TIRF Super-resolution microscope

在弗莱堡大学医学院生物化学和分子生物学研究所，九个研究小组研究细胞功能的分子机制，重点是膜的生物发生、运动和周转、线粒体组织、自噬与自噬小体的形成和动力学、核糖体组装和动力学。由于生化项目对先进的活细胞成像技术提出了很高的要求，因此迫切需要一种操作灵活、先进的显微镜。在我们的所有项目中，都涉及到小结构或小泡，这是广场显微镜不可能解决的。为了了解结构的动力学、组装和亚结构，我们需要先进的显微技术，包括超分辨率显微镜，它允许分析活细胞中的蛋白质复合体及其动力学，以及体外重组的蛋白质组装。特别是对于哺乳动物细胞的亚结构的分析，广域显微镜是有限的，因为它不能提供足够的对比度来看到小的点状物体，如自噬小体。我们申请了一个集成的超分辨率显微镜系统，它允许所有设置都有类似的工作流程，因此可以有效地培训研究生和博士后，并且易于使用和维护。该显微镜将满足生物化学和分子生物学研究所九个研究小组的主要需求(包括两项ERC拨款、一项DFG Koselleck项目、一项Heisenberg项目、一项Emmy-Noether项目、新SFB的七项项目，以及卓越CIBSS和研究生院的许多项目)。

At the Institute of Biochemistry and Molecular Biology of the Faculty of Medicine (University of Freiburg), nine research groups study molecular mechanisms underlying cell function, with a focus on membrane biogenesis, movement and turnover, mitochondrial organization, autophagy with formation and dynamics of autophagosomes, ribosome assembly and dynamics. Since the biochemical projects now have developed a high demand for advanced live cell imaging, a microscope with a flexible and advanced operation is highly needed. In all our projects, small structures or vesicles are involved, which are impossible to resolve by wide-field microscopy. To understand the dynamics, assemblies and substructure of the structures, we need advanced microscopy techniques including super-resolution microscopy, which allows to analyze protein complexes and their dynamics in living cells as well as protein assemblies of in vitro reconstitutions. Especially for the analysis of substructures also in mammalian cells, wide field microscopy is limiting, as it does not provide enough contrast to see small punctate objects such as autophagosomes. We apply for an integrated super-resolution microscopy system, which allows a similar workflow for all of the setups and therefore an efficient training of the graduate students and postdocs as well as an easy usage and maintenance. The microscope will fulfill a major demand of the nine research groups of the Institute of Biochemistry and Molecular Biology (including two ERC grants, a DFG Koselleck project, a Heisenberg project, an Emmy- Noether project, seven projects of the new SFB, and numerous projects in the cluster of excellence CIBSS and in graduate schools).