iterations and displayed as a maximal z-projection for z-sections. For image sequences, two dimensional blind deconvolution was performed using 5–30 iterations. Subsequent deblurring was performed with the same software. Moss Fixation Ethylene glycol bis[succinimidylsuccinate] (EGS) and m-Mal- eimidobenzoyl-N-hydroxysuccinimide ester (MBS) treatment were applied to plants growing in open chambers under the same media conditions as the closed chambers, but the protonemata were immobilized with 0.7% low melting point agarose. MBS and EGS were added to 30 mM and 100 mM respectively from 1006 DMSO stocks. Cells were treated with the crosslinkers for 15 min before observation. For fixation and phalloidin staining, cells were processed the same way as previously reported (Vidali et al., 2007) but using rhodamine phalloidin (Invitrogen) instead of Alexa-488 phalloidin. Growth Assay For the moss growth assay, one week old cultures of stable lines were protoplasted using established methods [11]. Plants were regenerated in top-agar (0.5% agar) in the presence of manitol for 4 days, then transferred to growth moss NH4 medium, PpNH4 (1.03 mM MgSO4, 1.86 mM KH2PO4, 3.3 mM Ca(NO3)2, 2.7 mM (NH4)2-tartrate, 45 mM FeSO4, 9.93 mM H3BO3, 220 nM CuSO4, 1.966 mM MnCl2, 231 nM CoCl2, 191 nM ZnSO4, 169 nM KI, 103 nM Na2MoO4) for 2 days and imaged. Cell walls were stained with a solution of 10 mg/ml calcofluor (fluorescent brightener 28, Sigma) in water for at least 15 min. Cellophane fragments with the cells embedded in top agar were inverted on 10 ml of staining solution, incubated for 1 min, and the cellophane was removed. An additional 10 ml of staining solution was added and mounted with a coverslip. The cells were incubated for at least 15 min and no more than 1 hr. Pictures were taken at 306 zoom with a 16 lens, as 36-bit RGB color images with a CCD camera (Leica DF300FX) on a fluorescence stereo- microscope (Leica MZ16FA). Filter combinations were for UV/ DAPI setting. The blue channel of the color images, correspond- ing to cell wall signal was digitally separated. The resulting 12-bit image was thresholded and the total area estimated. Solidity, a morphometric parameter, was evaluated by calculating the ratio of the plant area to its convex hull area; one corresponds to a perfectly solid object and numbers smaller correspond to more branched structures. A total of 30 to 60 plants was evaluated for each replicate. All image analysis was done using macros written for ImageJ (http://rsb.info.nih.gov/ij/). Macros are available upon request. Statistical analysis was done using ANOVA and post-hoc tests in KaleidaGraph (Synergy Software). To compare the average fluorescence per pixel to length in lily, pollen was bombarded with 3 mg of zmC13::Lifeact-mEGFP or zmC13::mEGFP. Images were collected using the 488 nm argon laser of a Nikon confocal microscope (Nikon D-Eclipse-C1) on an inverted stand (Nikon Eclipse-TE2000-S) and a 106 dry objective. Image analysis was performed using ImageJ (http://rsb.info.nih. gov/ij/). For the high resolution rate of growth analysis of lily, pollen was bombarded with 1.8 mg of zmC13::Lifeact-mEGFP then imaged 3 hours later. Pollen was imaged at 406 using the Nikon D-Eclipse-C1 microscope. Growth was tracked using MetaMorph software (Molecular Devices) and the distance from the origin was then plotted versus time. A linear regression was fit to the data and the slope represented the average velocity for the tube. To analyze the length distribution of pollen, the indicated amount of either zmC13::Lifeact-mEGFP or zmC13::mEGFP used to bombard pollen. The pollen was allowed to grow for Lifeact-mEGFP in Plant Cells 3 hours then fixed in 10 mM MgCl2, 100 mM PIPES, and 3.7% formaldehyde for 30 minutes. The pollen was then washed in LPGM, imaged and measured as described for the florescence vs. length assay. All statistical analysis was performed using Origin software (OriginLab, www.orginlab.com). Drug Treatments For the analysis of Latrunculin B effects on F-actin, moss was cultured in PpNO3 media for 3–5 days, on top of cellophane disks. Pieces of cellophane containing protonemata were cut, flipped, and the protonemata were placed in direct contact with an agar pad containing Hoagland’s medium and Latrunculin B at the indicated concentration. The cellophane was removed, 5 ml of liquid medium containing the same concentration of Latrunculin B were added, and a coverslip placed on top. The chamber was sealed with melted VALAP. Images were acquired with an interval of 10–20 min after chamber preparation. Control preparations contained DMSO at 0.2% in medium. Multiple cells and chambers were analyzed with identical results. For the Latrunculin B sensitivity assay in moss, cells were prepared in the same way as for the growth assay (see above). Protoplasts were plated on small cellophane circles on top of agar in 96 well plates; wells were filled to the top with agar to create a flat surface to deposit the protoplasts. Cells were plated in protoplast regeneration medium in the absence of Latrunculin B for 4 days. At day 4 the cellophane discs were transferred to regular PpNH4 medium containing different amounts of Latrun- culin B. Two days after transfer images were acquired from chlorophyll autofluorescence at a 306 zoom as 36-bit RGB color images with a CCD camera (Leica DF300FX) on a fluorescence stereo-microscope (Leica MZ16FA). Filter combinations were: excitation 480/40, dichroic 505 long pass, emission 510 long pass. The red channel of the color images, corresponding to chlorophyll fluorescence was digitally separated. The resulting 12-bit image was thresholded and the solidity estimated as mentioned above. Latrunculin treatments were performed in triplicate; a total of 7 to 38 plants was measured in each replicate. Dose response curves were fitted to the data using the sigmoidal fitting function of the program Origin (Microcal), using a logistic equation and a log10 scale for the concentration of Latrunculin B. The half maximal inhibitory concentration (IC50) was estimated from these fits. To compare the significance of the differences an ANOVA statistical test was used between the means obtained for each replicate. To calculate fractional solidity for each cell line and to plot the data, the following transformation was used: the minimum values obtained from the curve fitting were subtracted from the mean values; the resulting value was divided by the maximum value obtained by curve fitting. For lily, bombarded pollen was grown on a slide and imaged according to our standard procedure (see above). The growth media was then replaced with fresh media plus 2 nM Latrunculin using a pipette. The procedure was performed twice to ensure that all of the media had been replaced. Supporting Information Figure S1 Shows a maximal projection of the lily pollen tube shown in figure 5c. Fifteen sequential images taken in z-axis. Scale bar is 10 mm. Found at: doi:10.1371/journal.pone.0005744.s001 (0.41 MB PDF) Movie S1 Time lapse LSCM of Lifeact-mEGFP in a moss caulonemal cell. Two frames were acquired per second. Total elapsed time was 179.5 seconds. Scale bar is 5 mm. PLoS ONE | www.plosone.org 13 May 2009 | Volume 4 | Issue 5 | e5744