Strong immobilization of C. elegans en masse on cultivation plates by thermoelectric cooling for high-throughput submicron-resolution microscopy

Abstract

AbstractHigh-resolution in vivo microscopy has profoundly impacted our understanding of development and disease by revealing anatomy and molecular targets at the subcellular level. Unfortunately, many microscopy approaches remain low-throughput because current immobilization techniques for precise imaging require significant manual effort. To greatly improve the throughput of subcellular resolution imaging in the nematode Caenorhabditis elegans, we implement a novel cooling method to easily immobilize entire populations directly on their native cultivation plates. Previous studies immobilized animals in liquid media between 1-4 °C, which allows animal motion and blurs images. Counterintuitively, we find that cooling to 5-7 °C immobilizes animals much more effectively, enabling clear submicron-resolution fluorescence imaging. We demonstrate 64× magnification 3D image stacks and 2-min long timelapse recordings of the ASJ neuron without image blur. We estimate that the time spent on animal preparation and recovery under our cooling approach is 98% less than under a typical azide immobilization approach. Our simple, yet robust cooling platform enables high-throughput and high-resolution microscopy with minimal user effort. By obviating the need for individual animal manipulation, this approach could also empower automated imaging of large C. elegans populations within standard experimental setups and workflows.HeadlineStraightforward immobilization of entire C. elegans populations enables high-throughput microscopy at submicron resolution.