Single-Molecule FRET in Living Bacteria

Most single-molecule FRET experiments are performed in vitro, using tightly controlled conditions and well-defined concentrations of a limited number of interacting components. However, in order to understand biological mechanisms as they occur in vivo while simultaneously taking advantage of the extra information provided by single-molecule detection, there is a growing need for performing single-molecule fluorescence measurements in cellular contexts, and in particular in living cells.Towards this goal, we have developed physical methods for delivering fluorescent biomolecules in living Escherichia coli bacteria (one of the most common model organisms in biology) and observing single-molecule fluorescence and single-molecule FRET in the bacterial cytoplasm; we use both confocal and wide-field imaging approaches for detection, providing access to a large number of probed timescales. We have also been using localization-based super-resolution imaging approaches to study the subcellular localization, mobility and abundance of the internalized biomolecules.Our results using single-stranded and double-stranded DNA standards with different FRET efficiencies show that the FRET efficiency of the internalized DNAs agrees well with in vitro FRET measurements. Single-molecule FRET time-traces from the majority of internalized molecule show the characteristic spectroscopic signatures expected from a single FRET pair system. Ongoing work on other biomolecules, including doubly labelled proteins, should lead to the exciting prospect of visualizing sub-nanometer conformational changes at the single-molecule level in the natural milieu of live cells. Our approaches are general and should be useful for studying a large number of intracellular processes in bacteria.