Computer-assisted 3D kinematic analysis of all leg joints in walking insects

High-speed video can provide fine-scaled analysis of animal behavior. However, extracting behavioral data from video sequences is a time-consuming, tedious, subjective task. These issues are exacerbated where accurate behavioral descriptions require analysis of multiple points in three dimensions. We describe a new computer program written to assist a user in simultaneously extracting three-dimensional kinematics of multiple points on each of an insect's six legs. Digital video of a walking cockroach was collected in grayscale at 500 fps from two synchronized, calibrated cameras. We improved the legs' visibility by painting white dots on the joints, similar to techniques used for digitizing human motion. Compared to manual digitization of 26 points on the legs over a single, 8-second bout of walking (or 106,496 individual 3D points), our software achieved approximately 90% of the accuracy with 10% of the labor. Our experimental design reduced the complexity of the tracking problem by tethering the insect and allowing it to walk in place on a lightly oiled glass surface, but in principle, the algorithms implemented are extensible to free walking. Our software is free and open-source, written in the free language Python and including a graphical user interface for configuration and control. We encourage collaborative enhancements to make this tool both better and widely utilized.