Subcellular in vivo time-lapse imaging and optical manipulation of Caenorhabditis elegans in standard multiwell plates

Abstract

High-resolution in vivo time-lapse assays require repeated immobilization and imaging of whole animals. Here we report a technology for screening Caenorhabditis elegans at cellular resolution over its entire lifespan inside standard multiwell plates using repeated immobilization, imaging and optical manipulation. Our system does not use any fluidic or mechanical components, and can operate for tens of thousands of cycles without failure. It is also compatible with industrial high-throughput screening platforms and robotics, and it allows both chemical, and forward and reverse genetic screens. We used this technology to perform subcellular-resolution femtosecond laser microsurgery of single neurons in vivo, and to image the subsequent regeneration dynamics at subcellular resolution. Our single-neuron in vivo time-lapse analysis shows that neurite regrowth occurring over short time windows is significantly greater than that predicted by ensemble averaging over many animals.