Live Imaging of the Drosophila Pupal Eye Using Fluorescence Microscopy

Begin with a transgenic Drosophila pupa with an exposed eye.

Place the pupa on a jelly bead within a hydrated paper frame, surrounded by a jelly ring.

The pupa’s eye contains GFP-tagged junction proteins that highlight cell boundaries in the developing eye’s neuroepithelium.

Gently press a coverslip onto the pupal eye region.

Under a fluorescent microscope, GFP-tagged junctions fluoresce, displaying the neuroepithelium organization.

Take images at regular intervals across different depths and process the images to enhance contrast and minimize background noise. 

Align these images with increasing time intervals to track the developing neuroepithelium.

Initially, primary cells form boundaries around photoreceptor cell clusters, while interommatidial cells or ICs arrange into a single layer.

Later, the ICs differentiate into secondary and tertiary cells near photoreceptors.

Finally, excess ICs are removed via programmed cell death, forming a hexagonal lattice that structures the organized eye. 

Using forceps, carefully lift and remove the operculum to expose the pupal head. Next, tear along the side of the pupal case to expose the area around one eye. Gently remove the pupa from the adhesive tape.

Construct a small 15-square-millimeter frame from blotting paper, and punch a 5-millimeter hole in the middle. Immerse the frame in distilled water and place it in the center of a microscope slide. With a 30-cc syringe, squeeze out a uniform ring of petroleum jelly to surround the blotting paper frame.

Next, add a small bead of petroleum jelly directly onto the slide. Carefully arrange the pupa on its side, and support it with the petroleum jelly bead. Place a coverslip on top of the petroleum jelly ring so that it contacts the epithelium above the eye. Gently compress the preparation to seal the coverslip against the petroleum jelly, and generate a small, flat contact surface between the pupil eye region and the coverslip.

Image the pupal preparation using a fluorescent microscope. Capture serial sections through the apical domain of the eye neuroepithelium in the region of the adherens junction every seven minutes. Use appropriate deconvolution software to reduce background and enhance contrast of the serial section images.

For each z stack file in LAS AF software, navigate to the Tools panel, select 3D Deconvolution, and click Apply. Generate a maximum projection, or MP image, for each deconvoluted stack file. Align each MP image to highlight individual cell behaviors and reduce distractions caused by organismal growth, using the built-in algorithms in Photoshop CS5.

From the File tab of the Main menu, open Scripts, and select Load Files into Stack. Next, import the MP image files into the Load Layers panel. Make sure that the Attempt to Automatically Align Source Images option is not selected, otherwise the image data may be distorted. Once all MP files have loaded into the layers pane, check that all images are in chronological order, and that the earliest time point is at the top of the layer stack. Drag and drop them until they are ordered correctly if needed.

Then select Auto-Align Layers. Choose Reposition and click OK.