Single-molecule and super-resolution imaging of transcription in living bacteria

•Super-resolution imaging and tracking of RNA polymerase [RNAP] in living bacteria.•RNAP tracking reports on its intracellular mobility and spatial distribution.•RNAP spatial and diffusion profile is sensitive to antibiotics and growth media.•RNAPs on highly expressed genes are found in clusters at the nucleoid periphery.•RNAP spends most of its promoter search time bound non-specifically to DNA.

In vivo single-molecule and super-resolution techniques are transforming our ability to study transcription as it takes place in its native environment in living cells. This review will detail the methods for imaging single molecules in cells, and the data-analysis tools which can be used to extract quantitative information on the spatial organization, mobility, and kinetics of the transcription machinery from these experiments. Furthermore, we will highlight studies which have applied these techniques to shed new light on bacterial transcription.