Table 1. Categorization of migrating and stationary cells. Cell Type Migrating Cells Stationary Cells _L_o_n_g_e_s_t__D_i_s_t_a_n_c_e_ Total Displacement 0.71 0.1867 Standard Error 0.03 0.03266 The cells were categorized as migrating and stationary according to the ratio between the longest distance traveled and the total displacement of the cell during the experiments. This ratio is shown with standard error in the table. doi:10.1371/journal.pone.0004082.t001 Automated Analysis of FRET classified and divided into six clusters according to their RhoA FRET ratio values. The MATLAB function kmeans was used to classify the pixels into different clusters, by minimizing the within- cluster variation of the FRET values [37,38]. As a result, each cluster contained pixels with similar FRET ratio values. The MATLAB programs implementing these functions can be obtained by writing to the corresponding author of this paper. Results Image Registration Two classes of HeLa cells with significantly distinctive migration patterns were observed during experiments: migratory and stationary. To determine whether a HeLa cell was migratory or Figure 2. FRET images and their transformed maps on unit disks of the Raichu-RhoA biosensor (Venus/ECFP) in migrating and stationary cells. Panel (A) shows a sequence of time-lapse FRET images for a representative migrating HeLa cell. The white arrow points to the migration direction. Panel (B) shows the FRET images of the migrating cell in (A) transformed into a unit disk. The spatial temporal dynamics of the FRET images of the moving HeLa cell is also shown in Movie S1. Panel (C) shows a sequence of time-lapse FRET images for a representative stationary HeLa cell. Panel (D) shows the FRET images of the stationary cell in (B) transformed into a unit disk. doi:10.1371/journal.pone.0004082.g002 PLoS ONE | www.plosone.org 4 December 2008 | Volume 3 | Issue 12 | e4082