Introduction
The dimensions of most bacterial cells are in the micrometer range and thus come close to the diffraction limit of visible light of about 200-300 nm (Fig. 1A). Therefore, to resolve the details and dynamics of crucial bacterial activities, novel techniques are necessary. Recent developments in fluorescence super-resolution microscopy (SRM) are promising to move closer to the goal of observing the localization and motion of single proteins in living bacterial cells. SRM techniques have the potential to revolutionize the understanding of central processes in bacteria e.g., peptidoglycan assembly, the mode of action of DNA-binding proteins, the function of macromolecular machines involved in protein secretion, DNA replication or antibiotic resistance. In this review, we describe recent work showing the importance of fluorescence SRM for understanding complex molecular structures and functions in bacteria. Special attention will be paid to the MINFLUX nanoscopy technique, which is a promising approach to visualize the molecular motions and dynamic interactions of single molecules with a spatiotemporal resolution in the single-digit nanometer and low millisecond range. Applications of SRM methods including MINFLUX nanoscopy to observe individual components of a molecular machine, the bacterial type 3 secretion system (T3SS), will be described in more detail. While the focus of this review is on the visualization of molecular processes in bacteria, all of the described microscopy approaches are also applied in other cell types. A widely applicable workflow guiding researchers towards in celluloMINFLUX imaging at molecular scale has been described previously (Carsten et al., 2022).