Manipulation of C. elegans larvae in nanoliter droplets for high-resolution imaging

The study of first larval stage Caenorhabditis elegans (soil nematode) is of particular interest for research on developmental biology. Traditionally it has been labor intensive to study them because of their small size and high motility. We have overcome this challenge by developing a miniaturized platform that uses nanoliter droplets of a reversible hydrogel to offer advanced manipulation of individual animals.

HFSP Program Grant holder Hang Lu and colleagues
authored on Thu, 07 May 2015

Numerous post-embryonic developmental cellular events such as cell differentiation and cell migration and important developmental decision-making take place during the first larval stage of C. elegans. For example, commitment towards an alternative developmental path – the dauer - which dramatically affects the growth, behavior, and life span of the animal, is initiated at that stage. To facilitate experimental studies, high-resolution imaging is often used, which requires full immobilization of the specimen.

Traditional ways to prepare worms involve gluing or placing the worms on a pad and using anesthetics. However, the animals are small and particularly fragile at that stage, and handling them manually is a delicate task. Traditional techniques are therefore low throughput, time-consuming, and labor-intensive.  We previously developed microfluidic approaches that enable high-throughput screening in our lab, but these methods, based on cooling and geometrical restriction, have been better suited to older worms. Larval stage one (L1) animals are about 10 microns in diameter and they are extremely flexible and deformable. This represents a challenge for microfabricated devices because robust operation requires devices to be able to handle clogging by debris and channels smaller than 10 microns represent a large risk for clogging during normal operations.

 

We solved this problem by changing the strategy. Instead of directly handling the animals, we manipulated much larger objects – droplets that contained the larvae. In addition, the droplets were composed of a thermosensitive and reversible hydrogel, behaving like a liquid at low temperature and turning into gel at room temperature. Using the microfluidic tool box, we demonstrated manipulation of tens of individual L1s from encapsulation, transportation through a channel network to sorting. Full immobilization of the animals was achieved by stopping the flow and tuning the temperature to complete sol-gel transition of the hydrogel, creating an array of spatially well-ordered animals ready for imaging. We performed bright-field high-resolution imaging of the entire animal and fluorescence high-resolution imaging of fluorescent neurons with subcellular details. This works paves the way for high-throughput screening of L1 nematodes, which will promise exciting new insights for developmental studies.

Text by Hang Lu and Guillaume Aubry

Reference

Hydrogel-droplet microfluidic platform for high-resolution imaging and sorting of early larval Caenorhabditis elegans”. Aubry G., Zhan M., and Lu H, Lab Chip (2015) 15, 1424-31. DOI: 10.1039/c4lc01384k

Pubmed link