Single-molecule fluorescence of internalised biomolecules in live bacteria

In the last two decades emerging single-molecule fluorescence tools have been developed and adapted to study individual molecules in vitro under various conditions and to reveal biochemical and cellular processes in live cells with high precision. Single-molecule investigations in vivo rely mainly on fluorescent protein (FP) fusions with low quantum yield and photostability. Methods to introduce organic-labelled fluorescent molecules into cells have been achieved for mammalian cells via microinjection (not possible with bacteria) and are limited in throughput. We have developed a physical transfection method for delivering organic-fluorophore labelled biomolecules into live bacteria for single-molecule studies. We internalised labelled dsDNA and proteins (up to 100kDa) with high efficiency whilst maintain- ing cell viability. I was able to count the number of internalised molecules through photobleaching analysis. From this analysis, I was able to tune the concentration of internalised material from high concentrations compatible with localisation-based super-resolution imaging to lower concentrations compatible with single-molecule observation. Using localisation and tracking algorithms I followed the diffusion of individual molecules for up to 10 s and monitored the tracking paths within the cellular cytoplasm. This method allows us to observe molecules for up to 10 minutes (FPs ~ 5 s) under continuous illumination opening a new time regime to study biological processes. Our approaches are general and widely applicable to different microorganisms using biomolecules labelled with organic fluorophores already available for in vitro experiments.This thesis is not currently available in ORA