Phenotyping.The locomotion of sea elegance is an important tool in the discovery of genes and neurons involved in sea elegance behavior. Sea elegance undergo stereotyped locomotory behaviors such as forward and backward motions and various types of turns by changing their shape. To quantify these movements over long distances and at high spatial and temporal resolution, we have built a worm tracker to capture the motion of sea elegance as it crawls on the surface of an agar plate.
We have also developed software to isolate and analyze these movements quantitatively using custom programs written in the lab view and matlab. A challenge of all video tracking system is that they generate an enormous amount of data that's highly dimensional. Our image processing and data analysis program due with this challenge by reducing the worm shapes into a set of independent components, we have comprehensively reconstructed the worm behavior as a function of only three to four dimensions.
An agar plate is illuminated by a fiber light source and imaged with a camera. This system is mounted to an XY translation stage. The stage is moved by standard stepper motors, which are connected to a stepper motor controller.
The controller and camera are connected to the computer and controlled by custom programs written in lab view. The camera images the surface of an agar plate and identifies dark objects on a light background. A calibration mark for the automatic calibration process is made by poking the agar surface.
With a worm pick, the image quality is adjusted so that the computer program can quantify objects in real time. The gain, brightness and shutter speed of the camera can be adjusted to provide a dark object on a white background. The filtered binary image, which is used by the tracking program, can be checked.
The software has an auto calibration feature that calculates the calibration matrix by moving a test object. A fixed distance. The distances in pixels is calibrated to steps taken by the stepper motor by the calibration matrix.
After calibration, the system is ready to go and does not need to be recalibrated unless the magnification is changed or if the camera is repositioned. A copper ring will be used to corral the worms and keep them from migrating to the edge of the plate. Heat the ring first by placing it onto a heat block or equivalent.
The ring should be heated for approximately a minute. Place the ring onto a fresh agar plate and press down slightly to embed it into the agar surface. And an agar plate filled with NGM buffer will be used to wash L four and young adult stage worms of food residue.
After picking worms into the NGM buffer, let them swim for a few minutes. Carefully place one of the washed worms onto the tracking plate near the center of the ring. This plate will later be used for the worm tracker.
Place the prepared plate onto the worm tracker stage. Run the lab view program and select options if needed, such as location for images, types of images, measurements, and camera settings. Using the joystick, move the microscope until an image of the worm is in the field of view on the computer screen.
Then press track to engage the tracking program. Here we are showing a worm tracked in real time. The computer program measures the movements of binary filtered images as shown and can make measurements of this image in real time.
After tracking a reconstruction of the global trajectory can be made from the stepper motor movements while the local shape changes of the worm can be seen in detail. Worms crawl by making undulatory movements. We capture these movements by skeletonizing the worm into a single center curve, and then describing this curve as a set of angles.
We then decompose the skeletons into their fundamental shapes, otherwise known as eigen vectors. With these eigen modes, we can reconstruct the worm shapes and quantify the movements of the worm. Using the first two modes, we can specifically capture the phase position of the worm's undulatory cycle as seen in the second panel.
The first panel shows the joint distribution of modes one and two, highlighting a circular limit cycle. The phase movement in one direction corresponds to forward motion, and the phase movement in the other direction corresponds to backwards movement. Thus, the phase velocity corresponds to velocity of the worm.
In real space, the strong correlation of the phase and real world velocity can be seen in the last panel. Using the Eigen mode derived phase velocity, we can uncover some subtle details of sea elegance behavior that would be missed by eye. For example, the joint distribution of the phase velocity and phase clearly indicates that the worms leave and enter the forward state preferentially at specific phases of movement.
Here we have demonstrated an easy to use tracking system that record detail images of silicons behavior as a crawl on the surface and ACA plates. The amount of information contained in these images is vast and highly dimensional, and so we have also created a method to reduce the dimensionality of the data in the four fundamental measures. These measures are easy to interpret with respect to the worm behavior.
This work is generated to measure the behavioral worms at lower modifications, and the system can also be used to measure neuronal activity using gen genetically and coated regions. The system as a whole is flexible in design and can be used with other crawling systems such as Doof law, LOEs.
