Hochauflösende Fluoreszenz-Messstation mit konfokaler Mikroskopie und TIRF-Mikroskopie

作为启动资金的一部分，我们申请了一个用于TIRF和共聚焦显微镜的荧光工作站。该工作站的主要应用将是研究肌动球蛋白细胞骨架的动力学，无论是在体外分离的蛋白质复合物，但也在哺乳动物细胞系（如HeLa和Cos-7）和寄生虫。我们将集中在网格蛋白介导的内吞作用的不同阶段的肌球蛋白VI的功能和利什曼原虫中发现的肌球蛋白XXI的作用。我们将研究电机蛋白复合物的形成和定位在高的位置和时间分辨率上分离的单个蛋白质在体外，也在完整的细胞在TIRF模式的细胞膜附近。复合物的形成和运动的动力学更深的细胞内将使用共聚焦显微镜与快速共振扫描仪进行研究。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，并进行时间分辨FRET研究共定位的马达蛋白及其结合伙伴。光谱成像检测器与图像分析相结合，将有助于我们分离混合的荧光信号，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.