Subcellular Dynamics in Plants Figure 2. Dimensional appearance of periclinal and anticlinal cell walls in confocal and TEM images. (A) Fluorescence intensity curve recorded over a plasmalemmata-wall section of the periclinal wall of BRI1-GFP expressing Arabidopsis root cells. The full width at half maximum value (FWHM) of the Gaussian fitting revealed an apparent wall thickness of 425630 nm. (B) Fluorescence intensity curve recorded over a plasmalemmata- wall section of the anticlinal wall of the identical root cell described in A. The FWHM of the Gaussian fitting revealed an apparent wall thickness of 773633 nm. (C) Ultrathin TEM image of a tissue section cutting three different root cells. Two anticlinal and one periclinal cell walls (CW) are shown. CO, cortical cell; EP, epidermal cell; ER, endoplasmatic reticulum; EB, ER body; M, mitochondrium; V, vacuole. The arrow heads point to Golgi-derived vesicles. The bar represents 500 nm. doi:10.1371/journal.pone.0005716.g002 and 512 nm of BRI1-GFP expressing hypocotyl cells (Fig. 1B), the lifetime was characterized by a mono-exponential decay (Fig. 1C) typical for GFP (Figure S1) [26–27]. In contrast, the background autofluorescence, which was recorded at the identical wavelength range in wildtype hypocotyl cells, strongly deviated from the mono-exponential decay function (Fig. 1D). An acceptable fit to the experimental data with respect to the average lifetime and amplitude could only be achieved by a multi-exponential decay function (Fig. 1E, see Table S1 for additional information on the fitting parameters of all lifetime decay traces shown here). Furthermore, we were faced with the problem that the cell wall in the CSSM images appeared to be much thicker than when it was determined by transmission electron microscopy (TEM) (Fig. 2). However, one has to take into account the non-point- shaped dimension of the confocal laser beam focus. When this is considered, the measured full width at half maximum (FWHM) values of the Gaussian fits of the fluorescence intensity distribution of 425 nm for the periclinal cell wall (Fig. 2A) and 774 nm for the anticlinal cell wall (Fig. 2B) reflect a thickness of 83 nm and 432 nm, respectively (see Text S1 for calculation). The values of the periclinal cell walls are, therefore, consistent with those determined by TEM (about 45 to 100 nm; Fig. 2C; Table S5). The width of the anticlinal walls detected by our fluorescence intensity approach was still too large to correspond to our TEM measurements. However, to understand the values determined for the longer anticlinal walls, one has to take into account that a cell wall slanted to a few degrees appears to have a higher CSSM- measured thickness. For instance, imaging cell membranes bordering a cell wall with a real thickness of 100 nm at a slant of 30u generates a FWHM value of around 730 nm (see Text S1 for calculation). Thus, the prevailing larger FWHM values of the anticlinal cell wall measurements can be explained by their higher bending probability which results in higher slanting effects in the focus of the confocal microscope. Therefore, for an accurate determination of small changes in the expansion of plasmamem- brane-cell wall areas, the selected section has to be kept in tight focus during the entire analysis, which is enabled by our experimental CSSM setup. In addition, the differences of GFP and background fluores- cence with respect to their spectra and fluorescence lifetime enabled us to choose the appropriate experimental set-up for the distinction of subcellular compartments such as the plasmalemma and the cell wall (Fig. 3). Thus, we used the emission range at around 500 nm for recording GFP fluorescence and the range at around 600 nm for recording cell wall autofluorescence (hatched bars in Fig. 3A). Rapid BL-induced cell wall expansion in vivo We next addressed the problem of real-time measurement of possible physiological effects of BRI1-GFP at subcellular level in living plant cells. Former studies have shown that BL does not alter the fluorescence intensity, the number of vesicles in the endosomal pool and the intracellular distribution of BRI1-GFP in root cells [22]. This is in agreement with our observations (Fig. 4A to C, Fig. 5 and data not shown). However, the local fluorescence intensity of BRI1-GFP as a function of BL, time and a defined PLoS ONE | www.plosone.org 3 May 2009 | Volume 4 | Issue 5 | e5716