Untersuchung der Photoschädigung bei der hoch aufgelösten Lebendzell-Fluoreszenzmikroskopie

该项目旨在开发研究和最小化活细胞荧光显微镜应用中的光损伤的方法，特别关注基于单分子的超分辨率成像方法。为此，我们将开发定量的方法，细胞内产生的活性氧(ROS)的照射荧光标记细胞。ROS如单线态氧和过氧化氢是在荧光团被分子氧和硫醇(活细胞中的谷胱甘肽)猝灭其三重态时产生的。由于ROS会对活细胞造成光损伤，因此必须避免或至少减少它们的光诱导形成。我们打算使用细胞渗透性的非荧光探针来测试各种细胞内ROS检测试剂盒，例如被ROS快速氧化为高荧光探针的二氯二氢荧光素和二氯罗丹明。或者，我们将使用基因编码的高度特异的荧光蛋白(例如，循环排列的黄色荧光蛋白HYPER)来检测细胞器特异性的过氧化氢。活细胞中的不同靶蛋白(细胞质、核、线粒体)将使用化学标记和可光激活的荧光蛋白，用不同的有机荧光团进行基因标记。应用不同的激发几何形式，如广场和单面照明显微镜(SPIM)和实验条件(荧光团浓度、激发功率、剂量和持续时间、恢复阶段的影响、抗氧化剂的添加，如抗坏血酸和Trolox以及荧光团的修饰)，我们旨在研究基于单分子定位的超分辨率成像方法引起的光损伤的程度，并揭示如何将细胞光损伤降至最低，以实现长期的活细胞超分辨率成像。我们的结果将为避免活细胞超分辨率成像实验中的生理损伤提供定量的指导。

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.