Imaging single membrane proteines in a living bacterial cell by optical superresolution based on PALM (08 / 2010)
Single-molecule detection is used to localize and count the number of FoF1-ATP synthases in a living Escherichia coli cell in a collaboration project with R. Iino and H. Noji, Osaka University. Several hundreds of these enzmyes exist in a single bacterium according to recently published data (Xie and coworkers, SCIENCE 2010). We will utilize the possibility of switching single fluorophores on and off for counting each enzyme. By optimizing the on/off photophysics of the label, only one or a few FoF1-ATP synthases are detected in each movie image that has been recorded by a high speed, ultra sensitive EMCCD camera. Subsequently the center of each fluorescence spot in each frame of the movie is calculated. The resulting position map has a much higher localization precision than given by the physical resolution of the EMCCD camera pixels (see right image). This method is often called PALM, photoactivated light microscopy, or FPALM.
Controlled single-molecule switching is the basis of optical superresolution beyond the optical diffraction limit to resolve two adjacent point source-like fluorescence emitters. The movie shows flashing spots of single FoF1-ATP synthases in living E. coli cells which are immobilized on a poly-lysine coated cover glas. The brightfield image of the bacteria is shown to the left. The calculated localization map is shown to the right. Blue dots are the centers of the fluorescence spots over all frames of the entire measurement.
In the next step the diffusion coefficient of photoactivated FoF1-ATP synthase is calculated using a tracking algorithm and the data from the localization map. If the individual spots in each frame are well separated and by using the estimated limit for a fast diffusing single FoF1-ATP synthase in the bacterial membrane, the diffusion coeffiicient D is calculated from the mean-square-displacement.
Localized diffusion coefficient changes during the cell cycle, cell division, aging or in the presence of inhibitors are accessible by these measurements.