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

Abstract

Imaging, visual screens, and optical surgery are frequently applied to the nematode Caenorhabditis elegans at subcellular resolution for in vivo biological research. However, these approaches remain low-throughput and require significant manual effort. To improve throughput and enable automation in these techniques, we implement a novel cooling method to immobilize C. elegans directly on their cultivation plate. Previous studies cooled animals in microfluidics or flooded wells to 1-4 C. Counterintuitively, we find that cooling to 5-7 C immobilizes animals more effectively than lower temperatures. At 6 C, animal movement consists of bouts of submicron nose tip movement occurring at a sufficiently low magnitude and frequency to permit clear imaging. We demonstrate the ability to perform subcellular-resolution fluorescence imaging, including 64x magnification 3D image stacks and 2-min long timelapse recordings of the ASJ neuron without blurring from animal motion. We also observe no long-term side effects from cooling immobilization on animal lifespan or fecundity. We believe our cooling method enables high-throughput and high-resolution microscopy with no chemical or mechanical interventions.

Brain-wide representations of behavior spanning multiple timescales and states inC. elegans

“…For NeuroPAL recordings, animals were imaged as described above, but they were subsequently immobilized by cooling, after which multi-spectral information was captured. The slide was mounted back on the confocal with a thermo-electric cooling element attached to it, set to cool the agar temperature to 4°C (Wang et al, 2022). A closed-loop temperature controller (TEC200C, Thorlabs) with a micro-thermistor (SC30F103A, Amphenol) embedded in the agar kept the agar temperature at the 1 °C set point.…”

Assembly and operation of a cooling stage to immobilizeC. eleganson their culture plates

“…We show protocols for those three cooling immobilization strategies, which we term "slow", "fast", and "abrupt". Further details available from our companion publication, which fully characterized the strategies and animal movement 13 .…”

“…Note: Fast cooling strategy is the most basic immobilization method; however, agar plates idly occupy the stage for an extended time while reaching the T set . Also, when strong immobilization is needed and the T set is 6 °C, idle time is extended to around 1 hour 13 .…”

“…Our cooling stage approach can readily and reversibly immobilize an entire animal population with minimal user effort, eliminating 98% of animal processing time 13 . Below, we describe the procedures for building a cooling stage from scratch and operating it for immobilizing C. elegans on a typical upright microscope.…”

“…Thus, microfluidics are not widely utilized for C. elegans immobilization. Here, in conjunction with our recent publication 13 , we introduce a new cooling immobilization approach using a thermoelectric cooling stage ( Figure 1 ) to address these shortcomings. With the cooling stage, we can cool down a typical 60-mm polystyrene cultivation plate to any target temperature (T set ) between −8 °C to room temperature.…”