Photoactivated Localization Microscopy, or PALM imaging exploits the unique behavior of fluorescent proteins (FPs), whose spectral properties change upon exposure to specific wavelengths of light. Upon illumination with the activation light a random and sparse number of FPs are activated/photoconverted and emit fluorescence when exposed to the excitation light. The light emission from individual fluorophores can be recorded and used to localize and track single molecules with sub-diffraction precision, as in other SMLM super-resolution techniques. By concatenation of activation/detection/bleaching steps all the FPs in the sample can be counted, localized and tracked over time. PALM FPs can be appended to other proteins via genetic engineering and thus allow super resolution imaging of virtually any protein-of-interest within the cell, independently of the availability of antibodies or other affinity reagents.
In fast-growing Escherichia coli cells, RNA polymerase (RNAP) is spatially organized in large nucleoid-like patterns, which have been proposed to be active transcription centers for ribosomal RNA (rRNA). Here, we show how PALM super resolution imaging and single particle tracking (sptPALM) can be used to dissect the nanoscale organization and dynamics of E.coli RNAP and gain insights into the mechanisms of bacterial transcription regulation.
