Abstract | Keywords | Export | Availability | Bookmark

Choose an application

• Reference Manager
• EndNote
• RefWorks (Direct export to RefWorks)

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.