Development of a Combined Stimulated Emission Depletion and Scanning Ion Conductance Microscope for Correlated Multi-Parameter Super-Resolution Live-Cell Imaging of the Tips of Processes of Oligodendrocyte Progenitor Cells

开发联合受激发射损耗和扫描离子电导显微镜，用于少突胶质细胞祖细胞尖端的相关多参数超分辨率活细胞成像

• 批准号: 411517989
• 负责人: Professorin Dr. Irmgard Dietzel-Meyer, since 7/2021
• 金额: --
• 依托单位: RUBION, Zentrale Einrichtung für Ionenstrahlen und Radionuklide
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2018
• 资助国家: 德国
• 起止时间: 2017-12-31 至 2023-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/411517989?language=en
• 关键词: Development; Combined; Stimulated; Emission; Depletion

Oligodendrocytes are cells from the brain that ensure the fast propagation of action potentials. They originate from their progenitors, the oligodendrocyte progenitor cells (OPCs) which are present in the developing and the adult brain and form the myelin that enwraps and electrically isolates the axons of neurons. To develop and maintain the proper brain function, OPCs migrate to unmyelinated axons or axons with damaged myelin to form novel or replace damaged myelin. Axons with damaged myelin or without myelin occur in patients suffering from Multiple Sclerosis, and thus the understanding of the migration machinery of OPCs is of major relevance. OPCs are cells that exhibit two processes originating at opposite sites from the cell body, and the cell body migrates along these processes. Thus, it is crucial for the understanding of OPC migration to understand the mechanism of the protrusion of the processes. However, the interplay of cell membrane extensions and the cytoskeleton at tips of the processes of OPCs has not been investigated in detail yet. The major reason for this is the size of the OPC tips, which are too small to be investigated in sufficient detail by conventional fluorescence microscopy methods. Furthermore, to be able to link membrane protrusion dynamics to cytoskeletal dynamics, the simultaneous tracing of the unbiased cell membrane and the cytoskeleton in living cells is required. This can not be achieved by electron microscopy, since the sample preparation and measurement conditions are not suitable for living cells.In recent years, super-resolution fluorescence microscopy methods like stimulated emission depletion (STED) microscopy have been developed, which allow imaging at high resolution and which should allow the investigation of single, labelled proteins in the tips of OPCs at sufficient detail, even in living cells. Furthermore, Scanning Ion Conductance Microscopy (SICM) has been established as a tool to trace the membrane contours of living cells with only minimal bias at a resolution in the same range as provided by STED microscopy.The aim of this project is the development and subsequent evaluation of a combined STED/SICM. The instrument will allow to trace the cell membrane dynamics and cytoskeleton dynamics of living cells at high resolution. In this project, we will first build and characterize a combined STED/SICM. Subsequently, we will record proof-of-concept images of fixed and living cells. In a subsequent project, we will use this instrument to unravel the molecular mechanics governing the dynamics of the tips of OPCs.

少突胶质细胞是来自大脑的确保动作电位快速传播的细胞。它们起源于它们的祖细胞，少突胶质细胞祖细胞（OPC），其存在于发育中和成年脑中，并形成包裹和电隔离神经元轴突的髓鞘。为了发展和维持适当的脑功能，OPCs迁移到无髓鞘轴突或具有受损髓鞘的轴突以形成新的或替代受损髓鞘。患有多发性硬化症的患者中存在髓鞘受损或没有髓鞘的轴突，因此对OPCs迁移机制的理解具有重要意义。OPCs是表现出起源于细胞体相对位点的两个突起的细胞，并且细胞体沿着这些突起迁移。因此，理解OPC迁移的关键是理解突起的机制。然而，在OPCs的过程中，细胞膜延伸和细胞骨架的相互作用尚未得到详细的研究。其主要原因是OPC尖端的尺寸太小，无法通过常规荧光显微镜方法进行足够详细的研究。此外，为了能够将膜突起动力学与细胞骨架动力学联系起来，需要同时追踪活细胞中的无偏细胞膜和细胞骨架。由于样品制备和测量条件不适合活细胞，因此电子显微镜无法实现这一点。近年来，超分辨率荧光显微镜方法如受激发射耗尽（STED）显微镜已经发展起来，它可以在高分辨率下成像，并且可以足够详细地研究OPC尖端的单个标记蛋白质，即使在活细胞中。此外，扫描离子电导显微镜（SICM）已被建立为一种工具，以跟踪活细胞的膜轮廓，只有最小的偏差，在同一范围内的分辨率提供的STED microscopy.The项目的目的是开发和随后的评估相结合的STED/SICM。该仪器将允许以高分辨率跟踪活细胞的细胞膜动力学和细胞骨架动力学。在这个项目中，我们将首先建立和表征一个组合STED/SICM。随后，我们将记录固定和活细胞的概念验证图像。在随后的项目中，我们将使用该仪器来解开控制OPC尖端动力学的分子力学。