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Fluorescence correlation spectroscopy in the study of the properties of colloidal systems
Marková, Kateřina ; Lehocký, Marián (referee) ; Kapusta, Peter (referee) ; Pekař, Miloslav (advisor)
Because of their properties, hydrogels are a highly sought-after matrix for medical purposes. These properties are often conditioned by the structure, therefore the emphasis is put on a precisely defined polymer network. In the presented dissertation, different types of hydrogels were researched using fluorescence correlation spectroscopy (FCS) in conjunction with microrheology. The combination of aforementioned methods is rarely used when determining the properties of hydrogels, and therefore, the method had to be standardized and optimized. For these purposes, fluorescently labeled silicate nanoparticles with a neutral surface charge were selected. In the course of the method optimization, the limits of the device were discovered within which the measured diffusion coefficients could still be considered valid. Furthermore, we have determined the parameters that affect the MSD curve and thus also the correctness of the actual measured data. An aqueous solution of agarose was chosen as a model colloidal system, as it ranges from a viscoelastic liquid to a solid hydrogel depending on the concentration. On this colloidal system, the diffusion properties of the nanoparticles used were tested as well as the structural properties of the system itself. Limit concentrations were found when the diffusion coefficient could no longer be detected using the selected method. The limit was successfully partially extended by adjusting the correlation time, however, the dispersion of the diffusion coefficient values was very high in such case. The values measured in this way were compared with the calibration-free method of bifocal fluorescence correlation spectroscopy (2f–FCS). Furthermore, changes in the diffusion coefficient were determined depending on the type of sample preparation. Alongside these experiments, image analysis was also performed, which provided interesting results in conjunction with FCS. The last experiment, which provided information about the properties of both the embedded nanoparticles and the polymer system, consisted of washing out of the particles from the hydrogel structure. The maximum entropy method with an analysis using log-normal distribution of diffusion coefficients was selected as the last and the most advanced polymer network analysis of the, which defined the system in a new and an alternative way. The results obtained using these advanced analyses were similar to the data calculated using the mathematical model of anomalous diffusion. The description of properties using rheological modules was a completely new approach. The properties were obtained using a series of conversions from the measured autocorrelation curve. Therefore, a rheological module obtained from microrheological data was the output of the approach. The shape of the curve is comparable to classical (macro)rheology, but the numerical values are lower by an entire order of magnitude. The smallest particles in the entire concentration range behaved as if they were in a purely viscous environment while the largest particles defined the behavior of the system depending on the concentration from very viscous to viscoelastic. The last type of measurement was the study of hyaluronan using selected nanoparticles and its fluorescently labeled analogue. All the methods that were used in the study of agarose hydrogel were applied to the viscoelastic system of hyaluronan to verify their applicability.
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Study of fluorescence lifetime and spectral changes of nanoparticles in cell biology
Pelc, Pavel ; Janoušek, Oto (referee) ; Čmiel, Vratislav (advisor)
This work deals with the study of fluorescence lifetime and spectral changes of nanoparticles in cell biology. It describes the principle of fluorescence, fluorescence microscopy and laser confocal microscope Leica TCS SP8. The classic FLIM method, the Lambda Square mapping and the division of nanoparticles are introduced there. In the practical part, the created program for the evaluation of fluorescence lifetime and spectral changes is described. The program can show two-dimensional lambda maps, the fluorescence lifetime and spectral shift in the space area. In the final part of the thesis, an experiment with rhodamine nanoparticles is carried out and it is evaluated using the created program and then discussed.
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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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Intrinsic fluorescence of bacteria Cupriavidus necator
Marková, Kateřina ; Obruča, Stanislav (referee) ; Mravec, Filip (advisor)
This thesis focuses on autofluorescence of flavins in gram-negative bacteria Cupriavidus necator H16 and its mutant strain PHB-4. The main methods used were fluorescence microscopy and flow cytometry. To confirm the presence of flavins, excitation and emission spectra of the bacterial suspension were measured, which were compared with flavin standards. In the part of testing cells without stress response, the autofluorescence of bacteria in PBS buffer and cell suspensions stained with fluorescence probe BODIPY 493/503 was measured. The ratio of short fluorescence lifetime to long autofluorescence lifetime, and its dependence on fluorescence probe was compared with previous conditions. Autofluorescence of the supernatant was measured; it was found that the relative amplitude of long lifetime was multiple times higher than in the cell. In the part devoted to the stress response, this thesis was focused on the amount of dissolved oxygen in the production medium and the effect on bacterial autofluorescence. Then differently concentrated hydrogen peroxide was used, the best results were obtained from the concentration of 100 mM in media. For comparison a combination of hydrogen peroxide with ferro-ammonium sulphate was used, but there was no big difference. Sodium azide and antimycin A were selected as substances that directly influence on bacterial respiratory chain. Both compounds affected change in the ratio of the relative amplitudes, but the distribution of these lifetimes and the autofluorescence change over time was affected only by sodium azide.
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Correlation between steady-state fluorescence anisotropy measurement on fluorimeter and fluorescence microscope
Moslerová, Lenka ; Venerová, Tereza (referee) ; Mravec, Filip (advisor)
This Work concentrates on detection fluorescent anisotropy on a model system of fluorescent probe and glycerol. In this thesis, the ATTO 488 was used as a fluorescent substance and glycerol solutions of different concentrations were used to simulate different viscous environments. A fluorescence spectrofluorometer and confocal fluorescence microscope were used for the measurements. A linear encrease anisotropy with increasing viscosity of the environment was observed on fluorometer. The same trend was detected on fluorescence microscope. The values were compared and correlation factors were determined. The accuracy of the measurement was verified by calculations using the Perrin´s equation.
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Software FLIM system with pulse white light laser in confocal microscopy
Grund, Pavel ; Odstrčilík, Jan (referee) ; Čmiel, Vratislav (advisor)
The theoretical part of this master's thesis is focused on research of confocal microscopy and FLIM method. There are a principles and types of confocal microscopy and the use of broad-spectrum laser as a basic light source of these microscopes. It gives what the FLIM method and its use not only in cell biology. The practical part thesis includes the acquisition of three sets of fluorescence intensity images with use of applications tunable pulsed laser, function TimeGate and detection of hybrid detectors. For practical elaboration of this thesis is in the software Fiji created a plugin, which is the source code in the Java programming language. The types of plugins and their uses are described in the third chapter of the thesis. This plugin including the graphical user interface in the form of the dialog box, proceses the fluorescence intensity images and creates a graphical representation of data showing the fluorescence lifetime, so called pseudocolor map.
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Software FLIM system in confocal microscopy
Petrula, Jakub ; Odstrčilík, Jan (referee) ; Čmiel, Vratislav (advisor)
This bachelor thesis deals with the FLIM system utilization in confocal microscopy. It describes basic concepts which are closely related to the topic, such as principles of fluorescence, fluorescent dyes and principles of both fluorescence and confocal microscopy. Practical part involves work with the confocal microscope and set of intensity images acquisition of both the animal and plant cells. Final product of pratical part is fluorescence lifetime image. The thesis also describes an algorithm for generating a pseudocolor frames and created user interface.
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Study of UV-generated fluorescent zinc complexes by fluorescence spectroscopy
Havlíková, Martina ; Vaculovičová, Markéta (referee) ; Mravec, Filip (advisor)
This thesis focuses on the study of UV light-generated zinc complexes with cadmium and organic molecules SAM, SAH, CYS, HCYS and GSSG, specifically at 375 nm. Furthemore, the aim of the work is to characterize the precursors spectrally and temporally before and after irradiation in the transilluminator at 250 nm. Study of genesis these complexes was performed by FLIM. Thanks to this method, it was found that the formation of complexes occurs only with Zn:SAH, Zn:GSSG and Zn:Cd. The formation of complexes is influenced by the method of preparation. The spectral characteristic was performed on a fluorimeter where the increase in fluorescence intensity of the irradiated solution with the precursors was expected. These were turbid solutions where sedimentation of the particles was observed and the intensity of fluorescence was changed. In the Zn:SAM and Zn:CYS sample, the sedimentation increased in intensity, while in Zn:SAH and Zn:HCYS decreased. The Zn:Cd precursor solution was clear and there was no change in intensity. Zn:Cd showed the best spectral properties, while the Zn:SAM sample, whose excitation and emission maxima are very close to each other, appeared to be the worst. A sample with Zn:CYS and Zn:HCYS showed almost the same spectra and respective peak results. Based on lifetime characteristics by TCSPC, the sample with Zn:CYS, Zn:HCYS and Zn:GSSG, which showed 3 lifetimes, was best treated. Lifetime could not be unambiguously determined for SAM and SAH samples. Zn:Cd had 4 lifetimes
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