FIM Imaging and FIMtrack: Two New Tools Allowing High-throughput and Cost Effective Locomotion Analysis

The overall goal of the following methodology is to visualize and to quantify the locomotion of Drosophila Melan Gaster larvae, allowing high throughput screening in free moving conditions. This is achieved by creating a tracking surface on the FIM setup and using frustrated total internal reflection for indirect illumination. Larvae of different genotypes are then placed on the surface and crawling behavior in response to a heat gradient is recorded.

The videos are then analyzed by the FIM Track. Software and data obtained are used to generate plots describing differences in larva, locomotion patterns. The results show that precise, high throughput, flexible and easy to use quantifications of larva.

Locomotion can be done based on the FIM imaging technique and the associated FIM track program. The main advantage of this imaging technique of existing methods like using incident or transmitted light, is that only animals touching the surface are visible, resulting in a superb foreground background contrast with no disturbing Artifacts, though this method can provide insight into biology of oph lava. It can also be applied to study walking behavior flies and locomotion of other mono organisms such as sea elegance, plenaries, or even root growth of plants.

Visual demonstration of this method is critical to convince others that a complex technology is easy to use. The first step is to create a moist crawling surface for the FITR based imaging method or fim. To do this boil, 0.8%food grade auger in deionized ultrapure water.

Pour the hot auger onto an acrylic glass plate. Do not agitate the auger after boiling and pour constantly. To avoid bubbles at this temperature, a two millimeter thick slab should be created.

Use the remaining auger solution to fill standard six centimeter Petri dishes. These will be used to sort clean and habituate larvae prior to the experiment. Next, trim approximately two centimeters from the perimeter of the auger slab to obtain a plain square surface for recording.

Transfer the auger slab to the FIM setup directly after cooling. By gently pushing the auger over the edge of the acrylic glass plate, the auger should glide in place. You are now ready for imaging preparation to include an aversive auger barrier in the crawling surface.

Boil 2.5%food grade auger with three molar NACL in deionized ultrapure water. Next, cut a two centimeter wide notch surrounding the field of view into the previously poured crawling surface. When ready, fill the notch with salt auger 0.1 to 0.3 centimeters higher than the tracking surface.

Next, prepare the heat gradient device. The device is an aluminum plate covered with an insulating surface and perfused with water from pumps at both ends. To allow temperature control, turn on the heat gradient device one hour prior to use and place it above the setup to allow it to reach the desired temperature.

This time can be used for further preparations. When ready, transfer the crawling surface auger with a salt barrier to the setup. Place the radiator plate over the auger and adjust the spacing between the plate and crawling surface to two millimeters.

Establish a linear gradient from 34 degrees Celsius to 18 degrees Celsius by adjusting the temperature of the water circuits to one degree Celsius and 45 degrees Celsius, which is appropriate for this equipment. Allow the crawling surface to equilibrate in the gradient for 20 minutes. Before proceeding, test the temperature gradient with a TER to prepare the flies, retrieve them from a 25 degree Celsius incubator and drop a little water into the culture vials to drive late third in star larva movement.

Next, collect the largest larvae from the vial walls using a small paintbrush, two to five minutes before recording, transfer enough larvae for one video into a Petri dish that was prepared earlier. After the temperature gradient is established, set up the recording software. Specify the number of frames per video.

Define the saving path and set up for instant recording within 20 seconds. After placing the larvae on the crawling surface to begin image one of the larvae and adjust the illumination intensity to obtain good contrast. When ready, lift the radiator plate slightly and place the larvae at 34 degrees Celsius, two centimeters from the salt barrier.

Lower the radiator plate again and start recording in the dark to not disturb the animals. After recording, remove the larvae clean and moisturize the surface. To avoid spreading NACL, do not touch the salt auger while working, while the surface must be kept moist at all times, avoid excessive moisture, which can be seen as halos or drops surrounding larvae.

In recordings and disturbed tracking, allow the auger surface to equilibrate for one to two minutes before proceeding. During this time, save the collected images and gather new animals. To prepare for the next video, control the temperature gradient using a TER every five videos and adjust the temperature device if needed.

To begin tracking locomotion, adjust the tracking parameters using the preview option. Adjust millimeter per pixel according to camera and field of view. Next, adjust the frames per second based on the camera settings, use feedback given in the preview window to adjust the brightness thresholds so that all animals are detected correctly.

Next, adjust larval area size thresholds, single animals are highlighted by yellow circles colliding larvae are highlighted in red, and the area of each animal is given in blue. Start tracking by using the button in the bottom right after the tracking session, an image featuring larva tracks and a CSV file containing the calculated locomotion and posture features is stored in the image directory. Finally, use the FIM results viewer module to review and manually adjust the tracking results.

Define the crawling orientation in respect to the heat gradient to evaluate the data based on this stimulus. In the larva representation shown here, the head and tail point are marked between these points. An arbitrary odd number of spine points can be set with a radius.

In addition, the center of mass and the main body bending angle are calculated. Shown here are FI imaging and tracking results from different cameras. The images to the left capture three larvae crawling on a 10 centimeter by 10 centimeter tracking stage.

The mass trajectory and area of a single larva is shown to the right. The red arrow indicates the time point of the clipped image results from applying. A heat stimulus are shown here as different colored trajectories calculated via F track.

These high resolution application images of a third, second, and first in star larvae were obtained with a macro lens. Other organisms like sea can be imaged easily as well. Once mastered, about a hundred lab can be analyzed per hour per each tracking setup.

While attempting this procedure, it is important to remember to always record under the same conditions Following this procedure. Other methods like optogenetic stimulation or in vivo detection of neural activity could be included into the setup. After watching this video, you should have a good understanding of how easy it can be to efficiently analyze ular locomotion.