Live-Cell Imaging of Drosophila melanogaster Third Instar Larval Brains

Our research focuses on studying the impact of asymmetric cell division, or ACD, on stem cell proliferation and differentiation. We employ Drosophila neural stem cells, or neuroblasts, as a model to understand the mechanisms, regulations, and impacts of asymmetric cell division on development and neurogenesis. The recent developments in artificial intelligence have been making major strides in image quantification and analysis, which has significantly advanced the field of live-cell imaging.

Maintaining sample quality for a long-term movie is a longstanding challenge. While it's insightful to observe a sample for eight to 10 hours, logistical constraints like the sample's imaging requirements, and available imaging tools make it difficult. Our protocol addresses this by demonstrating long-term imaging without impacting the sample's quality.

Our technique could be easily adopted and applied to the field by newcomers. With some time and practice, one can feasibly generate short and long-term movies, which have the potential to yield insightful and meaningful data. Begin by crossing one to five-day old female virgin flies with one to seven day old adult male flies to produce progeny with the desired genotype.

To do so, place the flies in a fly cage with a meal cap. Use 10 to 15 female virgins with five to 10 males to get optimal yield. Incubate the fly cage containing flies at 25 degrees Celsius.

Change the meal cap daily to prevent overcrowding of larvae, which reduces the quality of the dissected brains. If the meal cap has more than 30 larvae, split it into half and replace one portion with a fresh meal cap cut in half. Incubate the meal caps with larvae at 25 degrees Celsius until the larvae reach the desired age.

Begin by pipetting around 400 microliters of dissection and imaging medium into each well of a three-well dissection dish. Under a microscope, wash 10 well-fed wild type larvae by gently holding them with dissection forceps and dipping them in and out of the dissection medium in the bottommost well to remove all food particulates. While continuing to work under the dissection microscope, transfer the clean larvae to the middle well.

Hold the larvae by the mouth hooks using one set of tweezers. With another set of tweezers, rupture one side of the larvae's cuticle to spill out the fat bodies. Using forceps, collect as much fat body from each larva as possible and transfer it to the top most well containing 400 microliters of dissection medium at room temperature.

Begin by pipetting around 400 microliters of dissection and imaging medium into each well of a three-well dissection dish. Working under a dissection microscope, wash the experimental larvae in dissection in imaging medium to remove food residues. While continuing to work under the dissection microscope, hold the larvae by the mouth hooks using one set of tweezers.

With another set of tweezers, carefully cut off approximately one third of the larvae from the posterior side. Next, while holding the larvae by the mouth hooks using one set of tweezers, use the other set of tweezers to brush the cuticle towards the mouth hooks gently. Simultaneously push inward with the tweezers, holding the mouth hooks until the entire larvae is turned inside out.

Invert the larvae to expose the central nervous system and other tissues while maintaining their connection to the cuticle. Locate the central nervous system or CNS to avoid accidental removal. Gently remove non CNS tissues using tweezers, keeping only the CNS and brain attached to the cuticle.

To release the brain from the cuticle by severing the axonal connections, using micro dissection scissors, cut gently beneath the brain lobes. Then cut the connections under the ventral nerve cord. Dissect larvae in batches to keep the dissection time under 20 minutes.

Transfer the dissected brain into a well containing the dissection and imaging medium. For imaging lasting over three hours, supplement the medium with fat bodies. To perform live-cell imaging of the third instar larval brain, begin by collecting the dissected brains and isolated fat bodies in the last well of a three-well dissection dish containing the dissection and imaging medium.

Place a gas permeable membrane on the back of a slide and press the split ring into the center. Using a 200 microliter micropipette, transfer up to 10 dissected brains with maximum possible fat bodies and 130 to 140 microliters of the medium onto the center of the membrane. Orient the brains according to the neural stem cell population to be imaged and the microscope type, ensuring the samples are close to the microscope's objective.

For example, to image neural stem cells in the central brain lobes, ensure the brain lobes are closest to the objective. Then gently place a glass cover slip over the solution on the membrane to spread the solution with the brains and fat bodies throughout the membrane. Hold the laboratory tissue at the cover slip edge to blot excess solution until the brains touch the cover slip without being squashed.

Next, immobilize the cover slip by applying molten petroleum jelly along its edges using a paintbrush and allow the jelly to solidify. Add 400 microliters of imaging medium to a well in a multi-well microslide. Transfer the previously dissected fat bodies to this well.

Then place up to 10 brains in a cluster near the center of the well. Orient the brains according to the neural stem cell population to be imaged and the microscope type. Allow the brains to settle in the well for two to five minutes.

Cover the microslide with the slide cover before transferring it to the microscope. Start the acquisition process using the lowest laser power and exposure time to minimize photobleaching. Imaging of larval brains expressing UAS-driven cherry Jupiter and endogenously tagged pins GFP using multi-well imaging slides and without fat body supplementation revealed that the pins formed a pronounced apical crescent in dividing neuroblasts to which the mitotic spindles consistently aligned during mitosis.

In those samples, the cell cycle length increased with increasing imaging time. Samples that were imaged in a fat body supplemented medium on a membrane-bound slide did not show an increase in cell cycle length. Furthermore, neuroblasts with four divisions were observed on the 10-hour membrane-bound slide.