Assembly and Operation of a Cooling Stage to Immobilize C. elegans on Their Culture Plates

We are developing a simple approach that can immobilize the entire Caenorhabditis elegans population directly onto cultivation plates. This strategy significantly accelerates imaging-related experiments. The sodium azide immobilization typically used for high-resolution imaging of the C.elegans population is extremely labor intensive, requiring individual animal mounting and handling.

This method can also reduce animal lifespan and viability, which can confound data. Traditional cooling method uses relatively low temperatures of two to four degree Celsius of employed microfluid, which requires extensive user training. We found that, with the coolings, that the warm temperatures, like six degrees Celsius, most strongly immobilize animals for high-revolution imaging.

Cooling immobilization of C.elegans is not widely used, but when used, it is usually combined with complex microfluidics that requires significant user training. Due to the shortcoming of the typical immobilization methodology, our improvement of cooling immobilization and accessibility enables high throughput and high-resolution imaging for researchers. Compared to labor-intensive mounting and anesthetizing of individual animals, our cooling approach can reversibly immobilize large C.elegans populations, eliminating 98%of processing time.

The lack of chemical anesthetics also minimizes harmful exposure to animals and researchers. A cooling stage allows for high throughput and high-resolution imaging directly on cultivation plates. The simplicity of this design enables other researchers to easily build their own devices and integrate them into their existing setups, to revolutionize the scope and the pace of their biological studies.

Our laboratory will adapt the cooling stage for various microscope configurations. Different designs will work with inverted microscopes, compound upright microscopes, as well as different imaging platforms. To begin, manufacture the copper plate from a 170 by 120 by 3 millimeter, 99.9%pure copper metal sheet, using a computer numerical control mill.

Using fine grit sandpaper, remove the sharp edges and dirty residue. To construct a water-cooling assembly, cut the silicone tube into three sections of 40, 50, and 80 centimeters. Plug the cut silicone tube sections into the radiator ports, pump tank, and copper cooling block, ensuring watertight connections to complete the water-cooling assembly.

Next, connect the pump tanks and the radiator's wires to the 12-volt power supply through the breadboard. Open the pump tank cap using a flathead screwdriver and add water until the pump tank is about 80%full. Do not cap the pump tank.

Power up the water-cooling assembly using the 12-volt power supply to facilitate the water flow inside the assembly and the blowing of the fans on the radiator. As water flows from the pump tank, the liquid level drops in the tank. Add water to the pump tank until it stabilizes at nearly two-thirds full.

Shake the radiator to remove air bubbles before placing the cap on the cooling tank. To begin, clean all the surfaces of the copper cooling block with 70%ethanol in the water-cooling assembly. Wear gloves and apply a thin and even layer of around 0.4 grams of thermal paste to one surface of the copper water-cooling block.

Ensure the surface orientation will prevent the tubes from crossing or bending. Similarly, clean the hot surface of the Peltier using 70%ethanol and apply the thermal paste to the surface. Connect the Peltier hot surface to the copper cooling block surface with thermal paste, following the orientation of the wire.

Apply pressure to secure it and clean the excess thermal paste. Then make sure the 12-volt power supply and the tunable power supply are off. Connect the red wire of the Peltier to the positive output and the black wire to the negative output of the tunable power supply using an alligator clip.

After rechecking the electrical and water-cooling assembly connections, switch on the 12-volt power supply and tunable power supply sequentially. Gradually turn the tunable power supply to 12 volts, so the current should be around six amperes with the suggested Peltier. After two minutes, measure the temperature with an infrared thermometer, without touching the cold surface, which should be colder than minus 35 degrees Celsius.

Begin by carefully cleaning the copper plate and the sapphire window with 70%ethanol. Use fine grit sandpaper to make rough copper plate surfaces smooth. Apply around 0.5 millimeter thin and even layer of thermal paste on three inner surfaces of the copper plate where the sapphire window will connect, and spread evenly.

Then lay the copper plate down while protecting the bench top with paper. Insert the sapphire window into the copper plate hole, ensuring the sapphire does not rotate during insertion. Remove excess thermal paste.

Adhere the four-inch wide tape to the top surface of the copper plate-sapphire window assembly, having a square depression area. Guide the adhesion slowly from one side to the other to avoid air bubbles during pasting. Cut out the area of the tape indicated with blue dashes from the assembly using a sharp blade, exposing the two thread holes, the square depression, and the 70-millimeter diameter area of the sapphire window.

Cut out the area adhered to the copper plate-sapphire window assembly. Once done, tape the bottom surface of the copper plate-sapphire window assembly and repeat the cutting procedure, exposing the two thread holes and the 70-millimeter diameter sapphire window. Apply about 0.4 grams of thermal paste to the square depression of the copper plate and to the cold surface of the Peltier already attached to the copper cooling block.

Connect the Peltier cold surface to the copper plate depression with downward pressure. Then clean the excess thermal paste. Mount the 3D-printed bracket on the top of the copper cooling block, and then use a hex key to tighten two 8-32 0.5 inch long screws to fix the bracket to the copper plate.

Place the copper plate into the-3D printed isolation base for thermal isolation from the benchtop or microscope base during operation. The cooling stage is assembled and ready to use. The cooling stage is designed for a typical upright microscope stage with easy insertion and removal.

When cooling immobilization is needed for imaging or screening, the cooling stage is placed on the microscope stage to finish the installment, and vice versa. A thermal couple thermometer, used to track the surface temperature of the plate placed on the cooling stage, showed that the stage cooled down the plate from 20 degrees to six degrees Celsius in six minutes, to one degree Celsius in 10 minutes, and eventually stabilized below minus seven degrees Celsius in around 40 minutes.