Light Spot Assay: A Method to Investigate Drosophila Phototactic Behavior

This protocol text is an excerpt from Sun et al., Light Spot-Based Assay for Analysis of Drosophila Larval Phototaxis, J. Vis. Exp. (2019).

1. Set-up of the imaging system

1. Clamp a high-resolution web camera with an iron clip, at about 10 cm above the light spot on the desktop (Figure 1).
2. Adjust the orientation of the camera lens towards the desktop. Connect the camera to a computer through a USB interface.
3. Place an agar plate on the desktop right beneath the camera.
4. Open the "Amcap9.22" software on the computer with Windows 7, and the light spot will be automatically shown in the window of AMcap. Move the camera slightly left or right to ensure that the light spot is near the center of the window. Ensure that the camera does not block the light path. The light spot should be complete and round.
   NOTE: The software can be found at http://amcap.en.softonic.com/.
5. Fix an 850 nm ± 3 nm band-pass filter with a clip at 5-7 mm right below the camera.
   NOTE: The diameter of the filter is about 2.5 cm, and the camera lens is less than 1 cm in diameter, so the filter can cover the visual field of the camera. With the filter below the camera, the light spot should not be seen in the window of AMcap.
6. Place three infrared-light-generating LEDs (central wavelength = 850 nm) evenly around the agar plate. Each LED should be about 5 cm away from the edge of the agar plate, and the lens face of the LED should be at a 70° downwards angle towards the agar plate. Connect the LEDs to the power through the AC-to-DC converter.
   NOTE: It is better to fix the positions and angles of the infrared light LEDs to ensure consistency of the brightness of the field in various experimental trials and facilitate later video processing.
7. Put a black board between the computer and the device. Set down the brightness of the computer screen to prevent the computer screen light from affecting the experiment.
   NOTE: Keep the environment dark when measuring wavelength or intensity of the light.

2. Setting parameters of imaging

1. On the menu of the AMcap software, choose Options | Video Device | Capture format, and set the pixel size of the captured video to 800 x 600 and frame rate to 60 fps.
2. Remove the filter from beneath the camera, put a ruler under the camera and adjust the camera focus to make the scale line clear and parallel to the width of the video field of view.
3. Click Capture | Set up | Video capture to select the save path, click Start recording, record the actual distance corresponding to 600 pixels, and calculate the ratio of each pixel to the actual distance.

3. Video recording of light avoidance behavior

1. Maintain a temperature of 25.5 °C through all experiments. Control room temperature with an air conditioner if required. Keep the humidity constantly at 60% with a humidifier.
2. Take a short video of the light spot position named "lightarea1". Then, move the 850 nm ± 3 nm filter back to cover the camera lens.
   NOTE: When recording larval behavior, the camera lens is covered by the 850 nm ± 3 nm filter so that the light spot is not shown in the video. The light spot can be reconstructed in videos with larvae later with Matlab. Do not change the position of the camera, and avoid changing the ratio of each pixel to the actual distance measured in step 2.3.
3. Turn on a light (i.e., a room light) far away from the experimental device. Turn down the light as low as possible, as long as the larvae can be clearly seen with the eyes. Take the larvae out of the culture medium with a spoon, gently pick a third-instar larva, and wash it clean with distilled water. Be careful to wash larva one at a time to avoid interference from hunger. A single experiment requires at least 20 larvae.
4. Transfer the larva to the center of the agar plate placed beneath the camera during step 1.3. Gently remove excess water from the larva with a brush or use blotting paper to remove water from the larva to prevent reflection of light under the lens. Turn off the room light and allow the larva to acclimate for 2 min in the dark environment.
5. Turn on the LED light to generate infrared light, and gently brush the larva to the center of the plate. When the larva begins to crawl straight, rotate the plate to make the larva head towards the light spot. Make sure that it crawls straight from the start, or else it may not obtain access to the light spot.
6. Click Capture | Set up | Video capture to select the save path, then click Start recording to record. Allow the larva to crawl towards the light spot, enter the light spot, then leave the light spot until it is nearly out of the field of view. Click Stop recording. If the larva turns away from the light spot before getting close, directly click Stop recording.
7. Move the filter away from the camera. Take a short video of the position of the light spot named "lightarea2" and compare it to "lightarea1" to ensure that the light spot position is not changed. If an obvious position change is observed, discard the data.