Fluorescence microscope (wide field system with inverse optics)

细胞内膜将真核细胞细分为特定的隔室，这使得不同的过程可以在空间上分离并以相当独立的方式同时发生。然而，对于细胞的生物发生来说，这种划分可能是一个巨大的挑战，原因有几个：(I)真核细胞包含两个基因组，在藻类和植物的情况下，甚至三个基因组的基因表达需要协调。(2)新合成的蛋白质必须从它们的合成部位运输到它们的最终功能目的地，在大多数情况下，它们必须跨膜转运。(3)全面的质量控制机制需要确保不同隔间中的所有这些过程是协调的，为此需要立即识别和解决局部问题。在几个研究项目中，我们分析了真核细胞的生物发生，从而特别关注包含基因组的细胞器中的过程，如线粒体和叶绿体。我们主要使用面包师酵母菌酿酒酵母和藻类莱茵衣藻作为简单的模式生物。然而，对于特定的实验，我们也使用培养的哺乳动物细胞。在这里，我们申请了一种具有反向光学的荧光显微镜来进行生命细胞成像。该仪器将被用来同时定位这些非常小的细胞中的几个单独的荧光标记蛋白，并研究它们在不同生理和应激条件下的细胞内分布。通过使用微流体室，细胞将被可视化几代，例如，阐明蛋白质聚集体的形成和遗传，以及测量细胞内细胞器及其基因组的动态形状、体积和分布。除了优良的光学系统和完全自动化的设备外，还需要用于图像分析的软件包，例如用于去卷积。从这些项目中，我们期望对特定的伴侣、组装因子、易位复合体和蛋白酶在线粒体和叶绿体的生物发生以及在不同(病理)生理条件下维持真核细胞功能的过程中的功能和相关性有详细的分子洞察。

Intracellular membranes subdivide eukaryotic cells into specific compartments which allows that different processes can occur simultaneously in a spatially separated and rather independent manner. For the biogenesis of cells, this compartmentalization, however, can be a big challenge for several reasons: (i) Eukaryotic cells contain two, in the case of algae and plants even three genomes from which gene expression needs to be coordinated. (2) Newly synthesized proteins have to be transported from the site of their synthesis to their final destination of function, for which, in most cases, they have to be translocated across membranes. (3) Comprehensive quality control mechanisms need to make sure that all these processes in the different compartments are coordinated for which it is required that local problems are immediately recognized and solved.In several research projects we analyze the biogenesis of eukaryotic cells, thereby particularly focusing on processes in genome-containing organelles such as mitochondrial and chloroplasts. We predominantly use the baker’s yeast Saccharomyces cerevisiae and the algae Chlamydomonas reinhardtii simple model organisms. For specific experiments, however, we also work with cultured mammalian cells.Here we apply for a fluorescence microscope with inverse optics for life cell imaging. This instrument will be used to localize simultaneously several individual fluorescence-labeled proteins in these very small cells and to study their intracellular distribution under different physiological and stress conditions. By use of microfluidics chambers the cells will be visualized for several generations, for example to elucidate the formation and inheritance of protein aggregates and to measure the dynamic form, volume and distribution organelles and their genomes within cells. In addition to an excellent optic system and fully automatization devices, software packages will be required for the analysis of the images, for example for deconvolution. From these projects, we expect detailed molecular insights into the function and relevance of specific chaperones, assembly factors, translocation complexes and proteases for the biogenesis of mitochondria and chloroplasts as well as for processes which maintain the functionality of eukaryotic cells under variable (patho)physiological conditions.