Evaluation of FRAP for D Measurements in Bacteria Figure 2. The ’’conventional’’ FRAP method. A–E: snapshots of a cell during data acquisition; A: the cell before photobleaching (panel labeled ‘‘pre’’); B–E: the cell during recovery after photobleaching (timestamp indicates the time after the photobleaching pulse). Dotted circle indicates the bleaching spot (B). Scale bar 2 mm. F–J: the fluorescence intensity along the dotted cross-section at given time points (corresponding to images on the left: A–E), where black is the normalized fluorescence intensity and red is the fit. K,L: the change of intensity in the course of the recovery at (K) the bleached pole of the cell (R1) and at (L) the opposite pole of the cell (R2). Black squares show the normalized data points and red lines the corresponding fits. doi:10.1371/journal.pone.0025664.g002 Fig. 2K–L. using the classical approach introduced by Axelrod [2]:, these fluorescent traces (Fig. 2K–L), yielded a diffusion coefficient D of 1.5 mm2/s, assuming a beam diameter of 1 mm. For each experimental condition, at least 20 individual cells were analyzed. The obtained diffusion coefficient values were plotted as histograms. Results and Discussion Pulsed FRAP and ‘‘conventional FRAP’’ yield similar diffusion coefficients Figure 3 presents histograms of distributions of diffusion coefficients of cytoplasmic GFP at different osmotic regimes. To PLoS ONE | www.plosone.org 3 September 2011 | Volume 6 | Issue 9 | e25664