Automatic tracking of individual migrating cells using low-magnification dark-field microscopy

Many fundamental biological processes, such as the search for food, immunological responses and wound healing, depend on cell migration. Video microscopy allows the magnitude and direction of cell migration to be documented. Here, we present a simple and inexpensive method for simultaneous tracking of hundreds of migrating cells over periods of several days. Low-magnification dark-field microscopy was used to visualize individual cells whereas time-lapse video images were acquired by computer for future analysis. We employed an automated tracking algorithm to identify individual cells on each video image allowing migration paths to be tracked using a nearest neighbour algorithm. To test the method, we followed the time-course of migration of 3T3 fibroblasts, endothelial cells and individual amoeba in the absence of any chemical stimulus gradient. All cell types showed a 'random walk' behaviour in which mean squared displacement in position increased linearly with time. We defined a 'migration coefficient' (D(mig)), analogous to a diffusion coefficient, which gave an estimate of cell migration rate. D(mig) depended on cell type and temperature. When amoebas were made to undergo chemotaxis, the cells no longer followed a random walk but instead moved at a near constant velocity (V(av)) towards the chemotactic stimulus.