The overall goal of this method is to show the live imaging of imaginal discs, which are popular experimental subjects for the study of a wide variety of biological phenomena including cell proliferation, differentiation, apoptosis, and competition. This method can help answer key questions in the developmental biology field, such as cell morphology and migration in specific tissue. The main advantage of this technique is that continual imaging can show more information than fixed tissue, such as cell migration.

Generally, individual new to this method will struggle because dissection and mounting are difficult. Visual demonstration of this method is critical as the rotation and transfer stage are difficult to learn because improper dissection results in poor resolutions. To begin the protocol, disinfect the equipment with 70%ethanol and allow the equipment to air dry.

Next, select a third-instar larva from a fly vial, and wash the larva in 1X phosphate-buffered saline, or PBS, to prevent contamination from the fly vial. Place the cleaned larva in one milliliter of culture medium at room temperature in a nine-well glass spot plate under a dissection microscope. Grasp the larva with a pair of forceps 1/3 of the way from the posterior end, and with another forceps, grasp the mouth hook.

Pull the two forceps in opposite directions to release the internal tissues. Then, remove the salivary glands, fat body, and cuticle from the eye-brain complex attached to the mouth hook. If the ventral nerve cord is still attached, cut it away with scissors.

Use one pair of forceps to grasp the mouth hook. Use the other pair of forceps to remove the tissue that connects the brain and eye discs in the dorsal region. Next, rotate the entire complex so the ventral side is facing up.

Remove the filament that connects the hook or disc to the brain. Then, remove the mouth hook, taking care not to damage the eye disc during the process. The eye disc is now free in the medium and only connected to the brain by the optical stalk.

Attach a double layer of O-rings to the center of a 42-millimeter by 0.17-millimeter coverslip to hold the agarose. Next, assemble the chamber with the coverslip. Place the coverslip on the stage acceptor.

Then, place the silicon O-ring on the coverslip. Then, set the base, lock the sealing locker, and place the cover. Using a 20-microliter micropipette with a 10 to 200-microliter tip, carefully transfer the dissected eye-brain complex to the center of the O-ring.

Remove most of the medium, and add 12 microliters of 0.7%low-melting, 37 degrees Celsius agarose to the sample. Vertical the end the agar to the sample can keep the tissue close to a coverslip and avoid to roll off the sample. Add one milliliter of culture medium at room temperature to the center of the O-ring.

Make sure that the agarose is glued to the O-ring to prevent the sample from drifting. Place the prepared chamber on the stage of an inverted confocal microscope for 30 minutes to equilibrate the temperature. Before long-term live imaging, check whether the tissue is intact by differential interference contrast microscopy.

Switch the objective to 40x for imaging. Finally, acquire 62-micrometer Z-stack images in 12 minutes at an optical interval of one micrometer. Acquire 40 cycles of scans over a total of 10 hours, and ensure that each cycle contains 12 minutes of imaging and three minutes of resting.

R3 and R4 were labeled with GFP. At the beginning of the 10-hour live-imaging session, there were eight rows of ommatidial clusters, and at the end, 14 rows of ommatidial clusters. Based on the relative positions of R3 and R4, ommatidial rotation occurred in the ex vivo cultured disc during live imaging.

In the beginning, the R3/R4 axis appears to be perpendicular to the equator. Then, it appears to rotate 15 degrees in three hours, 30 degrees in six hours, and 45 degrees in nine hours, suggesting that this method can sustain photoreceptor differentiation during 10 hours of live imaging. After watching this video, you should have a good understanding of how to properly dissect the Drosophila eye disc and to conduct the live image.