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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.

Bacterial Vivisection: How Fluorescence-Based Imaging Techniques Shed a Light on the Inner Workings of Bacteria