Subcellular Dynamics in Plants Figure 7. TEM images of ultrathin sections from different root cells of BRI1-GFP expressing seedlings. (A) Ultrathin section of a root cell from a seedling high-pressure frozen 30 min after the addition of 10 nM BL. (B) Ultrathin section of an independent root cell from a mock-treated, high-pressure frozen seedling. The cell walls of epidermal cells (upper cells) facing cortex cells (lower cell) are shown. PM, plasmalemma; CW, cell wall; PD, plasmadesmon. The bar represents 100 nm. For the statistical analysis of the cell wall measurements see Table S5. doi:10.1371/journal.pone.0005716.g007 ments of a single cell and between adjacent cells. Thus, membrane-bound GFP fusion proteins have the potential to be highly sensitive probes for determining such parameters at sub- cellular resolution in vivo. Ongoing experiments are aimed at discovering which parameters may influence the fluorescence lifetime of BRI1-GFP, aquaporin-GFP and other fluorophore- tagged proteins in vivo. After the addition of BL, we observed a significant change in the fluorescence lifetime of BRI1-GFP with time, which was not detected in BL-treated aquaporin-GFP expressing plants. These results can be interpreted to suggest that the activation of BRI1- GFP by BL could alter the physico-chemical properties of the plasmalemma or plasmalemma/cytoplasm interface, which do not affect the fluorescence lifetime of aquaporin-GFP. The observed changes would then be a cell physiological response initiated by BL-activated BRI1-GFP. This idea is substantiated by the fact that BFA inhibits the BL-induced change in BRI1-GFP fluorescence lifetime. As BL was shown to inhibit inwardly rectifying K+ currents in guard cell protoplasts of Vicia faba and to increase ATPase activity in Azuki bean epicotyls and maize roots, leading to proton extrusion [24,34–35], it is possible that the observed change in the fluorescence lifetime of BRI1-GFP might reflect a hormone-induced alteration in ion currents and membrane potential in Arabidopsis cells which depends on the presence of BRI1-GFP. The causal relationship between BL-induced BRI1- GFP activation, the change in BRI1-GFP fluorescence lifetime and cell wall expansion is currently under elucidation. Summary and concluding remarks Compared to pure imaging techniques with ultra-high spatial resolution, such as STED [4–5], PALM [6] and STORM [7], our application of ocFLM in combination with wavelength-selective fluorescence microscopy and a novel method of data analysis allows the recording of the spectroscopic and fluorescence lifetime data of GFP fusion proteins in vivo, providing new information about their molecular properties, cell physiological function and subcellular environment. This enables the unambiguous identifi- cation and clear intracellular localisation of GFP fusion proteins in plant cells. Furthermore, ligand-induced physiological responses in plant cells and receptor dynamics at subcellular resolution can be quantitatively recorded. The use of autofluorescence as ‘‘native’’ fluorophore avoids the use of additional chromophors or the PLoS ONE | www.plosone.org 8 May 2009 | Volume 4 | Issue 5 | e5716