Rediscovering Bacteria through Single-Molecule Imaging in Living Cells

Bacteria are microorganisms central to health and disease, serving as important model systems for our understanding of molecular mechanisms and for developing new methodologies and vehicles for biotechnology. In the past few years, our understanding of bacterial cell functions has been enhanced substantially by powerful single-molecule imaging techniques. Using single fluorescent molecules as a means of breaking the optical microscopy limit, we can now reach resolutions of ∼20 nm inside single living cells, a spatial domain previously accessible only by electron microscopy. One can follow a single bacterial protein complex as it performs its functions and directly observe intricate cellular structures as they move and reorganize during the cell cycle. This toolbox enables the use of in vivo quantitative biology by counting molecules, characterizing their intracellular location and mobility, and identifying functionally distinct molecular distributions. Crucially, this can all be achieved while imaging large populations of cells, thus offering detailed views of the heterogeneity in bacterial communities. Here, we examine how this new scientific domain was born and discuss examples of applications to bacterial cellular mechanisms as well as emerging trends and applications.