Microbes as single-cell organisms are important model systems to study cellular mechanisms and functions. In recent years and with the help of advanced fluorescence microscopy techniques, immense progress has been made in characterizing and quantifying the behavior of single bacterial cells based on molecular interactions and assemblies in the complex environment of live cultures. Importantly, single-molecule imaging enables the in vivo determination of the stoichiometry and molecular architecture of subcellular structures, yielding detailed, quantitative, spatiotemporally resolved molecular maps and unraveling dynamic heterogeneities and subpopulations on the subcellular level. Nevertheless, open challenges remain.

Here, I quickly review the past and current status of the field, discuss example applications from our own work and give insights in future trends [1, 2].

Even if not a microbiologist, you could be interested in this talk if you would like to get an update on current practices and tools with single-molecule sensitivity such as quantitative dual-color PALM imaging using dual fluorescent protein labeling in living cells, details of sample preparations like easy but precise drift correction by red-shifted beads or tracking of dense, highly dynamic single-molecule data. Interactive questions during the presentation are highly welcome!