Mund Markus, Kaplan Charlotte, Ries Jonas
European Molecular Biology Laboratory, Cell Biology and Biophysics Unit, Heidelberg, Germany.
Institute of Biochemistry, ETH Zurich, Switzerland.
Methods Cell Biol. 2014;123:253-71. doi: 10.1016/B978-0-12-420138-5.00014-8.
Conventional light and fluorescence microscopy techniques have offered tremendous insight into cellular processes and structures. Their resolution is however intrinsically limited by diffraction. Superresolution techniques achieve an order of magnitude higher resolution. Among these, localization microscopy relies on the position determination of single emitters with nanometer accuracy, which allows the subsequent reconstruction of an image of the target structure. In this chapter, we provide general guidelines for localization microscopy with a focus on Saccharomyces cerevisiae. Its different cellular architecture complicates efforts to directly transfer protocols established in mammalian cells to yeast. We compare different methodologies to label structures of interest and provide protocols for the respective sample preparation, which are not limited to yeast. Using these guidelines, nanoscopic subcellular structures in yeast can be investigated by localization microscopy, which perfectly complements live-cell fluorescence and electron microscopy.
传统的光学和荧光显微镜技术极大地增进了我们对细胞过程和结构的了解。然而，它们的分辨率本质上受到衍射的限制。超分辨率技术实现了高一个数量级的分辨率。其中，定位显微镜依赖于以纳米精度确定单个发射体的位置，这使得随后能够重建目标结构的图像。在本章中，我们提供了定位显微镜的一般指南，重点是酿酒酵母。其不同的细胞结构使得将在哺乳动物细胞中建立的实验方案直接应用于酵母变得复杂。我们比较了标记感兴趣结构的不同方法，并提供了不限于酵母的各自样品制备方案。使用这些指南，可以通过定位显微镜研究酵母中的纳米级亚细胞结构，这完美地补充了活细胞荧光和电子显微镜。
