(604f) High-Resolution Long-Term Live Imaging of C. Elegans Using Microfluidics

Continuously tracking development of C. elegans on single animal basis can provide insights into dynamic processes such as synaptogenesis, synaptic re-arrangement, and axonal growth cone development. However, such studies are experimentally complicated or impossible to perform on the basis of individual worms; this is largely due to i) negative effects of anaesthetics and most microfluidic methods on physiology and development, ii) need to individually track animals between imaging sessions, and iii) allowing animals to develop normally during experiments. We have developed a microfluidic device, which in combination with thermally controllable gels allows to i) repeatedly immobilize animals at physiological conditions, ii) perform high-magnification imaging while keeping track of individual specimen, and iii) culture animals between imaging cycles for days. We use an array of traps designed for C. elegans embryos; each trap is connected to a single culture chamber. Once all traps are loaded, we push embryos into chambers by pressurizing the device to bulge the traps open. Through a series of design features, we can culture animals for periods of 3 days to full adults starting from the embryo stage. The advantage of this device lies in the passive, valve-less control; animals remain trapped due to the design of the embryo trap and flow of medium without any active device control, and in the ability to observe early developmental larval stages. Immobilization of animals for high resolution imaging is achieved via formation of a thermally reversible gel from the solution of a biocompatible polymer Pluronic F127. The sol-gel transition occurs within a 1 °C temperature change and is tunable to match C. elegans physiological conditions. Precise temperature control is facilitated by microelectrodes patterned on glass that is in contact with the device. Image quality (e.g. amount of diffraction, photo-bleaching) when using the gel is comparable to that of standard methods with anaesthetics. We have verified the gel's biocompatibility with C. elegans; repeated and long-term exposure and immobilization show no significant effect on measured physiological traits such as pharyngeal pumping rate, number of progeny, and time to reach egg-laying. To our knowledge, our platform is the only system to date that facilitates longitudinal studies in the early stages of development on an individual-animal basis. It is also the first to enable live imaging of short-term (order of seconds) to long-term (hours and days) developmental events at arbitrary intervals on a single device.