Fast whole-body motor neuron calcium imaging of freely moving Caenorhabditis elegans without coverslip pressed.

Caenorhabditis elegans (C. elegans) is an ideal model organism for studying neuronal functions at the system level. This article develops a customized system for whole-body motor neuron calcium imaging of freely moving C. elegans without the coverslip pressed. Firstly, we proposed a fast centerline localization algorithm that could deal with most topology-variant cases costing only 6 ms for one frame, not only benefits for real-time localization but also for post-analysis. Secondly, we implemented a full-time two-axis synchronized motion strategy by adaptively adjusting the motion parameters of two motors in every short-term motion step (~50 ms). Following the above motion tracking configuration, the tracking performance of our system has been demonstrated to completely support the high spatiotemporal resolution calcium imaging on whole-body motor neurons of wild-type (N2) worms as well as two mutants (unc-2, unc-9), even the instantaneous speed of worm moving without coverslip pressed was extremely up to 400 µm/s.