Long-Term High-Resolution Imaging of Developing C. elegans Larvae with Microfluidics

Long-term studies of Caenorhabditis elegans larval development traditionally require tedious manual observations because larvae must move to develop, and existing immobilization techniques either perturb development or are unsuited for young larvae. Here, we present a simple microfluidic device to simultaneously follow development of ten C. elegans larvae at high spatiotemporal resolution from hatching to adulthood (∼3 days). Animals grown in microchambers are periodically immobilized by compression to allow high-quality imaging of even weak fluorescence signals. Using the device, we obtain cell-cycle statistics for C. elegans vulval development, a paradigm for organogenesis. We combine Nomarski and multichannel fluorescence microscopy to study processes such as cell-fate specification, cell death, and transdifferentiation throughout post-embryonic development. Finally, we generate time-lapse movies of complex neural arborization through automated image registration. Our technique opens the door to quantitative analysis of time-dependent phenomena governing cellular behavior during C. elegans larval development. [Display omitted] •Multichannel time-lapse imaging of C. elegans larvae of any stage for up to 72 hr•Cell-cycle timing statistics of vulval lineages for >100 animals•Fluorescent reporter expression during divisions, differentiation, and cell death•Automated image registration enables visualizing complex neurite outgrowth Keil et al. present a microfluidics setup, enabling long-term, high-resolution, time-lapse microscopy of up to ten C. elegans larvae simultaneously. They collect vulval cell-cycle timing statistics, measure intensities of fluorescent transcriptional reporters during cell-fate specification, transdifferentiation, and cell death, and visualize complex neurite outgrowth in automatically registered z stacks.