Strains were induced for 1 to 3 hrs at 30uC, then collected and resuspended in SC his2 ade+ G418+. Strains were grown for 2 to 3 hrs at 30uC to turn off expression of the MS2-CP-GFPx3 protein in order to decrease cytoplasmic GFP background. Immediately before imaging, 1 ml of culture was washed three times with 1 ml of SC his2 ade+ G418+ and concentrated in a volume of 100 ml. Gelatin pads composed of 25% solid gelatin (Sigma) dissolved in SC his2 ade+ G418+ were made by sandwiching between two pre- cleaned Superfrost microscope slides (Fisher Scientific). After solidification of the gelatin, one slide was removed, 2 ml of culture was applied to the pad to immobilize the cells, and a 22 mm622 mm #1.5 coverglass (VWR) was applied and sealed with molten valap (1:1:1 vaseline, lanolin, and paraffin wax). Fluorescent images were acquired using a 10061.40 PlanApo objective and a CoolSnap-HQ CCD camera (Photometrics) on a Nikon Eclipse 80i microscope. For experiments utilizing latrunculin B, cells were treated with 500 mM latrunculin B (0.5% DMSO final concentration) imme- diately before application to gelatin pads. As Sac6:mCherry- labeled actin patches began to recover after 45 min, cells were imaged for less than 30 min before new cells were treated with latrunculin B and imaged; cells treated with 0.5% DMSO alone served as a control. Diploid strains for assaying localization of MS2-GFP labeled mRNAs to FM4–64-labeled actin patches were grown as described above with the exception that G418 was left out of the growth media. After induction was halted, 100 ul of cells was pelleted and resuspended in SC his2 ade+ media plus 8 mM FM4–64. The cell suspension was quickly spotted on a gelatin pad, sealed with valap, and timelapse acquisition was started. DH-PSF Microscopy Cells were grown overnight in synthetic complete (SC) his- media supplemented with glucose (2% final wt/vol) and 40 mg/ml adenine hemisulfate. Cells were transferred into induction medium lacking methionine upon entry into early log phase and grown at 30uC for 3 h. Cells were then removed from induction medium for 3 h prior to imaging, washed three times in fresh non-inducing media, and then 2 ml of 106 concentrated cells was spotted onto an agarose pad. Agarose pads composed of 1.5% wt/wt solid agarose (Sigma type 1-A, low electroendoosmosis (EEO)) dissolved in SC his- ade+ were created by sandwiching between two plasma-etched 30 mm650 mm #1 glass coverslips. After removal of one of the coverslips and deposition of cells, a plasma-etched 25625 mm glass coverslip was placed on top of the cells to form the imaging interface and then sealed with paraffin wax. Because the stage drift in the microscope during the time scale of our experiments (5–10 s) is negligible compared to the motion of the mRNPs, fiduciary markers were not used. The DH-PSF 3D microscopy of single mRNP dynamics was performed exactly as previously described ([107] and accompanying Supplemental Methods) except that 50 ms exposure times were used to image BBC1 mRNP dynamics. To analyze the data, a tracking algorithm based on fitting the observed data to the sum of two Gaussians was employed. The fitting function contained the sum of two Gaussians plus an offset (for background), which allowed for the extraction of an (x, y) position from the midpoint between the two and the z-position from the angle between the two lobes relative to horizontal (see following discussion of DH-PSF calibration). For each frame after the first, the initial conditions used in the fit were taken from the fit in the previous frame. In this way, the particle is automatically tracked by the fitting code. Because only 0–2 mRNAs are found in each cell, it is highly unlikely that there was confusion in the identity of the particle of interest. PLoS ONE | www.plosone.org 16 February 2012 | Volume 7 | Issue 2 | e31912 Actin Patch mRNA Localization and Dynamics Similar to previous work [107], two criteria were then applied to remove spurious position measurements from the trajectories extracted from the fitting algorithm. First, any localization that fell outside the 2 mm depth of field was rejected because the DH-PSF is only designed to accurately measure z positions within this region. Second, points were probabilistically rejected by requiring that the calculated velocity between subsequent localizations is less than 4800 nm/s; this removes false localizations because of the high and variable background noise inherent in the MS2 labeling scheme. This velocity was chosen because it is approximately two standard deviations above the mean expected root-mean square velocity from the mean observed diffusion coefficients in these experiments. DH-PSF calibration A calibration of angle versus z position was performed by moving 200 nm fluorescent beads (Invitrogen Fluospheres 505/ 515) immobilized in 1% poly(vinyl) alcohol on a glass coverslip using an objective z-positioner (PIFOC p-721.CDQ stage with E625.CO analog controller, Physik Instrumente) in 30 steps spaced by 50 nm in z. Ten (10) frames were collected at each z position. Images of putative beads were identified by hand and cut into 9 by 9 pixel squares followed by manual identification of the two DH-PSF lobes. The data in the box were fit using the Matlab function fminsearch with least squares as the minimization function, exactly as described in Thompson, et al. [114]. We then obtained an angle vs. z position curve as well as x and y calibration curves. Because of aberrations in the system, an x and y correction is necessary for accurate three-dimensional localization. Because calibrations are performed using refractive index- matched media, they do not account for the spherical aberration that inevitably occurs in all mRNA imaging experiments in water. In previous work [107], the effect of spherical aberration on the estimation of z position was measured for imaging of mRNAs in yeast. As is now well known [133], spherical aberration affects the standard PSF differently above and below the focal plane. Briefly, by measuring the diffusion coefficients in x, y, and z both above and below the apparent focal plane in each trajectory we obtained b factors of 0.88 and 0.66 above and below the focal plane, respectively. Then the true z position, z, can be obtained from the apparent z position, z9, by the following formula 2 ð Þ 1 ð Þ z~ 0:66z’ z’v0 0:88z’ z’w0 A more detailed description as well as theoretical calculations can be found in the supplemental information of previous work [107]. Detection and characterization of confinement events In previous work [107], we built upon work by Saxton, et. al and Simson, et al. [113,134] and derived an equation for the probability that a purely random (Brownian) diffuser would stay within a given three-dimensional radius given an observed diffusion coefficient and time window. As discussed in the results, the logarithm of the probability, log(Y) can be approximated as a linear function given by log(Y)~0:301{4:286(Dtw=R2) where tw is the length of each subtrajectory, R is the maximum displacement in the subtrajectory, and D is the measured short time diffusion coefficient averaged over the entire trajectory. The maximum value of R, the diffusion coefficient D, and the subtrajectory time window, tw, are used to calculate the probability,