Untersuchung der Photoschädigung bei der hoch aufgelösten Lebendzell-Fluoreszenzmikroskopie

高分辨率活细胞荧光显微镜中光损伤的研究

• 批准号: 216430964
• 负责人: Professor Dr. Markus Sauer
• 金额: --
• 依托单位: Lehrstuhl für Biotechnologie und Biophysik
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2012
• 资助国家: 德国
• 起止时间: 2011-12-31 至 2014-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/216430964?language=en
• 关键词: Untersuchung; der; Photosch; digung; bei

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.

该项目旨在开发活细胞荧光显微镜应用中光损伤的调查和最小化方法，特别关注基于单分子的超分辨率成像方法。为了这个目的，我们将开发的荧光标记的细胞照射后产生的细胞内活性氧（ROS）的定量方法。ROS如单线态氧和过氧化氢是在荧光团激发和分子氧和硫醇（活细胞中的谷胱甘肽）淬灭其三线态时产生的。由于ROS引起活细胞的光损伤，因此必须避免或至少减少其光诱导形成。我们打算测试各种细胞内活性氧测定试剂盒，采用细胞可渗透的非荧光探针，如二氯二氢荧光素和二氯罗丹明，迅速氧化的活性氧高荧光探针。或者，我们将使用遗传编码的高度特异性荧光蛋白（例如，环状排列的黄色荧光蛋白Hyper）进行细胞器特异性过氧化氢检测。活细胞（细胞质、细胞核、线粒体）中的不同靶蛋白将使用化学标签和可光活化的荧光蛋白用不同的有机荧光团进行遗传标记。应用各种激发几何结构，如宽场与单面照明显微镜（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，添加抗氧化剂，如抗坏血酸和Trolox以及荧光团修饰），我们的目的是研究基于单分子定位的超-分辨率成像方法，并揭示如何将细胞光损伤最小化，以实现长期活细胞超分辨率成像。我们的研究结果将提供一个定量的指导，以避免在活细胞超分辨率成像实验中的生理损伤。