Light Spot Assay: A Method to Investigate Drosophila Phototactic Behavior

Overview

Drosophila larvae are light-sensitive and respond to changes in surrounding light. This video describes an assay that tests their ability to avoid light, called the light spot assay. The featured protocol demonstrates how to set up the assay with a blue light LED and shows how to record the animal's behavior in response to the light encounter.

Protocol

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.

Representative Results

Figure 1: Experimental set-up. (A) Schematic representation of the set-up for the light spot-based larval fast phototaxis assay. The blue lines represent the paths of visible light used as visual stimulation, and the red lines represent the paths of infrared light. Arrows indicate the direction of the light. The 850 nm band-pass filter allows infrared light to pass, but it blocks visible light. (B) An image of the set-up for the light spot assay. It should be noted that the image was taken under light conditions for better visualization. Please click here to view a larger version of this figure.

Materials

Name	Company	Catalog Number	Comments
850 nm ± 3 nm infrared-light-generating LED	Thorlabs, USA	PM100A	Compatible Sensors: Photodiode and Thermal Optical Power Rangea: 100 pW to 200 W Available Sensor Wavelength Rangea: 185 nm-25 μm Display Refresh Rate: 20 Hz Bandwidth: DC-100 kHz Photodiode Sensor Rangeb: 50 nA-5 mA Thermopile Sensor Rangeb: 1 mV-1 V
AC to DC converter	Thorlabs, USA	S120VC	Aperture Size: Ø9.5 mm Wavelength Range: 200-1100 nm Power Range: 50 nW-50 mW Detector Type: Si Photodiode (UV Extended) Linearity: ±0.5% Measurement Uncertaintyc: ±3% (440-980 nm), ±5% (280-439 nm), ±7% (200-279 nm, 981-1100 nm)
band-pass filter	Thorlabs, USA	DC2100	LED Current Range: 0-2 A LED Current Resolution: 1 mA LED Current Accuracy: ±20 mA LED Forward Voltage: 24 V Modulation Frequency Range: 0-100 kHz Sine Wave Modulation: Arbitrary
Collimated LED blue light	ELP, China	USBFHD01M	Max. Resolution: 1920x1080 F6.0 mm Sensor: 1/2.7" CMOS OV2710
Compact power meter console	Ocean Optics, USA	USB2000+(RAD)	Dimensions: 89.1 mm x 63.3 mm x 34.4 mm Weight: 190 g Detector: Sony ILX511B (2048-element linear silicon CCD array) Wavelength range: 200-850 nm Integration time: 1 ms – 65 seconds (20 seconds typical) Dynamic range: 8.5 x 10^7 (system); 1300:1 for a single acquisition Signal-to-noise ratio: 250:1 (full signal) Dark noise: 50 RMS counts Grating: 2 (250 – 800 nm) Slit: SLIT-50 Detector collection lens: L2 Order-sorting: OFLV-200-850 Optical resolution: ~2.0 nm FWHM Stray light: <0.05% at 600 nm; <0.10% at 435 nm Fiber optic connector: SMA 905 to 0.22 numerical aperture single-strand fiber
High-Power LED Driver	Minhongshi, China	MHS-48XY	Working voltage: DC12V Central wavelength: 850nm
high-resolution web camera	Thorlabs, USA	MWWHL4	Color: Warm White Correlated Color Temperature: 3000 K Test Current for Typical LED Power: 1000 mA Maximum Current (CW): 1000 mA Bandwidth (FWHM): N/A Electrical Power: 3000 mW Viewing Angle (Full Angle): 120° Emitter Size: 1 mm x 1 mm Typical Lifetime: >50 000 h Operating Temperature (Non-Condensing): 0 to 40 °C Storage Temperature: -40 to 70 °C Risk Groupa: RG1 – Low Risk Group
LED Warm White	Mega-9, China	BP850/22K	Ø25.4(+0~-0.1) mm Bandwidth: 22±3nm Peak transmittance:80% Central wavelength: 850nm±3nm
Spectrometer	Noel Danjou	Amcap9.22	AMCap is a still and video capture application with advanced preview and recording features. It is a Desktop application designed for computers running Windows 7 SP1 or later. Most Video-for-Windowsand DirectShow-compatible devices are supported whether they are cheap webcams or advanced video capture cards.
Standard photodiode power sensor	Super Dragon, China	YGY-122000	Input: AC 100-240V~50/60Hz 0.8A Output: DC 12V 2A
Thermal power sensor	Thorlabs, USA	M470L3-C1	Color: Blue Nominal Wavelengtha: 470 nm Bandwidth (FWHM): 25 nm Maximum Current (CW): 1000 mA Forward Voltage: 3.2 V Electrical Power (Max): 3200 mW Emitter Size: 1 mm x 1 mm Typical Lifetime: 100 000 h Operating Temperature (Non-Condensing): 0 to 40 °C Storage Temperature: -40 to 70 °C Risk Groupb: RG2 – Moderate Risk Group
Thermal power sensor	Thorlabs, USA	S401C	Wavelength range: 190 nm-20 μm Optical power range:10 μW-1 W(3 Wb) Input aperture size: Ø10 mm Active detector area: 10 mm x 10 mm Max optical power density: 500 W/cm2 (Avg.) Linearity: ±0.5%