Michael Hensel

Activation and function of virulence functions of bacterial pathogens are highly dynamic in time and space, and can show considerable heterogeneity between individual cells in pathogen populations. To investigate the complex events in host–pathogen interactions, single cell analyses are required. Fluorescent proteins (FPs) are excellent tools to follow the fate of individual bacterial cells during infection, and can also be deployed to use the pathogen as a sensor for its specific environment in host cells or host organisms. This Resources describes design and applications of dual fluorescence reporters (DFR) in cellular microbiology. DFR feature constitutively expressed FPs for detection of bacterial cells, and FPs expressed by an environmentally regulated promoter for interrogation of niche‐specific cues or nutritional parameters. Variations of the basic design allow the generation of DFR that can be used to analyze, on single cell level, bacterial proliferation during infection, subcellular localization of intracellular bacteria, stress response, or persister state. We describe basic considerations for DFR design and review recent applications of DFR in cellular microbiology.