Automated Analysis of FRET Figure 1. The diagrams depicting the region-tracking algorithm. (A) The centroid of the cell (C), a control point (P), and the boundary intersection at the convex hull (E, in left image) were used to compute the relative distance and orientation of the vector pointing from the centroid to the control point. (r,h) is the polar coordinate of the point on the unit disk (P9) corresponding to the control point (P) in the cell image (right image). (B) The registration process is shown from left to right. First, the cell is partitioned into a triangular mesh within its boundary. Second, the mesh was mapped to a unit circle by computing the relative distance and orientation of the vectors pointing from the centroid to mesh nodes (or control points). Finally, the control points and cell image were mapped to the unit disk by a piecewise linear interpolation within the triangular mesh. (C) The top panels show the images of a single cell undergoing expansion or translocation, with a selected ROI in the first frame. The lower panels show the unit disks transformed from the cell at different time points. The boundary of the transformed ROI in the first frame is used to track and quantify the signals from the same region in these different unit disks. doi:10.1371/journal.pone.0004082.g001 first cell image. Thereafter, a mask for the ROI was generated and mapped to the first unit disk by our image registration method. To track the average FRET signal in this region, the mask of ROI in the unit disk was assumed to remain at the same location in time (Figure 1C). Therefore, the same mask can be applied to all the unit disk images to track and calculate the time course of average FRET ratio on the selected region(s), which was computed by taking the ratio of the total CFP and YFP intensity within the region(s) in the cell (Figure 1C). The assumption of constant ROIs in the reference unit disks is accurate if the cells only translate in the imaging plane, or shrink/expand in the radial directions extending from the centroid. The cells were not expected to rotate around their centroids during the course of experiment. These assumptions are reasonable for slow migration cells within the time window of our experiments and consistent with the graded radial extension (GRE) model [36], where the cells were expected to extend or retract their lamellipodia locally perpendicular to the cell edge and along the actin fibers. The ROI(s) on the reference unit disk was mapped back to the original cell images to visually confirm that they can indeed track the actual region in migrating cells (Fig. 1C). To improve computational efficiency, only the nodes outlining the ROI on the unit disk, but not the whole mask, were transformed back onto the cell images. Cluster Analysis To examine the geometric features of subcellular regions with similar levels of RhoA activity, the pixels of the cell image were PLoS ONE | www.plosone.org 3 December 2008 | Volume 3 | Issue 12 | e4082