Fluorescence Imaging at Sub-Diffraction-Limit Resolution with Stochastic Optical Reconstruction Microscopy
Fluorescence Imaging at Sub-Diffraction-Limit Resolution with Stochastic Optical Reconstruction Microscopy
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DOI：
10.1007/978-0-387-76497-9_4
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发表时间：
2009-01-01
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影响因子：
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通讯作者：
Zhuang, Xiaowei
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文献类型：
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作者：
Dempsey, Graham T.;Wang, Wenqin;Zhuang, Xiaowei
荧光显微镜是生物学研究中必不可少的工具。然而，传统光学显微镜的一个主要缺点是分辨率相对较低，受数百纳米级的光的衍射的限制。近年来，已经发展了许多亚衍射极限分辨率的荧光成像技术，在横向和轴向维度上都实现了数十纳米的空间分辨率。本章重点介绍其中的一种方法，随机光学重建显微镜(STORM)，它利用可光切换的荧光探针来及时分离单个荧光团的空间重叠图像，并根据单分子图像确定的这些荧光团的精确位置构建超分辨率图像。这项技术的应用已经扩展到同时对不同颜色的荧光团进行三维成像和活细胞成像。本章介绍了多色和三维风暴对细胞结构成像的实现。它首先讨论了可光切换荧光探针的选择和标记感兴趣的细胞目标的方案。然后描述了用于执行风暴实验的仪器和方法，接着概述了用于创建风暴图像的分析例程。在本章的结尾处给出了该技术的应用以及一般协议和故障排除。
Fluorescence microscopy is an essential tool in biological research. One major drawback of conventional light microscopy, however, is its relatively low resolution, which is limited by the diffraction of light to several hundreds of nanometers. In recent years, a number of fluorescence imaging techniques with sub-diffraction-limit resolution have been developed, achieving a spatial resolution of tens of nanometers in both the lateral and axial dimensions. This chapter focuses on one of these methods, stochastic optical reconstruction microscopy (STORM), which utilizes photoswitchable flourescent probes to separate spatially overlapping images of individual fluorophores in time and construct superresolution images from the precise positions of these fluorophores determined from the single-molecule images. Application of this technique has been extended to imaging fluorophores of different colors simultaneously, in three dimensions, and in living cells. This chapter describes the implementation of multicolor and three-dimensional STORM to imaging cellular structures. It begins by discussing the choice of photoswitchable fluorescent probe and the scheme with which to label a cellular target of interest. The instrumentation and methods for performing a STORM experiment are then described, followed by an outline of the analysis routines used for creating a STORM image. Applications of the technique along with general protocols and troubleshooting are given at the conclusion of the chapter.