Over the past years major progress has been made in the field of quantitative biophysics regarding the self-organization of the cellular interior and protein dynamics by a multitude of fluorescence measurement techniques. Beyond mere dynamics, understanding cellular mechanisms and responses to external stress is of great importance for pharmaceutical and medical, as well as biophysical research. This thesis focuses on the spatially resolved stress-response of cellular fluids. Methods to quantify cellular stress are based on the locus-specific feedback given by nanoscopic molecular sensors inside living cells. These stress-translating molecules are known to change their photophysical properties through interaction with their micro-environment. The photophysical response to chemotherapeutica, oxidative and hyperosmotic stress induced by various stress-agents in different cell lines before and after drug treatment are analyzed by fluorescence lifetime and intensity measurements. Exploring the compartment-specific response of HeLa (human cervical carcinoma) cells to chemotherapeutical and oxidative stress with the help of the molecular rotor DASPMI reveals a significant decrease of mitochondrial viscosity after treatment with cisplatin as well as hydrogen peroxide. Additionally, a rapid and novel segmentation algorithm separating cellular compartments in phasor space is proposed. This phasor segmentation technique is based on the functionality of molecular rotors that shift their fluorescence lifetime contributions with respect to their surrounding and furthermore demonstrate negligible affinity to fluorescence quenching effects. CellRox-DeepRed, a sensor photoactivated by reactive oxygen species, reveals a decline of antioxidants in HPV (human papilloma virus)-positive HeLa-cells after the menadione-induced suppression of glutathione synthesis. In this context, several evaluation techniques are investigated, focussing on their reliability to distinguish small measurement differences in fluorescence lifetime data sets. Intracellular crowding measurements elucidate the ability of a crowding-sensor, FCrH2, to differentiate cellular fluids under different osmotic conditions. The folding and unfolding dynamics of this FRET-sensor, expressed in the nucleoplasmic and cytoplasmic region of HeLa-cells, provides information on the crowding-state of their surrounding. In this project the excellent sensitivity to hyperosmotic stress as well as the much lower but still measurable locus specificity of this molecular sensor is presented. In the scope of enabling spatiotemporal correlation measurements, a custom made microscope is described and characterized by means of geometrical as well as Fourier optics. The presented spectroscopic setup enables fast and scalable wide-field fluorescence applications utilizing the technique of inverted light-sheet microscopy.
|School:||Universitaet Bayreuth (Germany)|
|Source:||DAI-C 81/4(E), Dissertation Abstracts International|
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