Institute of Physical and Theoretical Chemistry, Goethe-University Frankfurt, Max-von-Laue-Str. 7, 60438, Frankfurt, Germany.
Janelia Research Campus, Howard Hughes Medical Institute, 19700 Helix Drive, Ashburn, Virginia, 20147, USA.
Sci Rep. 2018 Oct 3;8(1):14768. doi: 10.1038/s41598-018-33052-3.
Maintenance of the bacterial homeostasis initially emanates from interactions between proteins and the bacterial nucleoid. Investigating their spatial correlation requires high spatial resolution, especially in tiny, highly confined and crowded bacterial cells. Here, we present super-resolution microscopy using a palette of fluorescent labels that bind transiently to either the membrane or the nucleoid of fixed E. coli cells. The presented labels are easily applicable, versatile and allow long-term single-molecule super-resolution imaging independent of photobleaching. The different spectral properties allow for multiplexed imaging in combination with other localisation-based super-resolution imaging techniques. As examples for applications, we demonstrate correlated super-resolution imaging of the bacterial nucleoid with the position of genetic loci, of nascent DNA in correlation to the entire nucleoid, and of the nucleoid of metabolically arrested cells. We furthermore show that DNA- and membrane-targeting labels can be combined with photoactivatable fluorescent proteins and visualise the nano-scale distribution of RNA polymerase relative to the nucleoid in drug-treated E. coli cells.
细菌内稳态的维持最初源于蛋白质与细菌拟核之间的相互作用。研究它们的空间相关性需要高空间分辨率，特别是在微小、高度受限和拥挤的细菌细胞中。在这里，我们展示了使用一组荧光标记物的超分辨率显微镜技术，这些标记物可以短暂地结合固定的大肠杆菌细胞的膜或拟核。所呈现的标记物易于应用，具有多功能性，并且允许在不发生光漂白的情况下进行长期的单分子超分辨率成像。不同的光谱特性允许与其他基于定位的超分辨率成像技术进行多路复用成像。作为应用的示例，我们展示了与遗传基因座位置、与整个拟核相关的新生 DNA 以及代谢性停滞细胞的拟核的相关超分辨率成像。我们还表明，DNA 和膜靶向标记物可以与光活化荧光蛋白结合，并在药物处理的大肠杆菌细胞中可视化 RNA 聚合酶相对于拟核的纳米级分布。
