Hochauflösende Fluoreszenz-Messstation mit konfokaler Mikroskopie und TIRF-Mikroskopie

我们申请一个荧光工作站，用于TIRF和共聚焦显微镜作为我们启动资金的一部分。该工作站的主要应用将是研究肌动蛋白细胞骨架的动力学，既包括体外分离的蛋白质复合物，也包括哺乳动物细胞系（例如HeLa和Cos-7）和寄生虫。我们将重点关注肌球蛋白vi在网格蛋白介导的内吞作用的不同阶段的功能，以及在利什曼原虫中发现的肌球蛋白xxi的作用。我们将以高位置和高时间分辨率在体外分离的单个蛋白质上以及在靠近细胞膜的完整细胞上研究运动蛋白复合物的形成和定位。复杂的形成和运动的动力更深的细胞将研究使用共聚焦显微镜与快速共振扫描仪。共振扫描仪将使我们能够进行FRAP实验，研究蛋白质复合物的动态分布，并对运动蛋白及其结合伙伴的共定位进行时间分辨FRET研究。结合图像分析的光谱成像检测器将帮助我们分离混合荧光信号，并提高在多色荧光成像中检测单个荧光团的信噪比。

We apply for a fluorescence work-station for both TIRF- and confocal microscopy as part of our start-up funding. The main application of this work-station will be studies of the dynamics of the acto-myosin cytoskeleton, both on isolated protein complexes in vitro, but also in mammalian cell lines (e.g. HeLa and Cos-7) and in parasites. We will focus on the function of myosin-VI in different stages of clathrin-mediated endocytosis and on the role of myosin-XXI found in Leishmania parasites. We will study formation and localisation of motor protein complexes at high positional and time resolution on isolated single proteins in vitro and also in intact cells near the cell membrane in TIRF-mode. Dynamics of complex formation and motility deeper inside the cell will be studied using confocal microscopy with a fast resonant scanner. The resonant scanner will enable us to perform FRAP experiments, to study the dynamic distribution of protein complexes, and to carry out time resolved FRET studies for co-localisation of motor proteins and their binding partners. A spectral imaging detector combined with image analysis will help us to segregate mixed fluorescent signals and to improve signal-to-noise in detecting individual fluorophores in multi-colour fluorescence imaging.