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.

为了研究亚细胞结构和过程，荧光标记蛋白的高分辨率显微镜是一种必不可少的方法。为了研究动态质膜相关的过程，我最近在TechnischeUniversitätkaiserslautern（TUK）中建立的纳米生理研究小组特别取决于高分辨率的活细胞成像，以便能够随着时间的推移遵循所涉及的蛋白质。质膜结合蛋白的变化显着影响我们的细胞与环境的相互作用。质膜中掺入的受体蛋白的类型和数量确定，例如，哪些信号通路被激活以及在多大程度上激活。对于我们生物体的生存，我们的细胞必须动态反应它们不断变化的环境。实现这一目标的一种方法是通过内吞作用的靶向适应其表面蛋白质组。在此过程中，质膜区域选择的表面蛋白在一个复杂的蛋白质机械的帮助下起伏，形成将其货物运输到细胞的场所。例如，这种机制允许从表面上去除依从性蛋白，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的衔接蛋白的帮助下选择单个表面蛋白来进行内吞作用，以及其内在化的生理后果缺陷，无论是用于我们大脑的功能还是细胞迁移。细胞迁移要求细胞反复形成新的焦点粘合剂作为其前进端的锚点，然后必须在细胞移动时再次拆卸。内吞作用是从膜上去除粘合剂蛋白的可能过程，但是如何确切地了解复杂焦点粘合剂的受控拆卸。近年来，我们通过多种方式成功使用了高分辨率的活细胞显微镜，以详细研究内吞作用和局灶性粘合剂的调节。为了成功地在TUK上继续进行研究，我们正在与Kins和Storchová教授一起申请共聚焦旋转磁盘显微镜，他们也正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长期的多色实用细胞显微镜。旋转磁盘显微镜需要包括用于FRAP的扩展（光漂白后的荧光恢复），TIRF（总内部反射）和超级分辨率显微镜，以解决纳米尺度的亚细胞结构，我们正在充分研究纳米尺度的亚细胞结构，并可以很好地研究其组件的动力学。