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

少突胶质细胞是来自大脑的细胞，确保动作电位的快速传播。它们起源于它们的祖细胞，少突胶质前体细胞(OPC)，存在于发育中的和成年的大脑中，形成包裹和电隔离神经元轴突的髓鞘。为了发展和维持正常的大脑功能，OPC迁移到无髓鞘轴突或髓鞘受损的轴突，形成新的或替代受损的髓鞘。多发性硬化症患者存在髓鞘受损或无髓鞘的轴突，因此了解OPC的迁移机制具有重要意义。OPC是一种细胞，表现出起源于细胞体相反位置的两个突起，细胞体沿着这些突起迁移。因此，理解OPC迁移过程的突出机制对于理解OPC迁移是至关重要的。然而，细胞膜延伸和OPC过程尖端的细胞骨架之间的相互作用还没有详细的研究。主要原因是OPC尖端的大小，它们太小了，无法用传统的荧光显微镜方法进行足够详细的研究。此外，为了能够将膜突出动力学与细胞骨架动力学联系起来，需要同时跟踪活细胞中无偏向的细胞膜和细胞骨架。这是电子显微镜无法实现的，因为样品制备和测量条件不适合活细胞。近年来，像受激发射耗尽(STED)显微镜这样的超分辨率荧光显微镜方法已经发展起来，它可以进行高分辨率成像，并且应该能够足够详细地研究OPC末端的单个标记蛋白质，甚至在活细胞中也是如此。此外，扫描离子电导显微镜(SICM)已经被建立为一种工具，在与STED显微镜提供的相同范围的分辨率下，仅以最小的偏差跟踪活细胞的膜轮廓。本项目的目的是开发和随后评估STED/SICM的组合。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。在本项目中，我们将首先构建并表征一个组合的STED/SICM。随后，我们将记录固定细胞和活细胞的概念验证图像。在接下来的项目中，我们将使用这个仪器来解开支配OPC尖端动态的分子力学。

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.