Due to its awkward size, Drosophila is difficult to image. This method demonstrates imaging of the behavior and morphology of flies through use of a stereo microscope. This protocol can be used both for scientific research and teaching.
Demonstrating the procedure will be Ms.Li Tong and Ms.Weng Yujia, undergraduates doing research training under my guidance. To begin, make a fly behavior observation chamber, or FBOC, using a translucent plastic bottle cap of about 17 by 22 millimeters or by cutting a section of 17 millimeters from the thick end of a five milliliter pipette tip. Pour 1%agar into the FBOC to adjust its depth.
If food needs to be placed in the FBOC, pour the agar to obtain a depth of 12 millimeters. If food does not need to be placed in the FBOC, pour the agar to a depth of five millimeters to track the whereabouts of fruit flies more easily. If using a pipette tip to make an FBOC, place the cutoff pipette tip section in a 35 or a 60 millimeter Petri dish.
Pour the 1%agar gel into the Petri dish to a thickness of about five millimeter and wait for the agar to solidify and seal the bottom of the FBOC. Then pour the agar gel into the FBOC to the desired thickness. Make an FBOC base by boring a 10 millimeter deep hole in the center of a piece of ethylamine foam sheet with the same diameter as the FBOC.
Insert the FBOC into the hole. Make fly food if necessary with yeast medium, artificial diet, or pure sucrose or glucose, depending on the purpose of the observation. To visually determine whether flies are feeding, add food dyes to the food to a final concentration of 12.5 milligrams per 100 milliliters.
Pour the prepared food into a Petri dish to a height of eight millimeters. After solidification, place food on a rectangular drawn on paper and cut the food accordingly and place it on a piece of plastic and cut into a quadrangular pyramid or quadrangular frustum pyramid to allow recording of fly behavior from different angles as the flies land on the food. Cut a piece of plastic and use tweezers to place the plastic and food in the center of the FBOC.
Pour 1%agar gel into a clean empty bottle to a thickness of 1-2 centimeters and place it at room temperature for 1-2 hours. Transfer flies to the bottle and place it at 25 degrees Celsius for 36 hours or more. Chill and inactivate the flies in crushed ice, sort them on an ice pack and transfer them to the FBOC as previously described.
After transferring the flies to the FBOC, cover it with 30-40 millimeters of UV or clear filter for the camera to form an FBOC complex. Place the FBOC complex under the stereo microscope for observation. Illuminate the FBOC.
Mount mini LED video lights to flash hot shoe mount stands and place the lights on the left and right sides of the FBOC. Turn on the lights and set the color temperature to 5, 000 to 5, 600 Kelvin and the brightness to 100%Turn on the LED video lights and adjust the stereo zoom microscope until the edge of the FBOC can be clearly seen with the naked eye. Move the FBOC to the center of the field of view.
Attach the clamp of the universal telescope digital camera adapter to an eyepiece of the stereo microscope. Then attach a compact digital camera to the adapter securely by alternately turning the camera mounting screw and camera fixing screw. Turn on the digital camera and turn the horizontal/vertical fine-tuning knobs until the FBOC edge clearly appears in the center of the bright circular field of view on the camera's LCD screen.
Rotate the mode dial to aperture priority auto-mode. Press focus mode on the multi-selector, choose macro close-up and then press OK button. Move the zoom switch from the wide angle end to the telephoto end and zoom into the circular image until its central portion fills the full LCD screen.
Press the movie-record button and start recording the fly behavior. Turn the focus knob of the microscope until the flies in the FBOC are clearly visible. Choose the fly behavior of interest for observation or video recording.
Turn the zoom knob to zoom in and out to achieve the desired magnification for observation or video recording. The representative photograph taken through the UV filter is clear and sharp, very similar to the photograph taken when the culture vial is not covered. The quality of the photo taken through the glass of the Petri dish is very poor and partly blurred.
A photograph taken from the video recording showing the details of each part of the fly's body is shown here. Because the camera is connected to a zoom stereo microscope, it is very easy to shoot from panoramic to close-up shots using the zoom system. Using this protocol, fly behavior can be observed and documented for multiple viewing angles.
For example, the female fly is constantly rubbing the ovipositor with her hind legs during the process of laying eggs. This detail of egg-laying behavior cannot be seen clearly from the side. Using this simple and economical method, we can take a panoramic or close-up of the behavior and morphology of flies and obtain a good quality video or photo.
