pGFPCR plasmid was sufficiently high to allow measurements and thus no inducer was added to the medium. Prior to the microscopy, the cells were washed twice with the NaPGCl medium (NaPGCl = 95 mM sodium phosphate, pH 7.0, 50 mM glucose plus 125 mM sodium chloride), which has an osmolality equal to that of LB (DLBOsm = 0) and very low fluorescence. For measurements the cells were either kept in NaPGCl or osmotically upshifted by supplementing the medium with additional NaCl. The osmolality of all solutions was measured by determination of their freezing point (Osmomat 030, Gonotec). For microscopy, 2 ml of cells was placed on poly-L-lysine (1% w/v) coated cover slips and measurements were carried out immediately. Each sample was imaged for periods no longer than 25 min. For each osmotic condition, a minimum of 20 single cells was analyzed. All measurements were performed at 20 +/2 1uC. Measurements of diffusion coefficients Pulsed-FRAP measurements were carried out on a confocal microscope as described by van den Bogaart et al. [7]. Briefly, cells were first imaged with a confocal microscope and a low-intensity, diffraction limited laser beam was positioned in the middle of the cell. Subsequently, this laser beam was modulated using a shutter to apply short pulses, separated by time intervals without illumination to allow the fluorescence to recover. The fluorescent signal recorded during the pulses is influenced by the photo- bleaching of the GFP in the focal spot (decrease of fluorescence intensity) and by diffusion of the non-photobleached fluorophore into the focal spot (increase of fluorescence intensity). The fluorescence is linearly proportional to the concentration of GFP. The measured traces can be analysed using the Fick’s second law where the GFP concentration fluctuations inside the cell C(r, t): LC(r,t) Lt ~DDC(r,t), where C is the concentration of GFP, D is the LaPlace operator and r and t define the position and time point, respectively. We assume that the photo-bleaching rate is proportional to the intensity of the focused laser beam I(r) and thus obtain a bleaching constant B: LC(r,t) Lt ~BI (r)C(r,t): Finally, to obtain a diffusion coefficient (D), the traces are fitted numerically to a 2D diffusion model as described in detail by van den Bogaart et al. [7]. ‘‘Conventional FRAP’’ measurements were performed using an inverted microscope Observer D1 (Carl Zeiss, Jena, Germany) equipped with a Zeiss C-Apochromat infinity-corrected 1.2 636 water immersion objective and a motorized X-Y translating stage for fine positioning of the cells. The optical part of the set-up is depicted in Fig. 1 and is very similar to the one reported by Konopka and co-workers [6]. The laser beam (488 nm, argon ion laser, Melles Griot, Carlsbad, CA, USA) was split into two beams. The first beam was focused to a diameter of around 1 mm and was used for photobleaching. The second beam (‘wide’) had a diameter of around 100 mm in the image plane and was employed to monitor the fluorescence recovery. The fluorescence emission was collected through the same objective and separated from the excitation beam by a dichroic mirror (Chroma Technology, Evaluation of FRAP for D Measurements in Bacteria Figure 1. Optical scheme of the set-up used to perform conventional FRAP. Bs, beam splitter; M, mirror; NDFW, neutral density filter wheel; L1, L2, L3 lenses; Sh,shutter. doi:10.1371/journal.pone.0025664.g001 Rockingham, VT, USA) and further directed through a 488 nm notch filter (CVI, Melles Griot, Carlsbad, CA, USA). The fluorescence signal was collected by a Cool-Snap HQ2 CCD camera (Photometrics, Tucson, AZ, USA). The cells were measured as described by Elowitz [1] and Konopka [6]. To position the desired bacterium in the focal plane of the microscope and in the bleaching area, bright-field transmittance microscopy mode was used. A fluorescence microscopy image of the cell before photobleaching was recorded (Fig. 2A). Subsequently, an area at the pole of the bacterium was photobleached (Fig. 2B) with a short (100 ms) focused light pulse. Immediately afterwards typically 40 images of the recovery of fluorescence were collected, using fluorescence illumination with the ‘wide’ beam. The frame rate to monitor the fluorescence recovery was adjusted to the speed of diffusion, i.e. every 25– 50 ms for fast diffusion (normal osmotic conditions) and every 250 ms for severe osmotic upshift conditions. The analysis is described by Kumar et al. [4]. The images, recorded during the recovery phase, were analyzed numerically, using home written software as schematically depicted in Fig. 2G–J. A line was drawn through the longest axis of the bacterium and fluorescence intensity distributions along this cross-section (X-axis) were extracted from the image (Fig. 2B–J). To take into account intrinsic inhomogeneity of the fluorescence intensity along the X- axis, the ‘pre-photobleached’ distribution (Fig. 2F) was used to normalize all measured distributions. To obtain a diffusion coefficient, the software simulated the normalized fluorescence intensity distributions along the given cross-section during recovery (Fig. 2G–J). Following Elowitz [1], the one–dimensional diffusion approximation was assumed: LI (x,t) Lt ~D L2I (x,t) Lx2 where I is fluorescence intensity and D is the diffusion coefficient; with boundary conditions: LI (x,t) Lx ~0 at the bacterial poles, corresponding to zero flux of GFP through the cell membrane. For final renormalization the distribution of GFP prior to photobleaching (Fig. 2F) was used. For the cell shown in Fig. 2, we obtained a diffusion coefficient of 6 mm2/s. To illustrate the evolution of the fluorescence recovery, ‘single- spot’ fluorescence traces at positions R1 and R2 are depicted in PLoS ONE | www.plosone.org 2 September 2011 | Volume 6 | Issue 9 | e25664