Fourier-Based Diffraction Analysis of Live Caenorhabditis elegans

The overall goal of this fourier-based diffraction analysis is to characterize the locomotion of C elegans in real time as the species moves freely in a three dimensional space. This method can answer key questions in the field of neurobiology by describing species responses to different densities of solution or locomotory mutants. This method serves as a base line for fourier analysis of microscopic species.

It has the advantage that every microscopic species will have its own unique fourier spectrum. Though this method can provide insight into the locomotion of C elegans, it can also be applied to other micro organisms. Generally, individuals new to this method will struggle because finding the nematode in the cuvette, under the laser beam can be challenging.

We first had the idea for this method in attempting to learn about swimming behavior from the temporal diffraction signal, itself, a noisy signal. To begin, secure a helium neon laser near the back left corner of the optical workbench and connect it to a power source. Place a neutral density filter between the laser and the sample location, so that the laser beam travels through the filter before reaching the sample.

Using two front surface aluminum steering mirrors, build a periscope by securing the first mirror after the neutral density filter. Then, secure the second mirror about 10 centimeters below the first mirror to give room to steer the laser beam. Insert a cuvette between the mirrors and align the laser beam and cuvette so that the laser beam travels vertically through the cuvette.

Next, secure the photo diode directly across from the second mirror so that its sensor faces the mirror. Then, place a water filled cuvette on a stand. Adjust the height of the stand and the angles of mirrors one and two so that the laser beam travels through the cuvette and is directed near but not directly at the photo diode.

Use a lever to ensure the stand forms a level surface for the cuvette. If the stand is not level, make the appropriate adjustments until leveled. Finally, connect the photo diode to the digital oscilloscope using the USB cable provided and connect it to the computer.

Load the oscilloscope software and set the sample rate to at least eight hertz to resolve the trashing cycle of the worm sufficiently. Use a thin, flattened platinum wire pick to transfer four adult nematodes into a fresh NGM agar filled petri plate. Next, remove a disposable plastic cuvette from its package, being careful to only touch the cuvette on its ridged sides.

Then, use a micropipette to pipette distilled water into a cuvette until the cuvette is approximately 80%filled with distilled water. Place the petri dish containing the C elegans under a dissecting scope. Then, use the platinum pick to remove one mature C elegans from the petri dish and submerge the pick into the cuvette.

Move the pick in circles to dislodge the nematode if necessary. Handle the nematodes gently and do not shake the cuvette. Shaking the cuvette may cause the worms to be unresponsive.

To prevent bubbles from forming in the cuvette, fill the cuvette with distilled water until it slightly budges over the cuvette's top. Then, fill the cap with water and quickly put the cap onto the cuvette. Using lens paper, remove any water droplets that may have spilled over onto the cuvette.

Additionally, use lens paper to remove any small remaining droplets. Turn on the helium neon laser and adjust the frequency setting so that it produces a red beam. Then, turn on the sensor.

Hold the cuvette on its ridged sides and gently tilt it until the nematode is approximately in the center of the cuvette. Then, place the cuvette onto the stand in the optical system, centering the worm within the laser beam traveling from mirror one to mirror two. Next, place the photo diode in the diffraction pattern so that the location of the photo diode and the central maximum of the diffraction pattern do not coincide.

Then, rotate the neutral density filters so that there is a notable voltage output from the photo diode. Be sure to limit the light using the neutral density filter, to prevent the photo diode from saturation. A flat line indicates oversaturation or a lack of intensity.

Once the moving diffraction pattern is visible, collect data with the photo diode by clicking on the start button on the software, controlling the oscilloscope while monitoring the worm's movement. Continue taking measurements until the worm moves out of the laser beam and the diffraction pattern disappears. If the cuvette is scratched, dispose of it and the worm and begin again.

After about 20 seconds, stop the data collecting process by clicking the stop button on the software for the oscilloscope. Save each trial's data in comma separated value or text format. Collect at least 50 data sets for each phenotype and use eight to 10 animals per trial.

Examples of modeled sequential worm movement and corresponding diffraction patterns are shown here. The modeled diffraction patterns qualitatively resemble experimental patterns and indicate that the simulation successfully modeled the nematode. In addition, the temporal diffraction signature of the roller and wild-type c elegans can be shown quantitatively.

As seen here, each nematode thrashes at different rates and amplitudes. The average digital fourier transforms allow for the nematode to be identified by the amplitude of their diffraction frequency spectrum. The wild type worm spectrum is dominated by lower frequencies than the roller motion spectrum.

Here's an example of the w oscillation which can be thought of as two opposing sea motions. In contrast, the roller will mostly form a c to one side and appears like this. In examining the frequencies, the w motion is more complex, revealing more secondary low frequencies than the c motion.

Once mastered, one data set can be collected in about two minutes if the technique is performed properly. It takes about 20 to 30 minutes to set up the optics for a series of data sets. While attempting this procedure, it's important to remember to handle the C elegans with care, so as to preserve the integrity of their locomotion.

Following this procedure, we can apply other methods such as a tractor reconstruction and non-linear analysis to learn more about C elegans motion. After its development, this technique has paved the way for researchers in neurobiology to study in more depth locomotory mutants in C elegans. After watching this video, you should have a pretty good understanding of how to generate diffraction patterns for live nematodes and how to analyze them.

Don't forget that working with lasers can be extremely hazardous and taking precautions such as not having the lasers travel at eye level, should be taken while performing the procedure.