High-Resolution C. elegans Imaging Across All Larval Stages

In the lab we work on all kinds of developmental biology questions. I, myself, work more on the technical side, developing methods to better address these questions using imaging techniques. The C. elegans field has successfully adopted a huge variety of methods, ranging from CRISPR gene editing, transcriptomics and proteomics, all the way to super resolution microscopy. C. elegans is an amazing model, however, in the context of in vivo observation at high resolution, it comes with its challenges, requiring immobilization for best resolution, which in turn limits animal viability. The protocol presents one possible solution for long-term C. elegans imaging, allowing researchers to adopt the method in their own lab and study a wide variety of dynamic developmental processes directly in vivo. Within the lab, the method has allowed detailed observation of organ formation, for example, in the context of morphogenesis or following somatic cell divisions into adulthood. The method has since been adopted by numerous labs studying a diverse range of developmental processes.

[Presenter] To begin, attach the device to the support frame and mount it on the upright microscope. Fill a syringe with deionized water, then attach a 23-gauge needle and a long piece of one by 16-inch tubing with a hollow bent steel pin. Fill the tubing with deionized water from the syringe and insert the steel pin into the punched hole of the valve inlet to connect the tubing. Remove the syringe and the needle before attaching the tubing to the off-chip solenoid. Using the imaging software, turn on the solenoid and pressurize the device for several minutes to expel all air from the valve. Verify completion by visually checking that the air water interface appears dark and disappears into the PDMS material. Turn off the solenoid using the imaging software. Next, fill a one-milliliter syringe with the filtered bacteria solution and attach a 30-gauge needle and a long piece of one by 32-inch tubing to the needle. Press the plunger to fill both the needle and attached tubing with the bacteria solution. Then using tweezers insert the one by 32-inch tubing directly into the food inlet of the microfluidic device and place the syringe on the syringe pump. Press the syringe plunger using the thumbscrew at the back to fill the device with liquid. Block both the worm inlet and outlet with sealed steel pins and apply additional pressure using the set screw to remove any remaining air from the device. Then remove the blocked steel pin at the outlet and attach the waste container to the outlet. Push the syringe to ensure the waste container is properly connected and that no blockages exist in the system. Remove the second blocked steel pin from the inlet and push the syringe until a small drop of liquid appears at the worm inlet. Then using a 23-gauge needle, attach a longer piece of one by 16-inch tubing measuring approximately 15 to 20 centimeters to a one-milliliter syringe filled with S-basal buffer. Attach a straight 23-gauge steel pin to the other end of the tubing. Fill the needle and tubing with S-basal buffer from the syringe. Now, insert the steel pin at the end of the tubing into the tube containing the worms, and push a small amount of liquid through the tubing to ensure no air is left. Pull the worms into the tubing without pulling them into the syringe. Then push the syringe connected to the worms until a small drop of liquid appears on the steel pin and insert the steel pin into the worm inlet of the microfluidic device. Place the device onto a microscope at low magnification, either 5x or 10x, or onto a dissection microscope. Position the device such that the inlet is visible on one side of the field of view and the back of the trap channel inlet is visible on the other side. Gently push on the worm syringe's plunger. Next, push the animals toward the channel array. Once an animal faces the channel, push it into the channel and repeat for additional animals. After trapping sufficient animals, place the syringe, still attached to the worm inlet on the microscope stage where it will remain throughout the experiment. If loading was performed on a dissection microscope, transfer the device to the imaging microscope. Now, switch on the syringe pump and run it at a preset rate of one microliter per hour for 0.5 microliters. Program the syringe pump such that it runs at one microliter per hour for 0.5 microliters, after which the flow rate is increased by 100 microliters per hour for 0.5 microliters, and the flow pattern is repeated throughout the experiment. Place the device onto the microscope stage and ensure it is firmly held. Switch to the desired imaging magnification. Identify the animals and regions of interest within the trap channel array and set up the desired imaging conditions. Image at the desired imaging conditions, actuating the on-chip valve through the solenoid 10 seconds before image acquisition so the animals are held in place. The microfluidic device maintained high imaging quality across various microscopy modalities, including brightfield, epifluorescence, spinning disc confocal, and super resolution methods due to the use of a 170-micrometer thick cover glass. Development of Caenorhabditis elegans epithelial cells visualized from early L1 to the mid-L2 larval stage. Induction of the primary faded vulva precursor cells and their subsequent divisions from late L1 to the early L4 larval stage was evident. Vulva formation stages from early L3 to adulthood. Acquired images were enhanced through image deconvolution and registration, improving visual clarity Seen cell divisions occurred in a consistent and timely manner across all animals with all divisions completed within typical larval stage durations. Gonad length increased steadily during L2 and L3 stages with consistent measurements enabled by the straight orientation of animals.