Untersuchung der Photoschädigung bei der hoch aufgelösten Lebendzell-Fluoreszenzmikroskopie

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The project aims to develop methods for the investigation and minimization of photodamage in live-cell fluorescence microscopy applications with a particular focus on single-molecule based superresolution imaging methods. For this purpose, we will develop methods for the quantification of intracellular reactive oxygen species (ROS) produced upon irradiation of fluorescently labeled cells. ROS such as singlet oxygen and hydrogen peroxide are produced upon excitation of fluorophores and quenching of their triplet states by molecular oxygen and thiols (in live cells glutathione). Since ROS cause photodamage of live cells their photoinduced formation has to be avoided or at least reduced. We intend to test various intracellular ROS assay kits employing cell-permeable non-fluorescent probes, e.g. dichlorodihydrofluorescein and dichlororhodamine that are rapidly oxidized to highly fluorescent probes by ROS. Alternatively, we will use genetically encoded, highly specific fluorescent proteins (e.g. the circularly permuted yellow fluorescent protein HyPer) for organelle-specific hydrogen peroxide detection. Different target proteins in living cells (cytoplasm, nucleus, mitochondria) will be genetically labeled with different organic fluorophores using chemical tags and photoactivatable fluorescent proteins. Applying various excitation geometries such as wide-field versus single-plane illumination microscopy (SPIM) and experimental conditions (fluorophore concentrations, excitation power, dose and duration, influence of recovery phases, addition of antioxidants such as ascorbic acid and Trolox as well as fluorophore modifications) we aim to study the extent of photodamage induced by single-molecule localization based super-resolution imaging methods and to unravel how cellular photodamage can be minimized to enable long-term live-cell super-resolution imaging. Our results will provide a quantitative guideline for avoiding physiological damage during live-cell super-resolution imaging experiments.