Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

The overall goal of the following experiment is to monitor behavior development and neural activity of multiple senior he elegance. This is achieved by culturing the worms in food filled aros microfluidic chambers. Long-term calcium imaging is performed using an E-M-C-C-D camera and TTL triggered short exposure times.

The procedure can be scaled up by scanning multiple chambers sequentially or by filming several chambers at once. The results show larval and adult behavior as well as calcium transient of command inter neurons. The main advantage of this micro chamber technique is that behavior and development can be followed over a long time case with high quality imaging.

Visual demonstration of this method is critical as filling the micro chambers with the right amount of food and warmth is difficult to learn. To begin set up a microscope with a lid heater, automatic stage and E-M-C-C-D camera system. Fabricate polymethyl suboxane stamps in a microfluidics facility or by using a commercial foundry.

Cut the PDMS chip into 16 individual stamps using a scalpel. Next, expose both the stamp and a glass slide to air plasma for about one minute before attaching the stamp onto the slide. Working slowly to avoid melting the plastic.

Cut out a square area from the bottom of a 3.5 centimeter dish. Prepare several dishes at once. Prepare sterile aliquots of both high and low melting point aero prior to use.

Place three aliquots of high melting point aeros onto a heating block. In addition, melt one eloqua of low melting point aero before transferring it to a separate heating block. First, take the plastic dish with the square opening created earlier and place it upside down with the opening facing upwards.

Use a piece of double-sided sticky tape to cover the opening. Turn the dish around so that the sticky tape is on the bottom and place the dish onto a hard surface. Cut the opening free using a scalpel.

Next, fill the area that surrounds the opening with two milliliters of high melting point aros. Wait until the agros is solid. Once solidified, remove the protective film that covers the double-sided sticky tape on the other side.

This results in a fine ring of sticky tape that surrounds the outside of the opening. The agro serves as a moisture reservoir that will later surround the sample. Before constructing the micro chambers, prepare the PDMS stamp surface by exposing it to air plasma for 20 to 60 seconds.

Create two spacers of equal height by stacking five to nine glass slides. Place a single glass slide in between the two spacer stacks. Next place the glass slide that contains the PDMS stamp across the spacers.

Adjust the height of the spacers so that there is a 1.5 millimeter space between the molding surface and the glass slide. When ready, place a drop of hot high melting point aros onto the slide near the PDMS stamp and quickly slide the stamp horizontally into the liquid aros. Once the aros has solidified and appears opaque, pull the stamp off vertically with one move.

Using a fine platinum wire pick, transfer the eggs or worms together with OP 50 bacteria onto the aros. Use an eyelash to distribute one egg or one worm per chamber together with food. One aros pad should contain about 30 worms.

Cut the aros slab containing the micro chambers into a square so that it fits nicely into the opening of the dish. Use forceps to lift the slab and place it upside down onto a glass cover slip once dropped, do not lift or slide the cover slip. To avoid pushing contents out of their chambers.

The chambers are now sealed. Next, place the cover slip into the opening of the plastic dish. Gently press the cover slip down onto the ring made of double-sided sticky tape.

Take care not to break the glass. Turn the dish upside down and use low melting point aros to fill the gap between the agro slab and the aros reservoir for when the aros has solidified. Seal the dish with a lid and param.

After finishing the preparation of micro chambers, check them under a stereo microscope correctly. Filling chambers and controlling moisture are critical for success. To begin place the dish containing the micro chambers onto the microscope and focus on the sample Record 40 image frames in 20 seconds every half an hour for 24 hours.

Next, set up the scan so that it visits each worm using the stage. Aim to film about 30 worms in one run. Multiple worms can be imaged by zooming out.

Use a lower magnification to cover several micro chambers and film several adjacent micro chambers simultaneously. After the end of image acquisition, separate the data for each individual chamber by cropping a region of interest covering one animal. Frame, subtraction can be used to quickly assess mobility data.

After imaging calcium imaging is performed on a compound microscope equipped for widefield epi fluorescence. To limit light exposure, use a transistor transistor logic signal that triggers an LED to illuminate the sample. At the same time, the camera records a frame.

Run a burst movie for 24 hours. That images each worm every 15 to 30 minutes. First record 20 seconds with DIC.

Then 20 seconds with GFP fluorescence. And finally, an image of the MK two signal for control expression levels. For visual data inspection, use a false color map to enhance the visibility of small changes in fluorescence intensity.

Lastly, perform calcium data analysis using standard procedures. This example shows larval behavior. A chamber with larger dimensions allows development from an egg into an adult long-term, changes in behavior are shown here.

The dower larva seed here is an alternative life stage that is engaged during adverse environmental conditions. This adult worm has laid many eggs into the chamber. Finally, this image shows an adult hermaphrodite and a male mating inside an adult chamber.

Calcium imaging of the command inter neuron A VA for an L one larva is shown here. These images show calcium activity for the same type of neuron in an adult animal. Scaling up long-term imaging allows 30 worms to be imaged on one camera chip.

While attempting this procedure, it is important to fill the chambers with the correct amount of food and to ensure the correct motions of agarro. After watching this video, you should have a good understanding of how to use aros micro chamber imaging on various life stages of sea elegance.