The overall goal of this procedure is to rapidly and accurately measure flight performance in Drosophila. This is accomplished by first dropping flies into a flight cylinder. The second step of the procedure is to image the landing surface with a digital camera.
The final step is to analyze the data using imaging software by measuring the landing height of each fly. Ultimately, this procedure helps identify mutations and conditions such as aging or neurodegeneration, which impair flight ability and helps to quantify flight performance. The main advantage of this technique over existing methods, such as the original flight tester, is that it's more appropriate for high throughput screens for mutants and other conditions that impair flight.
We first had the idea for this method when we realized how much time was involved with testing a large number of genotypes using the original flight tester. Secure the flight cylinder to a ring stand using chain clamps. Leave approximately three centimeters underneath the cylinder for the way dish.
Load the way dish with a thin layer of mineral oil and slide it under the flight cylinder. Secure the funnel to a second ring stand. Adjust the height of the funnel so that the bottom of the funnel is flush with the top of the flight cylinder.
The narrowest part of the funnel must be narrower than a fly vial. The diameter of the drop tube should be slightly wider than a fly vial, so the vial is able to drop freely. Insert the drop tube into the top of the funnel and secure it using a claw clamp.
Next from a poly acrylamide sheet, cut a rectangular piece the length of the flight cylinder with a width slightly smaller than the inner circumference of the flight cylinder. Apply a thin layer of tangle trap adhesive to the sheet, leaving enough room at the top and bottom uncoated to hold the sheet. After the adhesive on the sheet has dried for an hour, slide the sheet into the flight cylinder with the adhesive inward.
Next, assemble the camera track using pine support brackets. Ensure that the bottom of the track can support the camera without blocking the lens. Then screw the stoppers into place where they do not interrupt the camera's view of the plastic sheet.
Collect vials of flies to be tested with no more than 20 flies per vial. Gently tap the flies to the vial bottom. Unplug the vial and insert it into drop tube.
Release the vial. When the vial hits the walls of the narrowing funnel, the flies that are ejected into the flight cylinder. Lift the drop tube to remove the empty vial and load more flies if desired.
Up to 200 flies loaded from 10 vials can be tested and imaged readily on a single poly acrylamide sheet. Now, remove the plastic sheet and place it on A flat white surface white poster board may be used if benchtops are dark colored. Assemble the camera track over the plastic sheet.
The camera should be sufficiently high above the sheet to have both the top and bottom of the sheet in the field of view. Slide the camera along the track while holding the capture button to acquire a panoramic image. The number of flies landing in the oil in the dish below the flight chamber can be counted manually.
For each trial between trials, flies can be removed from the sheet and the sheet can be reused. Open the image files in image J software crop the images if necessary to include only the landing surface area. The area coated entangle trap.
Convert the images to eight bit gray scale and create a threshold. To filter out the white background. Set the parameters to identify each fly.
Using the analyze particles menu, define the parameters. Used to identify a particle using the demonstration apparatus. An area of five to 90 pixels squared and a circularity of 0.4 to 1.0 will accurately identify all samples.
Now, measure the location of each fly using the generated list of coordinates for each particle. The X coordinate in pixels can be converted to centimeters. To calculate the landing height, import the table into a spreadsheet and proceed with analysis.
The flight performance of slow poke mutant flies, which have a known flight defect, was compared to wild type canton s flies. All the flies were three days old, raised at room temperature control flies consistently landed near the top of the cylinder. The spread of the landing was much greater for slow poke mutants.
The average landing height for cantons at 73 centimeters was significantly greater than that of slow poke mutants at 44 centimeters. Following this procedure, additional methods such as histology can be used to answer additional questions regarding the structural integrity of flight muscles, motor neurons, or neuromuscular junctions. After watching this video, you should have a good understanding of how you can rapidly assess flight performance in oph.
