Measurements of cell-generated deformations on flexible substrata using correlation-based optical flow

The optical flow algorithm presented here is a robust method that rapidly yields a high-density field of substrate displacement vectors based on two optical images. We found that one of the limiting factors, at least for inexperienced experimentalists, is the consistency of focusing or the drift in microscope focus. However, with properly collected images the standard error of the measurement was estimated to be on the order of +/- 0.10 pixels. Finally, although the discussion has been focused on the displacement of flexible substrata, a similar method should be applicable for detecting movements on other types of images, as long as the movement involves a certain degree of local coordination.