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).