Several microfluidic-based methods for long-term C. elegans imaging have been introduced in recent years, allowing real-time observation of previously inaccessible processes. The ex-isting methods either permit imaging across multiple larval stages without maintaining a stable worm orientation, or allow for very good immobilization but are only suitable for shorter experiments. Here, we present a novel microfluidic imaging method, which allows parallel live-imaging across multiple larval stages, while delivering excellent immobilization and maintaining worm orientation and identity over time. This is achieved by employing an array of microfluidic trap channels carefully tuned to maintain worms in a stable orienta-tion, while allowing growth and molting to occur. Immobilization is supported by an active hydraulic valve, which presses worms onto the cover glass during image acquisition, with the animals remaining free for most of an experiment. Excellent quality images can be ac-quired of multiple worms in parallel, with little impact of the imaging method on worm via-bility or developmental timing. The capabilities of this methodology are demonstrated by observing the hypodermal seam cell divisions and, for the first time, the entire process of vulval development from induction to the end of morphogenesis. Moreover, we demonstrate RNAi on-chip, which allows for perturbation of dynamic developmental processes, such as basement membrane breaching during anchor cell invasion.
The C. elegans heterochronic gene lin-46 affects developmental timing at two larval stages and encodes a relative of the scaffolding protein gephyrin