Background

Caenorhabditis elegans is an ideal model organism to observe dynamic changes at the cellular level using fluorescence labeling of target molecules owing to its transparent nature. Unfortunately, worms are sensitive to light and become very active when placed under a microscope. For optical calcium imaging, immobilizing the worms for a prolonged period is essential; however, this can be difficult due to movement artifacts.

Anesthetic agents, such as sodium azide or levamisole, are the most popular choice for immobilizing worms; however, they can cause physiological and behavioral variations in the organism (Fang-Yen et al., 2012; Lewis et al., 1980). As an alternative, gluing worms to a thin agar pad using cyanoacrylate adhesive has been used in calcium imaging and electrophysiological studies (Kerr et al., 2000). This method allows worms to recognize and respond to external stimuli more efficiently; however, it can be challenging to master, particularly gluing the worm only to the tail region. Often, worms become mired when glue sticks to the trunk and head and cannot be used for the experiment (Kerr et al., 2000). Additionally, it is difficult to measure calcium kinetics in the head neurons using this method because of the quick head turns of the worm.

Microfluidic chips have been employed to trap worms for extended kinetic measurements (Hulme et al., 2007). This expensive technique requires the use of precise microfluidic channels and fluid pumps. Polystyrene beads (0.1 μm size) on 5%–10% agarose pads have been used to immobilize worms for extended measurement (Kim et al., 2013). Despite its simplicity, bead-based immobilization triggers dopamine levels in worms (Dong et al., 2018; Raj and Thekkuveettil, 2022) and can compromise the interpretation of the results.

Here, we present a new protocol for immobilizing worms using sodium alginate gel. This method is quick, cost-effective, and causes minimal distress to worms. The gel creates a protective cavity around them, similar to that of microfluidic channels. The alginate gel is highly porous and enables olfactory signals to reach the worm. To prove the efficacy of this method, we applied it to a functional imaging application and showed that it can accurately measure calcium spikes in neurons during odor exposure.