An FIM-Based Long-Term In-Vial Monitoring System for Drosophila Larvae

Drosophila larvae are an insightful model and the automated analysis of their behavior is an integral readout in behavioral biology. Current tracking systems, however, entail a disturbance of the animals, are labor-intensive, and cannot be easily used for long-term monitoring purposes. Here, we present a novel monitoring system for Drosophila larvae, which allows us to analyze the animals in cylindrical culture vials. By utilizing the frustrated total internal reflection in combination with a multi-camera/microcomputer setup, we image the complete housing vial surface and, thus, the larvae for days. We introduce a calibration scheme to stitch the images from the multi-camera system and unfold arbitrary cylindrical surfaces to support different vials. As a result, imaging and analysis of a whole population can be done implicitly. For the first time, this allows us to extract long-term activity quantities of larvae without disturbing the animals. We demonstrate the capabilities of this new setup by automatically quantifying the activity of multiple larvae moving in a vial. The accuracy of the system and the spatio-temporal resolution are sufficient to obtain motion trajectories and higher level features, such as body bending. This new setup can be used for in-vial activity monitoring and behavioral analysis and is capable of gathering millions of data points without both disturbing the animals and increasing labor time. In total, we have analyzed 107 671 frames resulting in 8650 trajectories, which are longer than 30 s, and obtained more than 4.2 × 10⁶ measurements.