when in the movie it was born. The adhesions in blue have been detected for the longest time, while those in red and orange have been detected for the shortest amount of time. The simulated adhesions in columns 1–3 are all too faint to be reliably detected for the length of the simulation experiment, while those in column 4 are near the limit of detection. (B) The exponential distribution of adhesion longevity appears similar to that observed in the experimental data. The longevity of all the detected adhesions was correctly identified as 25 minutes. (C and D) The average adhesion intensity (C) and mean adhesion area (D) were correctly identified in the adhesions that were detected for their entire 25 minute lifespan. The red lines in C indicate the true values. (PNG) Figure S12 Evaluation of the tracking algorithm’s ability to follow adhesions of various sizes and speeds. (A) A sample frame from the simulated adhesion motion experiment where the adhesions were moved at 1 pixel per frame. The top row of adhesions of only a single pixel could not be followed. (B) As the movement speed of the simulated adhesions increases, only larger adhesions can be reliably tracked. (PNG) Figure S13 Evaluation of the rate and phase length detection algorithm using simulated focal adhesion images. (A and C) The predicted median assembly (A) and disassembly (C) rates were extracted correctly by the algorithm. (B and D) The predicted lengths of both the assembly (B) and disassembly (D) were also correctly identified by the algorithm. All the red lines indicate the expected values of the properties in each plot. (PNG) References 1. Geiger B, Spatz JP, Bershadsky AD (2009) Environmental sensing through focal adhesions. Nat Rev Mol Cell Biol 10: 21–33. 2. Lauffenburger DA, Horwitz AF (1996) Cell migration: a physically integrated molecular process. Cell 84. 3. 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(2004) Calpain- mediated proteolysis of talin regulates adhesion dynamics. Nat Cell Biol 6. 14. Paran Y, Ilan M, Kashman Y, Goldstein S, Liron Y, et al. (2007) High- throughput screening of cellular features using high-resolution light-microscopy; Application for profiling drug effects on cell adhesion. Journal of Structural Biology 158. 15. Zamir E, Geiger B, Kam Z (2008) Quantitative Multicolor Compositional Imaging Resolves Molecular Domains in Cell-Matrix Adhesions. PLoS ONE 3: e1901. Quantifying Focal Adhesion Dynamics Video S1 Example movie showing the results of track- ing the EGFP-Paxillin labeled adhesions. The left panel shows the normalized raw experimental data, while the right hand side shows each adhesion outlined in a different color. As the movie plays, the highlighting color remains the same for each unique adhesion. The scale bar is 10 mm. (MOV) Video S2 Example movie showing the results of track- ing the EGFP-PaxillinS178A labeled adhesions. 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