Live-Cell STED Microscope

对生理过程的机械理解需要结构-功能关系方面的信息。在分子水平上，这一目标被当前主要成像方法的有限分辨率所阻碍：传统光学显微镜的衍射有限的空间分辨率和电子显微镜提供的较差的时间分辨率。超分辨率显微镜（SRM）方法的最新发展产生了绕过衍射势垒的创新解决方案（2014年诺贝尔化学奖）。然而，目前我们面临的主要挑战是将这些技术转移到各种生理应用中。本提案旨在解决这一瓶颈，通过参与SRM的活细胞STED（受激排放耗尽）。所要求的仪器配置将高空间分辨率（3D < 100x100x100nm, 2D < 30x30nm）与荧光显微镜的分子特异性和实时成像能力相结合。我们已经组建了一个代表莱比锡大学医学和生命科学学院的研究人员团队，他们在分析神经系统在不同生物组织水平上的结构-功能关系方面经验丰富。这将使我们能够在广泛的神经生理学应用中优化纳米级亚细胞结构和实时蛋白质动力学的显微镜研究。我们预计这一策略将有助于提供有关大脑功能基本原理的新信息。

A mechanistic understanding of physiological processes necessitates information on structure-function relationships. At the molecular level, this objective is obstructed by the limited resolving power of the primary current imaging approaches: diffraction limited spatial resolution of conventional light microscopy and poor temporal resolution provided by electron microscopy. The recent development of super-resolution microscopy (SRM) methods has produced innovative solutions to bypass the diffraction barrier (Nobel Prize in Chemistry, 2014). At present, however, we face the main challenge of transferring these techniques to diverse physiological applications. The present proposal aims to address this bottleneck by engaging SRM by Live-Cell STED (stimulated emission depletion). The requested instrument configuration combines high spatial resolution (<100x100x100 nm in 3D, <30x30 nm in 2D) with the molecular specificity and live-imaging capacity of fluorescence microscopy. We have assembled a team of researchers representing the Faculties of Medicine and Life Sciences at Leipzig University who are experienced in analysing structure-function relationships of the nervous system at different levels of biological organisation. This will enable us to optimise the microscope for studies of nanoscopic sub-cellular structures and real-time protein dynamics in a wide range of neurophysiological applications. We anticipate that this strategy will help to deliver new information on fundamental principles of brain function.