High-Resolution C. elegans Imaging Across All Larval Stages

Caenorhabditis elegans has become one of the most widely studied and best-understood animal models in biology. Three features are key to C. elegans' success as a model organism: its invariant cell lineage, transparency, and genetic tractability. These render it ideal for a diverse range of microscopy-based studies directly in vivo. Live C. elegans larvae and adults often need to be immobilized during image acquisition. Traditional immobilization methods adversely affect animal development, especially in time-lapse imaging applications. Here, a detailed setup and operation protocol for a novel microfluidic imaging method is introduced, which addresses the limitations associated with traditional agar-pad-based immobilization and other microfluidic strategies. This approach enables simultaneous live imaging across various larval stages while preserving worm orientation and identity over time. To achieve this, a microfluidic trap channel array is employed, with its geometry precisely designed to maintain a stable worm orientation while accommodating growth and molting. Immobilization is facilitated by an active hydraulic valve that applies pressure to secure worms against the cover glass solely during image acquisition. This design allows high-resolution imaging with minimal effects on worm viability or developmental timing.