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Theoretical spectroscopy. Spectroscopic techniques

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In the first chapter, the aim and the outline of the thesis are given. The thesis will be focused on the possibility of using fluorescence detection of single molecules (SM) for biological applications. These applications became possible in the last years due to the development of experimental setups allowing the detection of single molecules not only at cryogenic temperatures, but also at room temperature. Another impulse was given by the possibility of obtaining information not only from immobilized single molecules, but also for diffusing molecules. Even though SM techniques just started to be exploited in biology, they already attracted many researchers, so an overview of the literature data on this topic is given. The advantages of SM detection are also discussed. In the second chapter, the theoretical principles of the confocal fluorescence microscopy are discussed, with particular emphasis on the multiparametric single molecule detection. How to obtain information from the fluorescence intensity trace on the macrotime scale (fluorescence correlation spectroscopy) and on the microtime scale (decay time measurements) is explained in detail. The experimental setup used for the measurements is also described. The experimental results are presented in three chapters. The SM detection requires the use of fluorophores with high extinction coefficient, high quantum yield, and improved stability to photobleaching. These are in fact the characteristics of the new perylene imide derivatives proposed in this thesis for biological studies. As it was recognized by Weiss (1999), “the development of better probes and the full photophysical characterization, on the single-molecule level, of existing dyes are crucial” for further biological applications. Without a deep understanding of the photophysical processes which can occur in the fluorescent molecules, the variation of their parameters (intensity, decay time etc.) in biological systems cannot be correctly interpreted. The photophysical properties of two new water-soluble fluorescent dyes based on the perylene diimide chromophore are investigated in chapter III at the ensemble and at the single molecule level. Water solubility was obtained by attaching hydrophilic substituents in the bay region. The fluorophores have absorption maxima in the green region of the visible spectrum and high fluorescence quantum yields (» 0.6). In addition, the two-photon absorption cross section of one compound has higher or comparable values with the ones reported in literature for other water-soluble dyes used in biological studies. The same compound has also an improved photostability in single molecule experiments when compared to other water-soluble dyes, probably due to a lower intersystem crossing rate constant than, for example, for rhodamine derivatives. Fluorescence correlation spectroscopy measurements in aqueous solution sustain this finding. The possibility of imaging the new molecules in living cells is demonstrated by one-photon confocal microscopy and by fluorescence lifetime microscopy with two-photon excitation. The differences in lipoplexes formation for normal and cholesterol-modified oligonucleotides are investigated in chapter IV. Data are recorded using a single photon counting card for single molecule experiments instead of a hardware autocorrelator. A separate analysis was done for the baseline fluorescence levels and for the fluorescent bursts in the same trace, an approach which was not use in previous studies of lipoplexes using fluorescence correlation spectroscopy. From the baseline fluorescence levels, the number of free and bound DNA molecules, the presence of tens-nm and/or of hundreds-nm sized lipoplexes could be estimated by applying various mathematical concepts. The analysis of the fluorescent bursts provided indication about the size of the µm-sized lipoplexes, the number of DNA molecules present in these large aggregates and the relative amount of lipids in each aggregate. An explanation for the higher transfection efficiency previously reported for the compound bearing 4 oligonuleotides attached to the cholesterol molecule was found in relation to the formation of bigger lipoplexes compared to the other investigated compounds. In chapter V, a new membrane probe containing the perylene imide chromophore with excellent photophysical properties (high absorption coefficient, quantum yield (QY) » 1, high photostability) is proposed for the study of membrane rafts. Visualization of separation between the liquid ordered (Lo) and the liquid disordered (Ld) phases can be achieved in artificial membranes by fluorescence lifetime imaging due to the different decay times of the fluorophore in the two phases. Rafts on µm scale in cell membranes due to cellular activation were also observed by this method. The decay time of the dye in the Lo phase is higher than in organic solvents where its QY is 1. This allows proposing a (possible general) mechanism for the decay time increase in the Lo phase, based on the local field effects of the surrounding molecules. For other fluorophores with QY<1, the suggested mechanism could contribute in addition to other effects reducing the non-radiative decay pathways leading to an increase of the fluorescence decay time in the Lo phase. In the second part of the chapter, a method for determining the size of the nm-scale rafts (similar to those proposed to exist in resting cells) in confocal fluorescence microscopy with single molecule detection is developed. The spatial information (i.e. rafts size) is obtained from the temporal scale of the exchange dynamics of PMI-COOH between the liquid disordered (Ld) and the liquid ordered (Lo) domains and from the temporal scale of the diffusion processes in the two phases. The presence of the dye in the Ld or in the Lo phase can be assigned due to its different fluorescence decay times. The time scale of the fluorescence decay time fluctuations (i.e. the exchange dynamics) is determined from the autocorrelation function of photon arrival times (Yang and Xie, J. Chem. Phys., 117 : 10965-79, 2002). By fitting the autocorrelation function with an exponential decay, the average time spent by the fluorescent molecule in a given phase can be derived. The diffusion coefficients in each phase are obtained from the fluctuations of the fluorescence intensity. The time spent by the dye in a Lo domain and the characteristic diffusion coefficient for the Lo domain are used to calculate the mean square displacement, which can be correlated with the raft size. In order to test the validity of the method and to establish the limits within which the method can be applied, the dye exchange is modeled as a Markov process between two states with two different decay times and the transition rate constants between the two states are varied. In the first chapter, the aim and the outline of the thesis are given. The thesis is focused on the possibility of using fluorescence detection of single molecules (SM) for biological applications. Even though SM techniques just started to be exploited in biology, they already attracted many researchers, so an overview of the literature data on this topic is given. The advantages of SM detection are also discussed. In the second chapter, the theoretical principles of the confocal fluorescence microscopy are discussed, with particular emphasis on the multiparametric single molecule detection. How to obtain information from the fluorescence intensity trace on the macrotime scale (fluorescence correlation spectroscopy) and on the microtime scale (decay time measurements) is explained in detail. The experimental setup used for the measurements is also described. The experimental results are presented in three chapters. The SM detection requires the use of fluorophores with high extinction coefficient, high quantum yield, and improved stability to photobleaching. These are in fact the characteristics of the new perylene imide derivatives proposed in this thesis for biological studies. As it was recognized by Weiss (1999), “the development of better probes and the full photophysical characterization, on the single-molecule level, of existing dyes are crucial” for further biological applications. Without a deep understanding of the photophysical processes which can occur in the fluorescent molecules, the variation of their parameters (intensity, decay time etc.) in biological systems cannot be correctly interpreted. The photophysical properties of two new water-soluble fluorescent dyes based on the perylene diimide chromophore are investigated in chapter III at the ensemble and at the single molecule level. Water solubility was obtained by attaching hydrophilic substituents in the bay region. One compound has an improved photostability in single molecule experiments when compared to other water-soluble dyes. The possibility of imaging the new molecules in living cells is demonstrated by one-photon confocal microscopy and by fluorescence lifetime microscopy with two-photon excitation. The differences in lipoplexes formation for normal and cholesterol-modified oligonucleotides are investigated in chapter IV. From the baseline fluorescence levels, the number of free and bound DNA molecules, the presence of tens-nm and/or of hundreds-nm sized lipoplexes could be estimated by applying various mathematical concepts. The analysis of the fluorescent bursts provided indication about the size of the µm-sized lipoplexes, the number of DNA molecules present in these large aggregates and the relative amount of lipids in each aggregate. In chapter V, a new membrane probe containing the perylene imide chromophore with excellent photophysical properties is proposed for the study of membrane rafts. Visualization of separation between the liquid ordered (Lo) and the liquid disordered (Ld) phases can be achieved in artificial membranes by fluorescence lifetime imaging due to the different decay times of the fluorophore in the two phases. Rafts on µm scale in cell membranes due to cellular activation were also observed by this method. In the second part of the chapter, a method for determining the size of the nm-scale rafts (similar to those proposed to exist in resting cells) in confocal fluorescence microscopy with single molecule detection is developed. The spatial information (i.e. rafts size) is obtained from the temporal scale of the exchange dynamics of PMI-COOH between the liquid disordered (Ld) and the liquid ordered (Lo) domains and from the temporal scale of the diffusion processes in the two phases. In order to test the validity of the method and to establish the limits within which the method can be applied, the dye exchange is modeled as a Markov process between two states with two different decay times and the transition rate constants between the two states are varied.

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Today, LEDs surround modern people everywhere: they are embedded in mobile devices, household appliances, and cars, not to mention their almost ubiquitous use in light advertising. It's no secret that the light efficiency of LED is increasing every year, and their cost at the same time is becoming more affordable. Over the past few years, compromises have constantly had to be made in the production of LEDs: achieving high light output led to a reduction in the service life of the device, and a high-quality color rendering index in a new light source was often combined with a relatively low brightness of the diode. They try to produce materials, that will show higher brightness and quality of emitted light at a lower cost than their current counterparts. Also, an increase in their energy efficiency will be an advantage too. There is no doubt that the prices of LEDs will decrease in the coming years. Several years ago, researchers have discovered a new phosphor, with which it is possible to reduce the cost of production and increase the efficiency of fluorescent and LED lamps. This discovery has a huge potential for the development of new generation fluorescent and LED light sources since new phosphors not only emit a lot of light but also are cheaper at production cost than currently common analogs. This phosphorus is based on highly luminescent clusters of silver atoms and the porous structure of minerals known as zeolites. Silver clusters consist of only a few silver atoms and at the same time have outstanding optical properties. Nevertheless, their scope of application is limited, since clusters tend to combine into larger particles, which lose their advantages. The scientists found a way to keep silver clusters at a distance from each other by placing them in a porous zeolite structure. As a result, it was possible to ensure the stability of silver clusters in operation and the preservation of their unique optical properties for a long time. Copper clusters are also known as good luminescent materials and their application faces the same problems as silver clusters, that can be solved in the same manner. The success of this development made metal clusters confined in zeolites a promising field for further investigation. In this thesis, we produced a material, where two metals were placed inside the zeolite to create mixed silver-copper clusters and its properties were investigated. Indeed, it was confirmed that some mixed clusters were formed, which have the properties of both silver, and copper and some unique properties too. There is still a long way to go, however, this work is a good base for further investigations.

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Microplastic pollution is one of the most critical environmental issues in the world right now. Many studies about human exposure to these micro-and nanoplastics (MNP) and the toxic effects they can cause are being conducted at this moment. Quantification and identification of these MNP lie at the bottom of solving this global problem. The most popular identification methods are Fourier-transform infrared spectroscopy and Raman spectroscopy. These techniques are lacking in non-destructive analysis with high sensitivity and low background noise. This thesis aims to develop an optical spectroscopy method to detect and identify microplastics after staining with Nile Red. The latter is a lipophilic solvatochromic dye. Staining will cause the particle to fluoresce, with its emission spectrum depending on its polarity. More polar plastics will have a more red-shifted emission maximum. Via hyperspectral analysis, the MNP can be identified based on this spectrum. Fluorescence lifetime imaging microscopy is also implemented. The fluorescence lifetime is the time that a fluorophore spends in the excited state, after absorbing a photon, before returning to the ground state. The lifetime depends on the fluorophore's micro-environment and therefore is also different for each MNP type. Differentiation is possible based on this lifetime value, but a graphical, fit-free phasor approach is also used. This allows for the mapping of the lifetime distribution in an image. When two different MNP types are present in one sample, they will each have a separate distribution cloud on the phasor plot, allowing for the graphical distinguishment of MNP. The plastics used are PP, PVC, HDPE, PET, and PS, these samples contain a range of shapes and sizes. A bought suspension of pure PS polymer particles of a fixed shape and size of 0,6 μm was also examined. The spectral data were processed in two ways. By calculating a ‘weighted average’ with the intensity values at each wavelength. This resulted in each MNP being attributed a weighted average value. After recording spectra of different particles, a statistical analysis, ANOVA and t-test, was done. This showed that the weighted averages of PP & HDPE and PS (self-made) & PVC do not differ significantly. The second option is through comparing the spectra themselves, using a relative similarity percentage. Because of the low similarity MNP gave when compared with themselves, only MNP with a significantly large similarity percentage could be distinguished. The same statistical tests were done on lifetime data. Measurements done on different particles of the same plastic showed that only HDPE & PET lifetimes did not differ significantly. Multiple measurements done on the same particle showed slightly different lifetimes. It could be concluded that HDPE & PET and PS (self-made and 0,6 μm) & PET cannot be differentiated. Finally, phasor plot analysis of samples containing two different plastics showed a possibility of recognizing MNP types based on the location of their phasor cloud on a phasor plot. When combining these two techniques, making use of the ’weighted average’ value and lifetime analysis, it could be possible to identify each plastic. The combination with similarly weighted averages, PP & HDPE give significantly different fluorescent lifetimes values and are possible to separate on a phasor plot. Still, both techniques have some flaws and improvement is possible.