The overall goal of this procedure is to measure thrashing frequencies of freely swimming microscopic organisms. This is accomplished by first preparing a sample of microscopic worms. Next, the optical setup is prepared.
Then the diffraction pattern introduced by the live organisms is recorded. Finally, the recorded data is analyzed to determine swimming frequencies. Ultimately, diffraction analysis is used to show changes in locomotion patterns of unconstrained worms.
The main advantage of this technique over existing methods like traditional microscopy, is that swim patterns can be observed as the species moves freely in a three-dimensional space. Though this method can provide insight into freely swimming sea elegance, it can also be applied to other transparent, small and microscopic organisms exposed to varying conditions. To set up C elegance for video analysis begin by using a flattened platinum wire pick to move 10 to 20 GR adult nematodes to fresh NGM.
Agar filled Petri plates containing small circular spots of e coli. Allow the nematodes to lay eggs for three to five hours, and then remove the adults. Thus establishing a small developmentally synchronized culture of 50 to 150 nematodes to grow the nematodes to early adulthood, incubate the Petri dishes at 20 degrees Celsius for four to five days.
On the day of the video analysis, use one milliliter of deionized distilled water to flush a plate of young adult nematodes, thus collecting 50 to 150 worms from the synchronized culture. Use a micro centrifuge to spin the worms to the bottom of the tube or allow them to settle by gravity for about 15 minutes. It remove the majority of the water from the tube and replace it with one milliliter of deionized distilled water to wash any adhering bacteria from the nematodes.
Using a micro pipette transfer five to 10 nematodes in water to a quartz vete. Assemble the optical setup as shown here, using a 543 nanometer helium neon laser for oversampling two front surface aluminum mirrors used to steer the laser beam through the vete, a vete holder, a projection screen, and a high speed camera capable of filming at approximately 240 frames per second. Next, use a piece of paraform to cover the top of the vete and invert it to mix the nematodes into the water column.
Place the vete in a vete holder so that the worms are initially near the top of the vete. Using the mirrors, steer the laser beam through the center of the vete. Because the nematodes are denser than water, they will slowly fall to the bottom of the vete while swimming within the water column on the projection screen, color the spot corresponding to the transmitted laser beam black to reduce scattering as the transmitted beam meets the projection screen.
Eliminating or reducing scattering from the transmitted beam will keep the CCD array of the camera from saturating due to the transmitted beam. To capture a measure of the size of the diffraction pattern, draw a line of about five centimeters on the projection screen next to the transmitted laser beam spot without interfering with the diffracted light image. Turn the room light off and record diffraction images on the screen as the worms pass through the laser beam.
To prepare the video data, install the following video analysis program and import the video into the program. Set the origin to coincide with the transmitted laser spot using the software. Track the angular displacement of the diffraction image formed by each nematode.
Copy and paste data into a spreadsheet and add 180 degrees or PI for all negative angles. To produce a continuous graph. Using the video analysis setup, place a photo diode with a small area off center in the diffraction pattern directly in front of the projection screen via A USB port.
Connect the photo diode to the digital oscilloscope that connects to the computer. Observe the thrashing patterns on the computer screen. Save data sets in ASCI or text format to analyze the data.
Use video or the photo to import the data into a data analysis program capable of fitting waveforms. Using chiqua minimization fit a sinusoidal curve to determine thrashing frequency. Average the swimming frequencies from various samples, and determine the variance.
For this study data were analyzed statistically using a single factor Inova, followed by the Bon Ferran. Multiple comparisons test A P value of less than 0.05 is considered statistically significant to model diffraction patterns. Begin by using a microscope to take images to ize an image.
Drag it into Mathematica. Use the binary eyes command to convert it into black and white. Alternatively, use the edge detect command to create a black and white image where the two trailing numbers control the coarseness of the resulting edges.
Next, using the image data command, convert the image into a matrix of zeros and ones. Use the forer command to forer, transform the matrix, then square the absolute value of the matrix, and view the resultant image, which is the diffraction pattern corresponding to the original image. In addition, use the log function to scale the contrast of the image.
Finally, compare the model diffraction patterns with the diffraction patterns obtained from freely swimming worms. In this example, sea elegance were studied in a quartz Q vet, one centimeter wide, five millimeters thick, and four centimeters tall. Sampling a single worm using video analysis.
The average swimming frequency obtained from video analysis in a five millimeter thick vet was about 2.5 hertz. Similarly, sampling a single worm using the realtime data acquisition method and a digital oscilloscope resulted in a swimming frequency of about 2.7 hertz. This procedure can be repeated for many worms.
A detailed study of freely swimming worms revealed an average swimming frequency of 2.37 hertz in a five millimeter vete. As expected, the swimming frequency is higher than that for a crawling worm. Using this diffraction method, the average swimming frequencies of a sea elegance, which is confined to a microscope slide, has been found to match the previously published value of two hertz.
Following the video. Data preparation and modeling of diffraction patterns demonstrated in this video allows for the modeling of swimming diffraction patterns. With the help of worm images obtained with a conventional microscope, the modeled diffraction patterns are used to simulate a swim cycle of the sea elegance.
A successful model consists of physically feasible success of swim patterns, matching the swimming frequencies. The worm should be in the same shape at the end of the swim cycle as it was in the beginning of a swim cycle. Once mastered, this technique can be done in less than a minute for each worm using the fast photo diet while observing the swim cycles on the computer screen.
After watching this video, you should have a good understanding of how to evaluate freely swimming nematodes.
