Visualizing Single Molecular Complexes In Vivo Using Advanced Fluorescence Microscopy

Summary

Here we demonstrate the protocols for performing single-molecule fluorescence microscopy on living bacterial cells to enable functional molecular complexes to be detected, tracked and quantified.

Abstract

Full insight into the mechanisms of living cells can be achieved only by investigating the key processes that elicit and direct events at a cellular level. To date the shear complexity of biological systems has caused precise single-molecule experimentation to be far too demanding, instead focusing on studies of single systems using relatively crude bulk ensemble-average measurements. However, many important processes occur in the living cell at the level of just one or a few molecules; ensemble measurements generally mask the stochastic and heterogeneous nature of these events. Here, using advanced optical microscopy and analytical image analysis tools we demonstrate how to monitor proteins within a single living bacterial cell to a precision of single molecules and how we can observe dynamics within molecular complexes in functioning biological machines. The techniques are directly relevant physiologically. They are minimally-perturbative and non-invasive to the biological sample under study and are fully attuned for investigations in living material, features not readily available to other single-molecule approaches of biophysics. In addition, the biological specimens studied all produce fluorescently-tagged protein at levels which are almost identical to the unmodified cell strains (“genomic encoding”), as opposed to the more common but less ideal approach for generating significantly more protein than would occur naturally (‘plasmid expression’). Thus, the actual biological samples which will be investigated are significantly closer to the natural organisms, and therefore the observations more relevant to real physiological processes.

Protocol

To begin this procedure, 50 μl of frozen stocks of fluorescent protein expressing Escherichia coli bacterial cells are first defrosted and grown aerobically with shaking in 5 ml LB growth media overnight at 37 degrees C. In the morning, 50 μl of this saturated culture is extracted and sub-cultured into minimal M63 glucose culture media, incubating at 30 degrees C for 4 to 6 hours. Here we demonstrate using two different cell strains, one of which expresses an electron-transporting cytochrome fused to GFP, the other which…

Discussion

Care must be taken not to “over shear” cell for looking at tethered bacteria, since this may impair the functionality of the flagellar motors. It is important to use cells for much longer than an hour once on the microscope slide since they may become oxygen depleted. Considerable optimization may be required to find the best microscope imaging conditions catered to your specific biological system under investigation. It may be wise to attempt the imaging using purified GFP alone to ascertain the correct intensity laser …