High-throughput submicron-resolution microscopy of Caenorhabditis elegans populations under strong immobilization by cooling cultivation plates

Despite its profound impact on biology, high-resolution in vivo microscopy largely remains low throughput because current immobilization techniques require substantial manual effort. We implement a simple cooling approach to immobilize entire populations of the nematode Caenorhabditis elegans directly on their cultivation plates. Counterintuitively, warmer temperatures immobilize animals much more effectively than the colder temperatures of prior studies and enable clear submicron-resolution fluorescence imaging, which is challenging under most immobilization techniques. We demonstrate 64× z-stack and time-lapse imaging of neurons in adults and embryos without motion blur. Compared to standard azide immobilization, cooling immobilization reduces the animal preparation and recovery time by >98%, significantly increasing experimental speed. High-throughput imaging of a fluorescent proxy in cooled animals and direct laser axotomy indicate that the transcription factor CREB underlies lesion conditioning. By obviating individual animal manipulation, our approach could empower automated imaging of large populations within standard experimental setups and workflows. [Display omitted] •Relatively warmer temperatures more strongly immobilize animals•Cooling enables high-resolution fluorescence imaging free from motion blur•Cooling approach reduces animal processing time for imaging by >98%•Rapid imaging permits high-throughput experiments for biological discovery Biological science instrumentation; Biological sciences research methodologies; Biological sciences tools; Techniques in neuroscience