Imaging Neuronal Synapses in Caenorhabditis elegans

Images of fluorescently tagged fusion proteins were captured in live C. elegans using a Zeiss LSM800 Airyscan confocal microscope (Carl Zeiss, Germany) with oil immersion lens 63× magnification (Carl Zeiss, Germany). Worms were immobilized on 2.5% agarose pad using a mixture of 7.5 mM levamisole (Sigma-Aldrich) and 0.225 M BDM (2,3-butanedione monoxime) (Sigma-Aldrich). Images were analyzed with Zen software (Carl Zeiss) and ImageJ (NIH, USA). Twenty to 26 Z-stack images were taken for each animal to encompass the cell bodies, axons, and synapses of the DA8 and DA9 neurons. The definition of DA8/DA9 synaptic overlap was defined by the distance between the most anterior DA9 synapse and the most posterior DA8 synapses (Mizumoto and Shen, 2013a (link); Chen et al., 2018 (link)). L4.4–L4.5 larval stage animals, judged by the stereotyped shape of the developing vulva (Mok et al., 2015 (link)), were used for quantification.