Figure 3. Comparison of pulsed-FRAP with ‘‘conventional FRAP’’. Diffusion coefficients of GFP in the cytoplasm of E. coli measured with ‘‘conventional FRAP’’ (gray bars) and pulsed-FRAP (black bars) under normal osmotic conditions (top panel, ‘‘+ 0 mM NaCl’’) and Evaluation of FRAP for D Measurements in Bacteria under conditions of osmotic upshift (lower panels, the extent of osmotic shock is indicated in the upper right corner of each panel). For the highest osmotic stress (lowest panel, ‘‘+500 mM NaCl’’) only ‘‘conventional FRAP’’ data are presented. The number of cells measured in each experiment was at least 20. doi:10.1371/journal.pone.0025664.g003 ease the visual comparison of the two methods, the corresponding data from pulsed-FRAP and ‘‘conventional FRAP’’ have been plotted below each other. We note that for pulsed-FRAP measurements of very low molecule mobility are difficult to probe. Pulsed-FRAP requires that the time between photobleach light- pulses is long enough for complete recovery of the GFP in the cell. In severely stressed cells (500 mM NaCl or more) diffusion is very slow and the recovery is not homogenous. In fact, as reported previously above 500 mM of NaCl [7] one observes apparent barriers for diffusion. For these so-called plasmolyzing cells, pulsed-FRAP is not suitable to obtain quantitative information on protein mobility. In general both pulsed- and ‘‘conventional’’-FRAP give quite broad distributions of diffusion coefficients, which has been also observed by others [1,6,7], not only for GFP but also for other macromolecules [13]. The wide range of distributions is in part due to errors in the measurements and data analysis, but a large part is due to population heterogeneity. Pulsed-FRAP yields somewhat broader distributions of diffusion coefficients, which might be a result of the changes of the cell position in respect to the focal volume during the measurement. Despite the difference in the methodologies, both techniques yield similar maxima in the distributions of values and the decrease in diffusion of GFP is proportional to the strength of the osmotic shock applied. Moreover, the diffusion coefficients of osmotically stressed cells increase in the presence of osmoprotectants (Fig. 3 panel ‘‘+250 mMNaCl+10 mM KCl’’). Differences between methods From a practical point of view ‘‘conventional FRAP’’ may be more suited to probe protein mobility in live cells than pulsed- FRAP. With ‘‘conventional FRAP’’ snapshots of the whole cell are recorded, which gives information about the distribution of the GFP inside the cytoplasm (i.e. whether it is homo- or heterogenous) and it can be easily observed if the cell moves during the measurement. In case of pulsed-FRAP the movement of cell cannot be observed until the end of the series of bleaching pulses, which could hamper data acquisition. Moreover, the data analysis on average takes only a few minutes with ‘‘conventional FRAP’’, while it can take up to hours to calculate a diffusion coefficient with pulsed-FRAP. On the other hand pulsed–FRAP maybe better suited for smaller cells or organelles. Pulsed-FRAP also proved suitable for measuring the mobility of very fast diffusing NBD-glucose, which has a poor photostability [13]. This would be technically challenging to perform with ‘‘conventional FRAP’’. The cell length of E.coli is typically 3 mm. The average diffusion coefficient of NBD-glucose in the cytoplasm of E.coli under normal osmotic condition is close to 50 mm2/s, which means that after photo- bleaching the fluorescence can fully recover within less than 30 ms. Since this dye is not very bright and quite photolabile, fast CCD imaging is insufficient to capture good quality FRAP data. Why are the reported diffusion constants different? In the recent literature, the diffusion coefficients for GFP in the E.coli cytoplasm, grown and analyzed under normal osmotic conditions, ranged from 3 to 14 mm2/s. This variation may be PLoS ONE | www.plosone.org 4 September 2011 | Volume 6 | Issue 9 | e25664