Time-lapse microscopy has proven indispensible for studying dynamic processes in living organisms and has uncovered significant individual-to-individual variability at the cellular level. However, time-lapse microscopy is currently rarely used to study C. elegans post-embryonic development. The major obstacle is the need to restrict movement of animals for imaging, as drug-induced or mechanical immobilization precludes the animal’s feeding and typically leads to growth arrest of larvae within hours. Here, we circumvent this problem by constraining the movement of animals in ~200x200x10μm chambers made of polyacrylamide hydrogel, with each chamber containing bacteria as food source to sustain development. We found that in such chambers C. elegans animals develop normally, as measured by body length extension, timing of the molting cycle and egg laying. Using arrays of microchambers, combined with fast image acquisition, we could perform fluorescence microscopy of developmental dynamics at the single-cell level and with ~10 minute time resolution for the full duration of post-embryonic development, in up to 50 animals simultaneously. We demonstrated the power of our setup in capturing a number of key developmental processes in C. elegans that span a significant fraction of the ~48 hours of post-embryonic development and hence have so far been inaccessible for time-lapse microscopy. In particular, we (i) studied the temporal regulation of seam cell divisions by imaging all cell divisions of the seam cell lineages, (ii) measured the trajectory of the distal tip cells during their entire ~30 hour migration and (iii) quantified the oscillatory gene expression dynamics of molting genes during all four larval stages. We observed significant variability, both between individuals and on the single cell level, in all these processes. Our setup should make it possible to use time-lapse microscopy as a routine tool to study C. elegans post-embryonic development.
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