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Preparation of luminescent nanoparticles for 3D imaging
Smolka, Rastislav ; Hrabal, Michal (referee) ; Vala, Martin (advisor)
The aim of this bachelor thesis was to study a suitable methodology for the preparation of fluminiscent nanoparticles and to characterize their optical properties associated with various physical properties of environment. Also, their potential use as a means of monitoring biological preparations in 3D microscopy using multiphoton absorption. In particular, we deal with nanoparticles prepared from N,N-diphenylamino-2,5-diphenyl-1,4-distyrylbenzene cyanide and their fluorescence and absorption spectra. Using the dynamic light scattering method and fluorescence spectroscopy, the dependence of the size of the prepared nanoparticles, the change in their fluorescence properties and the change in the quantum yield is observed, depending on the changing environmental properties. Data from multifoton absorption measurements demonstrate that this substance responds to multifoton excitation. It has been shown that this substance retains its unique fluorescence properties also in the form of nanoparticles and thus appears to be a suitable candidate for the observation of biological preparations using multifoton fluorescence microscopy. Therefore, the continuation of this work could be the mere determination of the total multifoton absorption cross section of these prepared nanoparticles.
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pH sensitive fluorescence probes
Marková, Kateřina ; Obruča, Stanislav (referee) ; Mravec, Filip (advisor)
The goal of this thesis was to suggest the suitable method for measuring bacterial cytosolic pH in bacteria strain Cupriavidus necator. Fluorescent microscopy was chosen to obtain scan of bacteria and time-resolved fluorescence was chosen to obtain a calibration curve. BCECF-AM was used as a pH-sensitive fluorescent probe. It was suggested that the external calibration is more suitable than internal one for the prokaryotic type of cells. Bacteria H16 shows a long fluorescence lifetime only on the granules containing PHB, while in PHB-4 the longest fluorescence lifetime occurs randomly throughout the cell.
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Characterization of coloid particles by excited-state proton transfer with advanced fluorescence techniques
Kotouček, Jan ; Mravec, Filip (referee) ; Pekař, Miloslav (advisor)
The deprotonation characteristics of fluorescent probes -naphthol and 8-hydroxypyrene-1,3,6-trisulphonic acid (HPTS) were studied in this diploma thesis, using steady-state and time-resolved fluorescence spectroscopy. Two cationic surfactants, Septonex and cetyltrimethylammonium bromide (CTAB), were studied. These surfactants were measured in the complex with hyaluronan (1.75 MDa, 1 MDa and 300 kDa). Steady-state fluorescence was used for determination of critical aggregation concentration of each surfactant and pKa*. Time-resolved fluorescence decays were used to calculate the average lifetimes and the deprotonation constants of naphthol and HPTS. The measurement with hyaluronan were compared with the polystyrenesulfonate (PSS) – surfactant system. The effect of hydration shell of hyaluronan on hyaluronan – surfactant complex formation results from the comparison of above mentioned systems. Large differences were found in the deprotonation characteristic between surfactants and even between individual molecular weights of hyaluronan. The measurement shows that the hydration shell is located near to the dissociated carboxyl groups of hyaluronan chain, where the interaction with the positively charged surfactants occurs. Furthermore, the aggregation number of Septonex was determined by quenching of pyrene using cetylpyridinium chloride (CPC) as a quencher. The aggregation number for 20 mM Septonex solution was determined as a value of 104 molecules. CPC was used for confirmation of the localization of -naphthol in the micelles of CTAB and polymer – CTAB, respectively.
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Native hyaluronan as delivery agent for hydrophobic molecules
Michalicová, Petra ; Márová, Ivana (referee) ; Pekař, Miloslav (advisor)
Hyaluronan is a chemical, which can be qualified as essential for vertebrates. It is a part of the extracellular matrix in most of tissues and also a major component of some other tissues. Besides of the mechanical functions this compound is important for many biological processes such as growth of tumor cells. The objective of this thesis was development of carrier systems containing native hyaluronan and hydrophobic drugs. For purposes of this work fluorescence probes (pyrene, prodan, perylene, DPH, mereocynine 540) instead of drugs were used. By using further mentioned sophisticated methods the properties of these systems were studied. The systems were prepared by freeze-drying. The effect of freeze-drying on support of interactions was observed by fluorescence spectrometry (steady-state and time-resolved). The stability of freeze-dried systems was determined by zeta potential, which was measured by electrophoretic light scattering. Cakes obtained by freeze-drying were analyzed by several methods. First one was effluence gas chromatography connected with FT-IR spectrometry. In this method the present of tertiary butyl alcohol in product was observed. The cakes were also analyzed by scanning electron microscopy, which can provide the information about the surface and elemental constitution of the material. The results of this work can shed light on the area of developing of drugs with targeted distribution of active compound.
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Fluorescence in hydrophilic polymers research
Kotouček, Jan ; Pilgrová, Tereza (referee) ; Venerová, Tereza (advisor)
This bachelor thesis deals with the use of fluorescence in the study of hydrophilic polymers such as polyvinylpyrrolidone, carboxymethylcellulose, bovine serum albumin, and hyaluronan. The first measurement was a fluorescence intensity of the anionic form of 3-hydroxy naphtholic acid which undergoes an intramolecular proton transfer in the excited state then the steady-state fluorescence anisotropy of 3-hydroxy naphtholic acid and also the lifetime of the fluorescent probe. The measured data were evaluated in terms of interaction of the fluorescence probe with hydration shell of polymer in an aqueous environment by determination of a binding constant of the system, polymer - fluorescent probe. The interaction between anionic form of the probe with polymer was found in BSA, PVP and CMC systems. There was no interaction of fluorescent probe with hydration shell of hylauronan due to charge repulsion between the probe and hyaluronan.
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Time-resolved fluorescence of system polymer-surfactant
Mondek, Jakub ; Čeppan, Michal (referee) ; Mravec, Filip (advisor)
In this diploma thesis was studied time-resolved fluorescence in polymer-surfactant system. At first aggregation numbers of cationic (cetyltrimethylammonium bromide), anionic (sodium dodecylsulfate) and nonionic (Triton X-100) surfactants were studied by steady-state and time-resolved fluorescence spectroscopy. These two methods were compared. Aggregation numbers by steady-state method were always lower than aggregation numbers measured by time-resolved method. Steady-state method of determination aggregation numbers is useless for surfactants with high aggregation number and for aerated samples. Addition of hyaluronan to surfactant system was studied. There was observed change in aggregation number after addition of hyaluronan and change in percentage of dynamic quenching after addition of hyaluronan. Hyaluronan affected aggregation number of cetyltrimethylammonium bromide and Triton X-100. Hyaluronan increased percentage of dynamic quenching in cetyltrimethylammonium bromide and in Triton X-100. Pyren in sodium dodecylsulfate was quenched by sphere of action with negligible percentage of dynamic quenching and addition of hyaluronan had no effect on quenching. As next goal of this thesis, the determination of the position of fluorescence probe pyrene in cetyltrimethylammonium bromide, sodium dodecylsulfate and Triton X-100 micelles was chosen. Position of pyrene changed with charge and structure of micelles. Next was studied how percentage of dynamic quenching by iodide compounds changes with different charge of micelle. In all cases majority of dynamic quenching was calculated.
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Excited-state proton transfer as a tool for characterization of coloid particles
Richterová, Veronika ; Pilgrová, Tereza (referee) ; Mondek, Jakub (advisor)
This bachelor thesis deals with the study of excited-state proton transfer in micellar systems. At first critical micelle concentration of surfactants CTAB, SDS and Triton X-100 was determined. Further the steady-state and time-resolved fluorescence of probes that undergo excited-state proton transfer with these surfactants was measured. As probes were chosen 1-naphthol, HPTS and 3HNA. Deprotonation rates of 1-napthol and HPTS and average lifetime of 3HNA were calculated from time-resolved measurement. Steady-state fluorescence was used for observation surfactants influence on excited-state proton transfer.
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Investigation of stability and dynamics of photosynthetic pigment - protein complexes using optical spectroscopy
ODERMATT, Mirjam
Isolated violaxanthin-chlorophyll a binding protein of Nannochloropsis oceanica was studied by optical spectroscopy, putting a specific focus on time-resolved fluorescence spectroscopy. Thereby, dynamic and kinetic information about the pigment-protein interactions with respect to non-photochemical quenching was collected utilizing pigment and intrinsic tryptophan fluorescence. Using this method, the investigation of the molecular environment of the pigment-protein complex during the process of qE was possible.
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Preparation of luminescent nanoparticles for 3D imaging
Smolka, Rastislav ; Hrabal, Michal (referee) ; Vala, Martin (advisor)
The aim of this bachelor thesis was to study a suitable methodology for the preparation of fluminiscent nanoparticles and to characterize their optical properties associated with various physical properties of environment. Also, their potential use as a means of monitoring biological preparations in 3D microscopy using multiphoton absorption. In particular, we deal with nanoparticles prepared from N,N-diphenylamino-2,5-diphenyl-1,4-distyrylbenzene cyanide and their fluorescence and absorption spectra. Using the dynamic light scattering method and fluorescence spectroscopy, the dependence of the size of the prepared nanoparticles, the change in their fluorescence properties and the change in the quantum yield is observed, depending on the changing environmental properties. Data from multifoton absorption measurements demonstrate that this substance responds to multifoton excitation. It has been shown that this substance retains its unique fluorescence properties also in the form of nanoparticles and thus appears to be a suitable candidate for the observation of biological preparations using multifoton fluorescence microscopy. Therefore, the continuation of this work could be the mere determination of the total multifoton absorption cross section of these prepared nanoparticles.
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Excited-state proton transfer as a tool for characterization of coloid particles
Richterová, Veronika ; Pilgrová, Tereza (referee) ; Mondek, Jakub (advisor)
This bachelor thesis deals with the study of excited-state proton transfer in micellar systems. At first critical micelle concentration of surfactants CTAB, SDS and Triton X-100 was determined. Further the steady-state and time-resolved fluorescence of probes that undergo excited-state proton transfer with these surfactants was measured. As probes were chosen 1-naphthol, HPTS and 3HNA. Deprotonation rates of 1-napthol and HPTS and average lifetime of 3HNA were calculated from time-resolved measurement. Steady-state fluorescence was used for observation surfactants influence on excited-state proton transfer.
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