C. elegans Movement Tracking: A Method to Assess Locomotion in Worms

- To assess locomotion in C. elegans, use a camera to record the worms' movement on an agar plate, ensuring even illumination of the plate during the recording. To restrict the worms' movement around the plate, use copper rings. Copper is aversive to C. elegans, so worms are corralled within the rings.

Place several copper rings onto one plate to simultaneously track worms in each ring. This allows the assessment of control and experimental animals to be performed under identical conditions, reducing day to day variability and allowing for side by side comparisons. Record the worms' movement over set time intervals, and use computer software to analyze features of the locomotive behavior, such as the speed of movement and distance traveled.

In the example protocol, we will see a demonstration of a movement tracking assay testing the effects of alcohol on the worm's locomotion speed.

- To begin, take two minutes or less to transfer 10 worms from each experimental group to the center of a copper ring on an acclimation plate. This is a plate with no ethanol. Avoid transferring food with the worms as it will reduce their movement. Also avoid damaging the agar surface because the worms will burrow. Record the time of loading onto the plate, and be sure to stagger the transfer of worms to each plate by over two minutes, so each plate can be filmed individually at the appropriate time interval.

Let each acclimation plate incubate at room temperature for 30 minutes. Then, transfer the worms to the assay plate, following the order used to place the worms on the acclimation plate. Using a thin-edged flattened worm pick without bacteria, collect the worms on top of the flattened pick using a scooping motion. Then, seal the plate with laboratory film to minimize loss of ethanol to vaporization.

- The speed at which the animals are transferred at this step is very important, because the transferred animals will be exposed to ethanol longer. So it is a good idea to rotate which strains are placed on the plate first across experimental replicates.

- A microscope with high resolution video camera is required to film the worms now. Even illumination, such as from a three-inch square backlight, is vital for the analysis. Then, to maintain contrast, image the plate media side up.

Prepare the image analysis software to capture 120 frame, 12-bit, grayscale, time-lapse movies in one frame per second. To reduce the size of the output file, while still retaining sufficient image resolution, use a two-by-two binning mode.

Now record two-minute movies from each plate, 10 minutes after the last worms were placed on that plate. Then make a second two-minute recording, 30 minutes after worms were placed on the plate. For this demonstration Image-Pro Plus software is used, as a wide variety of other software can be adapted to the technique. Analyze the movies as two-minute segments.

First apply a filter to the images that flattens the background and enhances the contrast of the worm objects. Under the menu, select Process, then Filters, then Enhancement, and then Flatten. Set the parameter for a dark background and a 20 Pix feature width.

Now, analyze the locomotion of the animals in each ring separately with a circular region of interest that overlaps the copper ring. Identify and track the worms with the Track Objects command under the Measure sub-menu.

In the Tracking Data Table window, use the Tracking Options button to allow specific tracks to be excluded and to limit any experimental artifacts. Under the Auto Tracking tab, set the track parameters to a velocity limit of 400 microns per frame. Set the Acceleration limit to Automatic.

Set the Minimum Total Track Length to 400 microns, and set the Predominant Motion Type to Chaotic. Then under the Track Parameter, set the objects to allow Partial Tracks to have a minimum length of 21 frames and to have a Tracking Projection Depth of one frame.

Now, to initiate the tracking process, first click the Find All Tracks Automatically function button. This brings up the Count/Size Options dialog box and the Tracking Dialog box. Select the Manual option for the Intensity Range Selection in the Count/Size dialog box. This provides an important threshold step. Then adjusts the Intensity Threshold sliders to create an inclusive range that highlights dark objects. A good starting point on the scale is between 0 to 1,500.

Now apply a size filter to exclude objects that are larger or smaller than a single worm. Go to the Measure menu, and then select Measurement sub menu. This opens the Count/Size options dialog box. There, set the area range to 28,000 to 120,000 square microns. And set the perimeter range to 600 to 2,500 microns. Make adjustments to these parameters when using worms of abnormal size.

Now, complete the tracking process by clicking Continue in the tracking dialog box. Double check that the output tracks with the movie footage. Make sure every worm is represented unless there is a clear reason to filter it. Then, manually delete tracks produced by non-worm objects.

Next, calculate the velocity of each worm by averaging the distance traveled between each frame or each second, and create an average velocity for the tracks in each population. Consider this final average to be one sample point for statistical analysis of the experiment.