsequently the system of cationic surfactant and hyaluronan has been investigated by 9-(2-carboxy-2cyanovinyl)julolidine, 4-(dicyanovinyl)julolidine and 1,3-bispyrenylpropane probes. The information about the microviscosity of the system gives the integral under the emission curve of 9-(2-carboxy2-cyanovinyl)julolidine and 4-(dicyanovinyl)julolidine and the ratio between excimer and monomer of 1,3-bispyrenylpropane. It has been discovered that the ionic strength influences the anisotropy of diphenylhexatrien and fluorescein only in the fist addition of salt (concentration 0,025 mol dm-3) and that addition of hyaluronan influences the anisotropy of diphenylhexatrien and fluorescein only in samples without addition of salt. Results of measurment with 9-(2-carboxy2-cyanovinyl)julolidine and 4(dicyanovinyl)julolidine describe the formation of aggregates of catinoc surfactant with hyaluronan and characterize these aggregates from the point of microviscosity.
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Energy transfer and hydrophobic domains in colloidal systems
Kučerová, Petra ; Čeppan, Michal (referee) ; Pekař, Miloslav (advisor)
In this thesis resonance energy transfer between perylene and fluorescein in tetradecyltrimethylammonium bromide (TTAB) solution was studied. The influence of addition of native or modified sodium hyaluronate on resonance energy transfer was also investigated. The addition of native sodium hyaluronate supports energy transfer at lower fluorescein concentrations and the addition of modified sodium hyaluronate influences the maximal value of energy transfer effectivity. Strong influence on the critical micelle concentration (CMC) values with the addition of sodium hyaluronate during CMC of TTAB determination was investigated. The data indicates not only micelles formation, but also formation of aggregates of sodium hyaluronate with TTAB. Aggregation numbers of TTAB with addition of native and modified sodium hyaluronate by the quenching of pyrene by cetylpyridinium chloride (CPC) was investigated. The addition of sodium hyaluronate into the solution of TTAB changes the average mean aggregation number. Solute exchange between micelles of TTAB and the influence of addition of modified sodium hyaluronate on this exchange was also investigated. No solute exchange between micelles in TTAB and in TTAB with added modified sodium hyaluronate was discovered during this experiment.
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Utilization of fluorimetry for detection of underground water tracers
Pokora, Zdeněk ; Čáslavský, Josef (referee) ; Kahle, Vladislav (advisor)
The thesis studies detection of fluorescein for coloration experiments in surface and underground water. The first part of the work deals with the adsorption of fluorescein on active charcoal from water and desorption by means of different desorption solutions. The results of measurements are used for practice of coloration experiments in karst research. In the second part of thesis it is researched the option of automated record of fluorescence concentration and its detection by means of laser induced fluorescence with confocal microscope.
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Use of fluorescent techniques in study of aerosol deposition
Lippay, Josef ; Jedelský, Jan (referee) ; Mravec, Filip (advisor)
Several experiments were designed for utilization of fluorescence spectroscopy as a method of aerosol particle detection in a model of lungs. One of the experiments was to arranged use luminescent properties of DEHS (bis(2-ethylhexyl)decandioate) for calculating aerosol deposition. The outcome of this experiment was confirmation of clusters existence, which causes luminescence of DEHS. But the luminescence is not enough dependent on concentration and as such is not suitable for calculation of aerosol deposition. As the next experiment DEHS-fluorescein particles were generated by condensation monodisperse aerosol generator (CMAG), where water was used instead of isopropyl alcohol as a solvent. By this alteration the negative influence of DEHS was eliminated, which caused results refinements of aerosol deposition. Generation of fluorescein sodium salt particles by small-scale powder disperser (SSPD) was designed as a last experiment. The lower deposition efficiency measured by this method was caused by particles polydispersion. Photo records were used for documentation of Hot-spots. Outcomes of this study are new knowledge of fluorescence spectroscopy utilization for study of aerosol deposition and possibilities of fluorescent aerosol particles generation. Acquired data can serve for knowledge extension of possible detection methods for aerosol particles in the model of lung and can serve for validation of numerical simulations.
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Fluorescenec spectroscopy in study of aerosol-forming substances
Lippay, Josef ; Jedelský, Jan (referee) ; Mravec, Filip (advisor)
To study the spectral characteristics of a substance DEHS several experiments were suggested. Closer investigation was made on study of the effect of different environments on the spectral properties of the substance DEHS. It was found that the highest positive effect on the fluorescence of the substance DEHS have a medium with hydroxyl groups. IPA was evaluated as the best medium for further experiments. It was found that the medium xylene decreases intensity of the fluorescence to the detection limit. Spectral characterization of the nondegraded and degraded DEHS was performed. It was found that for the degraded DEHS shift to higher wavelengths occurs due to degradation. Possible contamination of the DEHS with substances causing luminescence has not been proven.
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Resonance energy transfer in the environment of the hydrogel matrix
Janča, David ; Venerová, Tereza (referee) ; Mravec, Filip (advisor)
The diploma thesis deals with resonance energy transfer in hydrogel matrix enviroment. In the theoretical part, all the important features of these systems and their components are summarized and all the principles for explaining the method used are described. The work also includes a short literary background research. In the experimental part, the resonance energy transfer was measured in micellar solutions and hydrogels in 0.15 M NaCl medium. The hydrogels were formed on the basis of interaction of the surfactant with the oppositely charged polyelectrolyte. As a positively charged surfactant, carbethopendecinium bromide (Septonex) was chosen and hyaluronan as the negatively charged polyelectrolyte. Perylene with fluorescein and perylene were selected as fluorescent pairs for RET. It has been found that increasing concentration of the acceptor molecule increases the intensity of RET, in other word the rate of acceptor binding to the donor. In the study of fluorescent pair of perylene with DiO was observed, that it was not appropriate to use fluorescence probe DiO at higher concentrations. Experiments were carried out to determine whether RET changes in the time from the point of mixing the donor and acceptor fluorescence probes together. This phenomenon has not been confirmed. In hydrogels, the effect of increasing surfactant concentration and molecular weight of hyaluronan was observed. The resulting analysis showed that the molecular weight of hyaluronan has a significant effect on RET efficiency in hydrogels.
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Binding of organic dyes to proteins. Applications in practical course in biochemistry.
Hynková, Anna ; Hudeček, Jiří (advisor) ; Koblihová, Jitka (referee)
In this submitted thesis the possibility of creating a new laboratory task for advanced practical courses in biochemistry, concerning the binding of a low-molecular compound (dye) to a protein macromolecule, was experimentally verified. First intention was to modify the existing task "Dialysis kinetics" with a binding of fluorescein to a protein. However, the experiments have proved, that fluorescence measurements of this kind are not reproducible in the conditions of practical courses laboratory and absorbance measurements have low sensitivity. Therefor a whole new task was created: "Binding of bromophenol blue to serum albumin", in which the stoichiometry of this binding is studied using so-called Job plot. After the optimization of procedure a laboratory task instructions were created, which are attached to this thesis. Keywords: fluorescein, bromophenol blue, serum albumin
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