Figure 6. BL-induced expansion of the cell wall in BRI1-GFP expressing hypocotyl cells. (A) FWHM values of the anticlinal wall autofluorescence from hypocotyl cells recorded over the subcellular section indicated by the white line (10 mm) shown in the confocal image inlet at 0 min and 30 min after addition of 10 nM BL. (B) FWHM values of Calcofluor-stained anticlinal wall from hypocotyl cells recorded over the subcellular section indicated by the white line (11 mm) shown in the confocal image inlet at 0, 15 and 30 min after addition of 10 nM BL. For the determination of the FWHM error in A and B see Material and Methods. For the statistical analysis of the cell wall autofluorescence and Calcofluor fluorescence measurements see Tables S3 and S4. doi:10.1371/journal.pone.0005716.g006 Discussion Specific GFP versus unspecific compartment fluorescence As shown in Figure 1, there is a significant, cell-type specific background fluorescence signal which mainly derives from the wall of the plant cell. This unspecific background fluorescence can be differentiated from the GFP signal by its spectra and also by its characteristic fluorescence decay pattern. Whereas GFP fluores- cence shows a prototypical monoexponential decay function, background fluorescence can only be described by a multi- exponential decay function. This suggests that the unspecific Subcellular Dynamics in Plants background fluorescence at the excitation wavelength of 473 nm originates from different components inside the cell wall, which have different excited-state lifetimes. Thus, the combination of wavelength- and time-resolved fluorescence spectroscopy enables us to distinguish unambiguously between a specific signal and background fluorescence in a cell type-specific manner, dispensing with the use of additional fluorescent dyes, which might interfere with cellular processes. These differences in lifetime also allowed us to establish a procedure for the efficient background correction and, thus, the improvement of the signal-to-background ratio in living plant cells (patent application no. 10 2009 005 953 from 19.01.09; F. Schleifenbaum, K. Elgass, M. Sackrow, K. Caesar, K. Berendzen, K. Harter, A. Meixner, manuscript in preparation) Measurement of cell wall extension at subcellular resolution Our data demonstrate that the cell walls significantly expand within a few minutes of BL treatment in BRI1-GFP expressing cells. A slight expansion response was also observed in aquaporin- GFP expressing and wildtype seedlings which was without clear overall statistical significance. However, when determined at single cell level, almost every cell showed a BL-induced wall expansion. This suggests that BRI1-GFP is necessary for the rapid BL- induced expansion of the cell wall but its over-expression is required to produce a significant effect. A more detailed analysis of many cells from independent seedlings showed that there is a linear relationship between BRI1-GFP fluorescence intensity and the degree of cell wall expansion (K. Elgass, A. Meixner, K. Harter, unpublished). BL is known to alter the biophysical properties of the cell wall, such as its relaxation via BRI1-mediated transcriptional and post-transcriptional activation of cell wall- loosening enzymes [24,34]. However, such an early BL-responsive spatio-temporal and BL-dependent expansion has not yet been reported in the cell wall sections of living Arabidopsis cells. From the resolution point of view, it should, in principle, also be possible to determine cell wall expansions of the observed dimension by TEM. However, it is not technically possible to produce a series of TEM sections at the identical subcellular region before and after the addition of BL. Thus, different seedlings have to be used for the comparative physiological analysis using TEM. The set of difficulties is documented by our own TEM analysis, which demonstrates a high variability of cell wall thicknesses between individual cells and even individual periclinal walls. The high variability is probably due to the different age of the cells, which have different wall dimensions, and the difficulty to exactly localize the site of the measurement along the wall. For instance, the wall thickness at the edge of a cell is different form the width in the middle. In addition, although we used cryofixation and freeze-substitution for the preparation of the probes, we can not entirely exclude the possibility that this treatment may influence the water status and spatial dimension of the cell wall. On the one hand, we conclude that it is almost impossible to determine weak, dynamic changes in cell wall expansion using a TEM approach. On the other hand, these findings unequivocally demonstrate the potential of our approach for the analysis of the dynamics of subcellular processes in vivo. Changes in the close environment of membrane-bound GFP fusion proteins The observed differences in BRI1-GFP and aquaporin-GFP fluorescence lifetime suggest strong gradients of physico-chemical parameters, such as membrane potential, pH-value, osmotic conditions and refraction index [30], within subcelluar compart- PLoS ONE | www.plosone.org 7 May 2009 | Volume 4 | Issue 5 | e5716