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.

该项目旨在开发用于调查和最小化光损伤的方法，以特别关注基于单分子的超分辨率超分辨率成像方法。为此，我们将开发用于定量荧光标记细胞后产生的细胞内活性氧（ROS）的方法。在激发荧光团和通过分子氧和硫醇（在活细胞中）激发其三胞胎状态的ROS（例如单线氧和过氧化氢）会产生。由于ROS会引起活细胞的光损伤，因此必须避免或至少降低其光诱导的形成。我们打算使用可使用细胞可渗透的非荧光探针的各种细胞内ROS分析套件，例如二氯二氢氟乙烯和二氯二胺被ROS迅速氧化为高度荧光探针。另外，我们将使用遗传编码的高度特异性荧光蛋白（例如，圆形置换的黄色荧光蛋白超级）进行细胞器特异性的过氧化氢检测。活细胞中不同的靶蛋白（细胞质，核，线粒体）将使用化学标签和可光活化荧光蛋白在不同的有机荧光团中遗传标记。应用各种激发几何形状，例如宽场与单平面照明显微镜（SPIM）和实验条件（荧光团浓度，激发能力，剂量和持续时间，恢复阶段的影响，恢复阶段的影响，添加抗氧化剂，抗氧化剂，例如抗胆酸和Trox，以及基于基于液位修饰的抗氧化剂），我们的定位方法是单身方法和范围 - 单个方法和范围 - 为了揭示如何最小化细胞光损伤以实现长期的实时超分辨率成像。我们的结果将提供定量指南，以避免在活细胞超分辨率成像实验中生理损害。