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.
Representative Results:
When the protocol is done correctly the images of the cells viewed in brightfield is very distinct, with the perimeters of the cell bodies dark against a white/gray cell body (Figure 1a). In fluorescence using immobilized cells, we can see distinct spots intensity, of typically 250-300 nm in width (Figure 1b). Healthy, tethered cells will be seen to rotate around the point of tether attachment in brightfield images. Under fluorescence excitation some molecular complexes in our case might also be seen at the point of attachment, indicating a localization of the tagged protein with the flagellar motor. These spots are individual molecular complexes and the number of them seen will depend upon the illumination mode used and how many of the complexes are actually present in the cell at any one time. The mobility of the spots depends upon the specific biological system under study. If the density of spots is initially very high, as is the case with the labeled cytochromes used here, then performing an initial FRAP bleach can improve the imaging contrast.
Figure 1. (A) Brightfield and (B) TIRF image (false color) for an immobilized Escherichia coli cell expressing a protein fused to green fluorescent protein (GFP) which is known to be involved in the flagellar motors of bacteria. Please click here to see a larger version of figure 1.
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 excitation required for your particular microscope system.
The authors have nothing to disclose.
We acknowledge the kind donations of bacterial strains from the groups of Prof. Judith Armitage (University of Oxford, UK) and Prof. Conrad Mullineaux (Queen Mary University of London, UK). IMD is jointly funded by the Dept of Biochemistry (Oxford University) and OCISB; AR is funded by an Engineering and Physical Sciences Research Council (EPSRC) DTC studentship; ND is funded from the Biotechnology and Biological Sciences Research Council (BBSRC); MCL is funded by a Royal Society University Research Fellowship.