Subcellular Dynamics in Plants Figure 8. BL-induced changes in the cell wall width of aquaporin-GFP expressing Arabidopsis cells. (A) Confocal image of root tip tissue before (left) and 30 min after addition of 10 nM BL (right). Fluorescence intensity curves were recorded over the plasmalemmata-cell wall sections indicated by the white, alphabetically numbered lines and the FWHM values of their Gaussian fits calculated. (B–F) FWHM values of the plasmalemmata-cell wall sections indicated in A before (0 min) and 30 min after addition of 10 nM BL. (G) FWHM values of Calcofluor-stained anticlinal wall from hypocotyl cells recorded over the subcellular section indicated by the white line (8 mm) shown in the confocal image inlet at 0, 15 and 30 min after addition of 10 nM brassinolide. (H) FWHM values of the anticlinal wall autofluorescence from hypocotyl cells recorded over the subcellular section indicated by the white line (5 mm) shown in the confocal image inlet at 0 min and 30 min after addition of 10 nM BL. For the determination of the FWHM error in B to H see Material and Methods. For the statistical analysis of the aquaporin-GFP fluorescence measurements see Table S6. doi:10.1371/journal.pone.0005716.g008 expression of further markers which might interfere with the plant cell’s physiology. This is a marked advantage of our approach over STED, PALM and STORM. In addition, with an image acquisition time of approximately 3 h, PALM and STORM do not allow the fast recording of subcellular processes. We, therefore, propose that high resolution ocFLM combined with wavelength- selective fluorescence microscopy will not only lead to new experimental abilities in plants but also is a valuable novel tool for the accomplishment of cell biology in any cell system. Materials and Methods Plant material and growth conditions BRI1-GFP [22] and aquaporin-GFP [28] expressing seedlings were grown for 5 days at 22uC and under a regime of 14 h light and 10 h darkness on agar containing 0.5 MS media. For measurements seedlings were carefully removed from the plates and transferred onto microscopic slides and covered with water. The BL solutions at the indicated concentrations and Calcofluor (1% w/v) were applied to side of the microscopic slide and allowed to diffuse to the seedling sample. For the response of the cell walls in presence of BFA, the seedlings were pre-incubated for 1 h in 50 mM BFA solution. Optical and spectroscopic measurements The measurements were performed with a homemade CSSM, based on a Zeiss Axiovert, and equipped with a pulsed 473 nm diode laser operating at a repetition rate of 10 MHz as source for excitation light and a high numerical aperture oil immersion objective (Zeiss Plan-Neofluar, 1006/1.30) [19–20]. The system was equipped with an avalanche photodiode (APD, SPCM-AQR- 14) as a spectrally integrating detector to record fluorescence intensity. Spectra were obtained by a spectrograph (Princeton Instruments Acton Spectra Pro 300i, 300 grid grating) coupled to a thermo-electric cooled CCD-camera (Roper Scientific). Lifetime decays were recorded using a time-correlated single-photon counting board (Timeharp 200, Picoquant) for data acquisition and the APD as a detector. Fluorescence intensity images were obtained by raster-scanning the sample and detecting emission intensity for every spot on the sampled area. Distinct spots were addressed to record the corresponding spectra. The pulsed 473 nm diode laser (Picoquant LDH-P-C470), operating at a repetition rate of 10 MHz served as source. The setup was equipped with a 480 nm long pass filter (Semrock Razor Edge LP02-473RU-25) to block back-scattered excitation light, with a 500 nm bandpass filter (Semrock BrightLine BL500/24) to detect GFP-fluorescence or with a 600 nm bandpass filter (Semrock BrightLine BL607/36) to detect autofluorescence. The lifetime data were recorded sequentially to invesitgate the emission lifetime of the GFP label and the autofluorescence background independently from each other. Acquisition time was 5 seconds per measurement. The pixel size is diffraction-limited and the distance between each measurement point was 250 nm. Processing of fluorescence intensity images was accomplished with the WSxM software (Nanotec Electronica) [36]. The intensity decay curves were deconvolved from the instrument PLoS ONE | www.plosone.org 9 May 2009 | Volume 4 | Issue 5 | e5716