The rise in fluorescence-based imaging techniques over the past 3 decades has improved the ability of researchers to scrutinize live cell biology at increased spatial and temporal resolution. In microbiology, these real-time vivisections structurally changed the view on the bacterial cell away from the “watery bag of enzymes” paradigm toward the perspective that these organisms are as complex as their eukaryotic counterparts. Capitalizing on the enormous potential of (time-lapse) fluorescence microscopy and the ever-extending pallet of corresponding probes, initial breakthroughs were made in unraveling the localization of proteins and monitoring real-time gene expression. However, later it became clear that the potential of this technique extends much further, paving the way for a focus-shift from observing single events within bacterial cells or populations to obtaining a more global picture at the intra- and intercellular level. In this review, we outline the current state of the art in fluorescence-based vivisection of bacteria and provide an overview of important case studies to exemplify how to use or combine different strategies to gain detailed information on the cell’s physiology. The manuscript therefore consists of two separate (but interconnected) parts that can be read and consulted individually. The first part focuses on the fluorescent probe pallet and provides a perspective on modern methodologies for microscopy using these tools. The second section of the review takes the reader on a tour through the bacterial cell from cytoplasm to outer shell, describing strategies and methods to highlight architectural features and overall dynamics within cells.

中文翻译：

---

细菌活体解剖：基于荧光的成像技术如何揭示细菌的内部运作

在过去的 3 年里，基于荧光的成像技术的兴起提高了研究人员以更高的空间和时间分辨率检查活细胞生物学的能力。在微生物学中，这些实时活体解剖在结构上改变了对细菌细胞的看法，从“酶的水袋”范式转向这些生物与其真核生物一样复杂的观点。利用（延时）荧光显微镜的巨大潜力和相应探针的不断扩展托盘，在解开蛋白质定位和监测实时基因表达方面取得了初步突破。然而，后来很明显这种技术的潜力可以进一步扩展，为将焦点从观察细菌细胞或种群内的单个事件转变为在细胞内和细胞间水平获得更全面的图片铺平了道路。在这篇综述中，我们概述了基于荧光的细菌活体解剖的最新技术，并概述了重要的案例研究，以举例说明如何使用或组合不同的策略来获得有关细胞生理学的详细信息。因此，手稿由两个独立（但相互关联）的部分组成，可以单独阅读和查阅。第一部分重点介绍荧光探针托盘，并提供有关使用这些工具进行显微镜检查的现代方法的观点。评论的第二部分带领读者参观了从细胞质到外壳的细菌细胞，