Confocal spinning disk microscope with FRAP and TIRF

对于亚细胞结构和过程的调查，荧光标记蛋白质的高分辨率显微镜是一种必不可少的方法。对于动态质膜相关过程的研究，我最近在德国工业大学（TUK）成立的纳米生理学研究小组特别依赖于高分辨率活细胞成像，以便能够随着时间的推移跟踪相关蛋白质。质膜结合蛋白的变化显著影响我们的细胞与环境的相互作用。结合在质膜中的受体蛋白的类型和数量决定了例如哪些信号通路被激活以及激活到何种程度。对于我们的有机体的生存至关重要的是，我们的细胞对不断变化的环境做出动态反应。实现这一点的一种方法是通过内吞作用对其表面蛋白质组进行靶向适应。在这个过程中，选定的表面蛋白质周围的质膜在复杂的蛋白质机制的帮助下内陷，形成一个囊泡，将其货物运送到细胞中。这种机制允许，例如，从表面去除粘附蛋白，从而调节细胞迁移或内化突触后谷氨酸受体，从而实现突触可塑性。我们正在研究如何在特定衔接蛋白的帮助下选择单个表面蛋白进行内吞作用，以及它们内在化的生理后果是什么，无论是对我们大脑的功能还是对细胞迁移。细胞迁移需要细胞在其前进端反复形成新的粘着斑作为锚点，然后随着细胞继续移动，粘着斑必须再次分解。内吞作用是从细胞膜上去除粘着蛋白的可能过程，但复杂粘着斑的受控分解是如何发生的尚不清楚。近年来，我们已经成功地使用高分辨率活细胞显微镜在各种方式来研究的过程中的内吞和调节局灶性粘连的细节。为了继续我们在TUK的研究，我们与Kins教授和Storchová教授一起申请共聚焦旋转圆盘显微镜，他们也在研究动态亚细胞过程，因为这种类型的显微镜特别适合快速和持久的多色活细胞显微镜。旋转圆盘显微镜需要包括FRAP（光漂白后的荧光恢复），TIRF（全内反射）和超分辨率显微镜的扩展，以解决我们正在调查的纳米级亚细胞结构，并研究其组件的动力学。

For the investigation of subcellular structures and processes, high-resolution microscopy of fluorescently labelled proteins is an essential method. For its studies of dynamic plasma membrane-associated processes, my recently established research group for Nanophysiology at the Technische Universität Kaiserslautern (TUK) is particularly dependent on high-resolution live cell imaging to be able to follow the involved proteins over time. Changes in plasma membrane-bound proteins significantly influence how our cells interact with their environment. The type and number of receptor proteins incorporated in the plasma membrane determines, for example, which signaling pathways are activated and to what extent. For the survival of our organism it is essential that our cells react dynamically to their constantly changing environment. One way to achieve this is the targeted adaptation of their surface proteome via endocytosis. In this process, the plasma membrane around selected surface proteins invaginates with the help of a sophisticated protein machinery to form a vesicle that transports its cargo into the cell. This mechanism allows, for example, to remove adhesion proteins from the surface and thus to modulate cell migration or to internalize postsynaptic glutamate receptors and thus enable synaptic plasticity. We are investigating how individual surface proteins are selected for endocytosis with the help of specific adaptor proteins and what physiological consequences defects in their internalization have, be it for the functioning of our brain or for cell migration. Cell migration requires that cells repeatedly form new focal adhesions as anchor points at their advancing end, which then have to be disassembled again as the cell moves on. Endocytosis is a possible process to remove adhesion proteins from the membrane, but how exactly the controlled disassembly of the complex focal adhesions takes place is not yet understood. In recent years, we have successfully used high-resolution live cell microscopy in a variety of ways to study the process of endocytosis and the regulation of focal adhesions in detail. In order to continue our studies at the TUK successfully, we are applying for a confocal spinning disk microscope together with Prof. Kins and Prof. Storchová, who are also studying dynamic subcellular processes, as this type of microscopy is particularly well suited for both fast and long-lasting multi-colour live cell microscopy. The spinning disk microscope needs to include extensions for FRAP (fluorescence recovery after photobleaching), TIRF (total internal reflection) and super resolution microscopy, in order to resolve the nanometer-scale subcellular structures we are investigating sufficiently well and also to study the dynamics of their components.