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，少突胶质祖细胞（OPCs），这种细胞存在于发育中的大脑和成年的大脑中，形成髓鞘，髓鞘包裹并电隔离神经元的轴突。为了发展和维持正常的脑功能，OPCs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。髓磷脂受损或未受损的轴突发生在多发性硬化症患者中，因此了解OPCs的迁移机制具有重要意义。OPCs是在细胞体的相反位置表现出两个过程的细胞，并且细胞体沿着这些过程迁移。因此，理解突起的机制对于理解OPC迁移至关重要。然而，细胞膜延伸和细胞骨架在OPCs过程尖端的相互作用尚未被详细研究。造成这种情况的主要原因是OPC尖端的尺寸太小，无法通过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，需要同时追踪活细胞中的无偏细胞膜和细胞骨架。这不能通过电子显微镜实现，因为样品制备和测量条件不适合活细胞。近年来，超分辨率荧光显微镜技术，如受激发射损耗（STED）显微镜已经发展起来，它可以在高分辨率下成像，并且可以对OPCs尖端的单个标记蛋白质进行足够详细的研究，甚至在活细胞中也是如此。此外，扫描离子电导显微镜（SICM）已被确立为一种追踪活细胞膜轮廓的工具，在与STED显微镜相同的分辨率范围内，只有最小的偏差。这个项目的目的是开发和随后评估一个综合的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，我们将首先构建和描述一个综合的STED/SICM。随后，我们将记录固定细胞和活细胞的概念验证图像。在后续的项目中，我们将使用该仪器揭示控制OPCs尖端动力学的分子力学。

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.