The overall goal of this procedure is to measure and evaluate the vertical path velocity, and forces exerted by a freely swimming nematode using coherent monochromatic radiation. This is accomplished by first setting up a coherent monochromatic shadow experiment for freely swimming sea elegance. Then an individual adult sea elegance is placed into the shadow experiment viewing chamber, which is a vet.
The shadow of the worm as it descends through the laser beam is recorded as a movie, and the movie is used to analyze the motion of the animal. Hence, the path of a freely swimming nematode and the associated forces generated are determined. The main advantage of this technique over existing methods, such as traditional microscopy in a horizontal plane, is that the motion of freely swimming microorganisms can be investigated using a wide range of monochromatic light at a low cost.
This method can answer key questions about forces generated by or responded to by nematodes in a liquid environment. We first had the idea for this method when we first started exploring the idea of vertical movement for microscopic species. Visual demonstration of this method is critical as the procedural steps are difficult to learn because it takes some practice to get the laser alignment correct.
Furthermore, some time in the bio lab is necessary to learn how to prepare the worms, Assemble the experimental setup to create shadow images. Use two or more mirrors to steer the helium neon laser into the Galilean beam expander where it should increase to 12 millimeters. Have one or two pin holes in the beam path to ensure its alignment from the expander.
Direct the beam to a mounted quartz Q vet. After the beam passes, the Q vet magnify it using a plano convex lens with a positive focal length of 75 millimeters. Then position a screen about 120 centimeters from the lens, which is the optimal resolution distance.
Position, a high speed camera where it can image the screen such as next to the Q vet. A dissecting microscope for picking worms should also be near the qve. Temporarily secure a transparent ruler with millimeter divisions to the center of the QVAD holder on the screen near the edge of the camera's view.
Use the ruler's lines to mark the scale distance for one millimeter. Do this for each wavelength used as refraction will vary due to chromatic aberrations in the QV and changes in the magnification as previously described. In JoVE, have young adult nematodes ready for the qve.
Also have prepared nematodes dead from exposure to chloroform. Begin by filling the Q Vet with distilled water to one millimeter from the rim. Start a recording with the room lights on, so the line on the screen is initially visible and then turn off the room lights.
Then collect a nematode on a platinum wire pick. It's important to place the worms into the vet by slightly touching the pick to the surface of the water without agitating the medium. The projected image of the worm and the water column is inverted.
Worms that are descending with gravity will appear to move upward on the screen. For each worm record about 20 seconds and record about 50 worms per condition. First, import the video into the video analysis program.
Set the scale using the length markings in the video to determine the magnification factor. Then start tracking the linear displacement of the head of the shadowed nematode. Select at least 10 data points over the path traveled one every 0.3 seconds or so.
The vertical velocity is then calculated from the derivative divided by the magnification factor. It is critical to create separate curves for the horizontal and the vertical displacement. Since gravity only acts in the vertical direction.
If the data points form a generally straight line, the path is linear. Some head movement deviation can be ignored. Next, from the analysis menu, create a linear regression line by fitting a straight line to the data.
The slope of the line is the vertical speed of the worm. If a worm moves non-linearly, select curve fit from the analysis menu. From there, fit the curve to a second or third order.
Polynomial for each curve in the path fit a curve to the data. By sliding the brackets on each side of the graph, the mathematical expression of the curve is presented. A relative error of up to 15%here is acceptable.
Add additional curves as needed to cover the data points and try to spline the functions. The curve fits should have the same slope where they overlap. The first and second derivatives of the curve provide velocity and acceleration.
Thrust is calculated using three micrograms as the worm's mass when drifting, there was no distinguishable difference in the descending rates of living and dead see elegance. The rate of downward drift for both was 1.5 millimeters per second. However, live nematodes capable of changing direction and swimming against gravity, curves are fit to the vertical displacement of the nematode and acceleration is calculated from their second derivatives.
The forces that act on the worm until the worm reaches the turnaround point are gravity, drag, buoyancy, and thrust. The net force equals their vector sum. Consider the vertical force components.
The gravitational force is equal in magnitude to the buoyancy force, assuming the worm is about as dense as water, so the equation can be simplified until the turnaround point. Drag plus thrust are constant. Drag is initially larger, but gradually reduces to zero while thrust increases at the turnaround point, there is no vertical drag thrust is pointing up during the worm's.
Vertical ascent drag increases in the downward direction, so the thrust force must be larger than drag plus weight. The nonlinear path of this worm was analyzed. It changed directions.
Looking at its displacement, it slowed down and started to turn around at 68 seconds. However, the upward acceleration decreased continuously until the net acceleration hit zero at 68.5 seconds. Then it began accelerating in the negative direction to descend again.
Thus, when the velocity was zero, the worm had significant upward thrust calculated to be 1.32 Pico newton's Once mastered, the data can be taken within 10 minutes if the worms are ready to be picked off the auger. While attempting this procedure, it's important to remember to keep the camera recording rate constant for all data taken because we need to adjust the frame rate in the video analysis program Following this procedure. Other studies can be performed like single wavelength studies in order to answer questions like what forces are exerted in various directions, vertical and horizontal.
For example, This optical technique could be used by other biologists interested in locomotion properties of small organisms like Proti or other aquatic una. After watching this video, you should have a pretty good idea of how to analyze the motion of freely swimming microorganisms using the magnification of shadow images, and then a video analysis program.
