Willig Katrin I, Nägerl U Valentin
Cold Spring Harb Protoc. 2012 May 1;2012(5):pdb.prot069260. doi: 10.1101/pdb.prot069260.
The confluence of innovations in transgenic labeling and light microscopy techniques in recent years has greatly advanced our understanding of dynamic cell biological events underlying neuronal function and plasticity. Increasingly, it has become possible to perform fundamental experiments inside the relevant subcellular compartments of a neuron embedded in three-dimensional living tissues. Overcoming the limiting role of diffraction in far-field light microscopy, nanoscopy is advancing our ability to see and manipulate cellular events well below the diffraction barrier of ∼200 nm. The first concrete and implemented concept of nanoscopy was STED (stimulated emission depletion) microscopy. This article gives an example of the power that STED microscopy holds for neuroscience research. It provides a method for live-cell time-lapse imaging of the dynamic morphology of dendritic spines of pyramidal neurons. Imaging is performed in an organotypic hippocampal slice culture system, with yellow fluorescent protein (YFP) used as a volume marker for the synaptic structures. In addition, the article describes the basic elements needed to assemble a custom-built STED microscope capable of live cell imaging and how to use it for physiology experiments.
近年来，转基因标记和光学显微镜技术的创新融合极大地推动了我们对神经元功能和可塑性背后动态细胞生物学事件的理解。越来越有可能在嵌入三维活组织的神经元的相关亚细胞区室内进行基础实验。克服远场光学显微镜中衍射的限制作用，纳米显微镜正在提升我们观察和操纵远低于约200 nm衍射极限的细胞事件的能力。纳米显微镜的第一个具体且已实施的概念是受激发射损耗（STED）显微镜。本文给出了STED显微镜对神经科学研究具有的强大作用的一个实例。它提供了一种对锥体神经元树突棘动态形态进行活细胞延时成像的方法。成像在器官型海马切片培养系统中进行，黄色荧光蛋白（YFP）用作突触结构的体积标记物。此外，本文描述了组装一台能够进行活细胞成像的定制STED显微镜所需的基本元件以及如何将其用于生理学实验。
