Automated Analysis of FRET Figure 6. The cluster analysis of RhoA activity in a representative migrating cell. (A) The six subcellular regions with different RhoA activities were computed using the K-means cluster analysis. These regions are color-coded according to their RhoA activities, with cold and hot colors indicating low and high levels of RhoA activity, respectively. The migrating direction is indicated by the white arrow. (B) The three regions with the highest RhoA FRET ratios from panel (A) are selected and demonstrated. The white dotted lines outline a wedge in the cell. The coordinate system used for the polar analysis is shown in the lower right corner. (C) Time-lapse images showing the three clusters with high FRET ratio before PLoS ONE | www.plosone.org 8 December 2008 | Volume 3 | Issue 12 | e4082