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The overall goal of this procedure is to demonstrate an efficient method for generating live imaging movies of drosophila pupil eye development. This is accomplished by first removing the O perm of the pupil case and tearing along the side to expose the neuro epithelium of one eye. The second step is to assemble an imaging rig and to mount the pupa on a small bead of petroleum jelly.

Next serial sections of the neuro epithelium in the region of the adhesion belt are acquired using a standard fluorescence microscope. The final step is to stabilize the footage and make color adjustments in standard image editing software. Ultimately, live cell imaging of the drosophila neuro epithelium is used to elucidate the cell behaviors that pattern.

The eye Visual demonstration of this method is critical as partially dissecting a pupa and then correctly mounting it in the imaging rig can be tricky because pupi are rather fragile at this stage. In addition, correct orientation of the pupa and correct placement of the cover slip above the eye epithelium is critical for successful imaging. Demonstrating the procedure will be Mark Hellerman from our laboratory.

To begin the experiment, select white pre puy from previously crossed genotypes and place them in 1.5 milliliter centrifuge tubes. Next place a 10 centimeter squared piece of tissue soaked in distilled water into a clean plastic tip box. Insert the micro centrifuge tubes into the tip box.

The wet tissue will prevent desiccation of the pupi, then incubate at 25 degrees Celsius for 17 to 25 hours. After incubation, place a piece of fresh double-sided tape onto a black cigar dissection dish. Position the pupa dorsal side up on the tape and make sure the head of the pupa is attached to the double-sided tape without adhering the thoracic and abdominal regions of the pupa.

Using forceps, carefully lift and remove the A perm to expose the pupil head. Next, tear along the side of the pupil case. To expose the area around one eye gently remove the pupil from the adhesive tape.

Construct the small 15 square millimeter frame from blotting paper and punch a five millimeter hole in the middle. Immerse the frame and 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 cover slip on top of the petroleum jelly ring so that it contacts the epithelium above the eye. Gently compress the preparation to seal the cover slip against the petroleum jelly and generate a small flat contact surface between the pupil eye region and the cover slip image, the pupil preparation. Using a fluorescent microscope, capture serial sections through the AAL domain of the eye, neuro epithelium in the region of the adherence junction every seven minutes.

Use appropriate deconvolution software to reduce background and enhance contrast of the serial section images for each Zack file and L-A-S-A-F software. Navigate to the tools panel. Select 3D deconvolution and click apply.

Generate a maximum projection or MP image for each de convoluted 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 CS five 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 okay. Out of focus, regions of an initial MP image may be corrected to yield a uniformly in focus retinal field. To begin image processing, use the crop function in LAS.

Saf manually restrict the initial and final slices, so they span only the adhesion belt within the initially out of focus region. Click apply to generate a new stack file that is optimized for this region. Next, generate a new maximum projection for the locally optimized stack and export it as a TIF file in Photoshop.

Open scripts and select load files into stack. Then input the initial MP file and the optimized MP file into the load layers panel. Again, ensure that the attempt to automatically align source images option is not selected and select okay.

Select both layers and choose auto blend layers to produce a uniformly in-focus retinal field. Save this composite image as a TIF file. Use a transform tool to rotate the images so that the dorsal ventral axis of the retina is aligned to the Y axis of the movie frame.

Use the level adjustment of individual frames to enhance contrast between the cell membrane and the cell body. Then select make frames from layers to convert the images into a movie. Note that layers are added to the animation pane in sequential order beginning from the bottom of the stack.

To reverse the order of the stacks, select all of the frames and then select reverse frames. Select the frame delay time of 0.8 seconds for each frame and choose QuickTime. Export and select the desired video format.

Finally, under render options, select the frame rate to custom and enter a frame rate of 15. Then click render. A pronounced developmental gradient was observed across the eye with this technique, which prompted detailed descriptions of eye patterning.

During primary wrapping, two cells were recruited as primaries, which rapidly encircled the photoreceptor bundles connecting to each other to form stable junctions. Undifferentiated intero material cells lay in a disorganized pattern as the primaries wrapped and sealed. Each photoreceptor bundle local cell movements reorganized.

The inial cells grouped at the dorsal and ventral sides of each oma. Inter cell intercalation rearranged cells from two rows into a single row. Following inter intercalation tertiary competition was captured under the microscope.

Competing cells occupied the tertiary niche for five to 10 minutes before displacement. Eventually, a single cell established the stable tertiary niche. During final pruning, a surge of apoptosis reduced the number of ICS to the minimum required to efficiently generate an enter material cell lattice across the compound eye.

Once mastered, the imaging of PPE can be done in four hours if performed correctly. Subsequent image processing takes around one hour.