Internalization of Fluorescent Biomolecules for Long-Lived Single-Molecule Observation in Living Bacteria

We present a complete toolbox for the internalization and single-molecule study of singly- or multiply-labeled fluorescent biomolecules (such as DNA and protein) into living E.coli cells. This technique allows use of organic fluorophores, which are much smaller, brighter and more photostable than genetically encoded fluorescent proteins (e.g. GFP), and provide better labeling flexibility (using in vitro site-specific labeling) and wider spectral range. As such, our methods enable experiments that have so far been precluded due to the inability to internalize fairly large molecules such as globular proteins through the cell membranes of micron-size bacterial cells.Our internalization method, based on electroporation, has allowed us to observe and track fluorescent molecules in living E.coli on the second-to-minute timescale providing >100-times longer observation spans compared to GFP. Aided by the quantized photobleaching of fluorophores, we have characterized the number of internalized molecules, which ranged from 1 to 1000, depending on the size and the amount of electroporated molecule. We also characterized the diffusion behaviour of the internalized molecules, obtaining information on diffusion coefficients, heterogeneity and paths.By internalising doubly-labeled DNA molecules, we observed single-molecule FRET in single bacteria for the first time. Systematic in vivo single-molecule FRET measurements with DNA standards (exhibiting FRET efficiencies from ∼20 to ∼90%) show all the hallmarks of single FRET pairs, with efficiencies that agree well with those obtained in vitro. We further demonstrated the technique with labeled proteins, successfully internalizing singly- and doubly-labeled proteins of different sizes. Currently we are extending this method to achieve live-cell super-resolution imaging. Our toolbox should be very helpful for addressing a wide range of questions regarding the structure, interactions and dynamics of bacterial systems in their natural context.