Spahn Christoph, Glaesmann Mathilda, Gao Yunfeng, Foo Yong Hwee, Lampe Marko, Kenney Linda J, Heilemann Mike
Institute of Physical and Theoretical Chemistry, Goethe-University Frankfurt, Max-von-Laue-Str. 7, 60438, Frankfurt, Germany.
Mechanobiology Institute, T-Lab, National University of Singapore, 5A Engineering Drive 1, Singapore, 117411, Singapore.
Methods Mol Biol. 2017;1624:269-289. doi: 10.1007/978-1-4939-7098-8_20.
Despite their small size and the lack of compartmentalization, bacteria exhibit a striking degree of cellular organization, both in time and space. During the last decade, a group of new microscopy techniques emerged, termed super-resolution microscopy or nanoscopy, which facilitate visualizing the organization of proteins in bacteria at the nanoscale. Single-molecule localization microscopy (SMLM) is especially well suited to reveal a wide range of new information regarding protein organization, interaction, and dynamics in single bacterial cells. Recent developments in click chemistry facilitate the visualization of bacterial chromatin with a resolution of ~20 nm, providing valuable information about the ultrastructure of bacterial nucleoids, especially at short generation times. In this chapter, we describe a simple-to-realize protocol that allows determining precise structural information of bacterial nucleoids in fixed cells, using direct stochastic optical reconstruction microscopy (dSTORM). In combination with quantitative photoactivated localization microscopy (PALM), the spatial relationship of proteins with the bacterial chromosome can be studied. The position of a protein of interest with respect to the nucleoids and the cell cylinder can be visualized by super-resolving the membrane using point accumulation for imaging in nanoscale topography (PAINT). The combination of the different SMLM techniques in a sequential workflow maximizes the information that can be extracted from single cells, while maintaining optimal imaging conditions for each technique.
尽管细菌体积小且缺乏细胞区室化，但它们在时间和空间上都展现出了惊人程度的细胞组织。在过去十年中，出现了一组新的显微镜技术，称为超分辨率显微镜或纳米显微镜，这些技术有助于在纳米尺度上可视化细菌中蛋白质的组织情况。单分子定位显微镜（SMLM）特别适合揭示关于单个细菌细胞中蛋白质组织、相互作用和动力学的广泛新信息。点击化学的最新进展有助于以约20纳米的分辨率可视化细菌染色质，为细菌类核的超微结构提供了有价值的信息，尤其是在短代时情况下。在本章中，我们描述了一个易于实现的方案，该方案允许使用直接随机光学重建显微镜（dSTORM）来确定固定细胞中细菌类核的精确结构信息。结合定量光活化定位显微镜（PALM），可以研究蛋白质与细菌染色体的空间关系。通过使用纳米尺度形貌成像的点积累（PAINT）对膜进行超分辨率成像，可以可视化感兴趣的蛋白质相对于类核和细胞圆柱体的位置。在一个连续的工作流程中结合不同的SMLM技术，可以最大限度地从单个细胞中提取信息，同时为每种技术保持最佳成像条件。
