マイクロ流体デバイスを用いた機械的刺激と線虫の高解像度イメージング

The overall goal of this technique is to measure how nematodes and their neurons respond to external mechanical stimuli using a microfluidic chip setup with pressure actuators. This method can answer key questions in the fields of mechanobiology and sensory biology such as how cells, tissues and animals respond to mechanical stimulation. The main advantage of this technique is that it sufficiently reduces the worm's mobility in a non-invasive way to allow high-resolution imaging while still having access to the worm's cuticle for mechanical stimulation.

This method can also be used to study the influence of mechanical cues on development. It could even be applied to other systems such ex vivo organ exponse or other animals of similar size to C.Elegans. Set up a microscope system for simultaneous excitation of GCaMP and RFP.

One option is a discreet light source that transmits only cyan and yellow light. For viewing, use a 10-times objective and high-magnification objective. Also, send the image to a computer via digital camera.

Next, include a florescence cube, and if needed, an excitation filter. Add a dichroic mirror to the cube that reflects cyan and yellow light and transmits green and red light. Set up a beam splitter with a long-pass dichroic mirror with a cutoff at 570 nanometers.

Also, provide a band-pass emission filter for green light at 525 nanometers with a 50-nanometer width and a band-pass emission filter for red light at 632 nanometers with a 60-nanometer width. Project both beams into the camera's field of view. Make sure the green part is projected to the top half of the screen, and the red part to the bottom.

Always use microfluidic chips that are less than one month old. For the setup, first load the gravity flow reservoir with filtered M9.Position the reservoir about 60 centimeters above the chip and connect it to a chip outlet. Next, connect the other outlet to a waste container with two inputs, and connect the second input to the waste container to a peristaltic pump.

Make all the connections with polyethylene tubing. Next, assemble interconnects, 50-millimeter tubing lengths with metal tubing connectors on both ends. Press-fit and interconnect onto each of the six actuation fingers and into the worm inlet.

Now, position the chip under the optics. Be sure to leave the interconnects attached to the chip as the PDMS will wear out quickly with repeated manipulations. It is vital that the PDMS actuator and the tubing connecting the chip to the air reservoir are properly sealed to ensure good chromatic actuation of the diaphragm.

Before loading animals, check for loss of pressure in the microfluidic chip. Measure the deflection of the diaphragm by performing several actuation cycles at the desired pressure. Then, compare the quantitative values with theoretical predictions.

If the measured values are not as expected, check for leaks in the tubing or tubing connectors. The PDMS may also have a different elasticity due to an alternative ratio of base polymer and curing agent, or the PD mass could be old and excessively crosslinked. Have prepared age-synchronized-to-young-adult or adult-day-one C.elegans as described by Porta-de-la-Riva and company in their Jove publication from 2012.

Now, pick two to five young adults or one-day-old hermaphrodites. Choosing animals of the correct size is critical. Small animals will not get trapped in the channel, and overly large animals will be difficult to remove and can clog the device.

Pull the picked worms in a drop of filtered M9.Then, draw them into a length of tubing using a one-milliliter syringe without pulling them into the syringe body itself. Next, connect the tubing to the interconnect at the worm inlet of the chip. Then, activate the gravity flow by opening the valve to the reservoir and starting the pump.

Now, observe the trapping channel at four-times magnification and gently push the animals into the device. After loading the animals into the waiting chamber, use the plunger to gently flow one of them to the front of the trapping channel such that its head enters the tapered shape of the channel. If the worm does not fill the entire cross-section of the channel from the nose to near the end of the body, remove the worm.

Many times, worms that are too small will go straight through the chip without being trapped. If an animal that got caught in the trapping channel needs to be removed, simply press the plunger of the syringe until the worm disappears from the channel and then load a new worm. Once a worm is properly loaded, switch to florescence mode and increase the magnification.

To prevent saturation, be sure to adjust the excitation intensity based on the florescence intensity. Check whether the neurite of a neuron of interest lies across the diaphragm of one of the actuators. That actuator must also be anterior to the cell body of the neuron.

If not, adjust the position of the animal by pulling or pushing on the plunger. If that does not help, remove the worm and load a new one. Also, if there happens to be autofluorescence around the neuron, replace the worm.

Once a worm is properly loaded, focus on the cell body of the neuron of interest, then determine which actuator the chip is on its anterior side and connect this actuator to the programmable pressure pump using the associated interconnect. If the experiment requires measuring the distance between the actuator and the neuron, fit both into the field of view with the channel wall parallel to the upper and lower edge of the image. Next, define a pressure protocol using the programmable pressure pump.

Always start by taking at least 50 images at zero kilopascals to define a baseline. Then, program the stimulus waveform and pressure. Now, run the imaging and pressure protocol.

While recording, the neuron of interest must be the brightest spot in a 10-square-pixel area. It cannot move more than 10 pixels in two sequential images, and it must stay in the field of view. While recording, you may observe changes in the florescence intensity when the pressure and the actuators change.

This indicates a successful stimulation of the neurons. Between stimuli, include 10 seconds at a constant pressure of zero kilopascal. It is possible to simultaneously record signals from multiple neurons in the field of view so long as they are separated by at least 10 pixels during the entire recording.

To keep worms for further study, disconnect the outlets of the chip toward the gravity flow and the waste container. Then, gently press the plunger until the entire worm is pushed through the trapping channel into the flow channel, and continue to press the plunger until the animal appears in a droplet outside of the chip. Now, disconnect the syringe with the tubing and aspirate the worm in the droplet onto an agar plate.

To remove and sacrifice a worm in the channel, press the plunger until the entire worm is pushed through the trapping channel and into the flow channel. Then, the worm will flow out of the chip and into the waste container. After setting up the microfluidic chip, the deflection of the diaphragm was tested.

The measured values were reproducible with slight variation not exceeding one micron at 450 kilopascal of pressure. After inserting the worms into the inlet of the chip, the skin of a single animal inside the trapping channel was presented to the six actuators. With this design, the PLM neuron usually cannot be immobilized completely, so it free to move laterally and vertically.

Although successful activation of PLM neurons is possible, recording calcium transients from them is difficult due to the tail movement. Once in place, the animal was stimulated using one of the six actuators positioned to deliver mechanical stimuli to each of the six TRNs. One of the three different two-second stimuli were presented.

A hold at 275 kilopascals, a ramp to 275 kilopascals or a buzz consisting of a 75-kilopascal 10-hertz sine superimposed with the 275-kilopascal step. 10-second gaps with no pressure separated the stimuli. Pressure ramps and pressure steps activated TRNs only if the stimuli achieved pressures above 400 kilopascals, whereas the buzz stimuli robustly activated all TRN.

Once mastered, this technique can be performed within five minutes per worm. It is important to remember that a poor seal between the chip and the coverslip, or between the chip and the interconnectors will cause the device to leak and fail. Following this procedure, other methods like voltage imaging or targeted delivery of mechanical stimuli to different neurons can be performed in order to characterize the mechanical response of nociceptors.

Also, because the animals can be recovered following this procedure, the technique could be used to screen for mutants defective in their response to mechanical stimuli. Don't forget that working with compressed gases and chemicals can be extremely hazardous. Take precautions such as wearing personal protective garments and working in fume hoods when necessary to avoid those hazards.