Confocal spinning disk microscope with FRAP and TIRF

配备 FRAP 和 TIRF 的共焦转盘显微镜

• 批准号: 461336323
• 金额: --
• 依托单位: Rheinland-Pfälzische Technische Universität Kaiserslautern-Landau
• 依托单位国家: 德国
• 项目类别: Major Research Instrumentation
• 财政年份: 2021
• 资助国家: 德国
• 起止时间: 2020-12-31 至 无数据
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/461336323?language=en
• 关键词: Confocal; spinning; disk; microscope; FRAP

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.

对于亚细胞结构和过程的研究，荧光标记蛋白质的高分辨率显微镜是一种重要的方法，对于动态质膜相关过程的研究，我最近在凯泽斯劳滕工业大学（TUK）成立的纳米生理学研究小组尤其重要。依赖于高分辨率活细胞成像，能够随着时间的推移跟踪所涉及的蛋白质的变化，显着影响我们的细胞与其环境相互作用的受体蛋白质的类型和数量。例如，质膜决定了哪些信号通路被激活以及激活的程度，对于我们有机体的生存来说，我们的细胞对其不断变化的环境做出动态反应至关重要，实现这一目标的一种方法是对其表面蛋白质组进行有针对性的适应。在这个过程中，选定的表面蛋白周围的质膜在复杂的蛋白质机器的帮助下内陷，形成囊泡，将其货物运输到细胞中，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接头蛋白的帮助下选择单个表面蛋白进行内吞作用，以及它们内化的缺陷会对我们的功能产生什么生理后果。细胞迁移需要细胞在其前进末端反复形成新的粘着斑作为锚点，然后随着细胞的内吞作用而必须再次分解。从膜上去除粘附蛋白的过程，但复杂的粘着斑的受控分解到底是如何发生的尚不清楚。近年来，我们已成功地以多种方式使用高分辨率活细胞显微镜来研究该过程。为了在 TUK 成功地继续我们的研究，我们正在与 Kins 教授和 Storchová 教授一起申请共焦旋转圆盘显微镜。还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，转盘显微镜需要包括 FRAP（光漂白后的荧光恢复）、TIRF（总荧光恢复）的扩展。内反射）和超分辨率显微镜，为了解析纳米级亚细胞结构，我们正在充分研究并研究其成分的动力学。