Studying Membrane Protein Trafficking in Drosophila Photoreceptor Cells

Take a dark-adapted, anesthetized, transgenic, white-eyed fly.

The fly expresses eGFP-tagged TRPL ion channels in the photoreceptors of the eye.

In darkness, TRPL localizes to the membranes of the rhabdomeres— the microvilli of the photoreceptors. 

Using compressed air, push the fly headfirst into a pipette tip.

Trim the tip and push the fly backward.

Trim it again and push the fly forward until only its head protrudes.

Immobilize the tip in modeling clay adhered to a slide and place it on a fluorescence microscope stage, with the eye facing the microscope objective radially. 

Add a drop of water to the objective, then lower it until it touches the eye.

Capture images of the photoreceptors.

Measure the fluorescence intensities in the rhabdomeres, cell body, and background.

Illuminate the fly with orange light to induce TRPL translocation to the cell body.

Repeat the imaging and fluorescence measurements.

Compare the images to observe reduced rhabdomeral fluorescence post-illumination, indicating TRPL translocation to the cell body.

To prepare a fly in a non-lethal variation, transfer the ice anesthetized fly headfirst into a 200-microliter pipette tip, and carefully push the fly toward the tip with compressed air. Then, using a scalpel, cut off the pipette tip just in front of the head. And using tweezers, carefully push the fly a few millimeters into the pipette tip.

Cut off the pipette tip again. And push the fly back toward the tip with the compressed air, so that only the head of the fly protrudes from the pipette tip. After adhering a piece of plasticine onto an object slide, press the pipette tip into it so that the left or the right eye faces upward. Ensure that the eye is oriented correctly under the microscope. For image acquisition, select a water-immersion objective.

In the case of a non-lethal variation, use a laboratory pipette to adhere a large drop of chilled water to the underside of the water immersion objective. Carefully place the object slide with the prepared fly onto the microscope stage. Then, lower the water immersion objective manually until it contacts the water surface, or the fly's eye touches the drop.

Then, switch the light path toward the microscope camera, and generate a live image. Readjust the focus for the camera and evaluate the orientation of the eye, considering that the eye must face the microscope objective radially. Use the lookup table software to detect oversaturation.

In the case of non-pigmented flies, adjust the exposure time such that the brightest pixels are just below the saturation limit for every image. Record an image and save it as a raw file to archive all corresponding metadata of the recording. Then, export the image in a dot TIFF format.

To quantify relative EGFP fluorescence in the rhabdomeres of water-immersion micrographs, adjust ImageJ settings by clicking on Analyze. Then, set measurements, and check only the box for mean gray value. Import the dot TIFF image by clicking on File, then Open. Choose a representative region of the image in focus, and enlarge it to 200% to 300% by repeatedly pressing Control and Plus together.

Next, select the Oval tool. And while pressing the Shift key, generate a circular selection in the image that is significantly smaller than one fluorescent rhabdomere. Then, look for the exact size displayed below the toolbar in the ImageJ main window. To measure the fluorescence intensities within the circular selection, move the circle to the first rhabdomere with the arrow keys on the keyboard, and click Analyze, then measure.

A result window listing the measured gray value will pop up. Continue with measurements of rhabdomeres 2 to 6, and also measure the background signal. In the case of non-pigmented flies, make additional measurements of the corresponding cell body areas. Measure the fluorescence of two more ommatidia, resulting in three technical replicates. Mark the analyzed ommatidia by using the pencil tool and save this image for documentation.

Select and copy the measured gray values from the result window and paste them into spreadsheet software for further calculations. Sort the fluorescence intensity values according to their origin into the category's rhabdomere, cell body, and background, and calculate the mean intensity from each category. Then calculate the relative amount of EGFP present in the rhabdomere, using the first formula for non-pigmented eyes, and the second for pigmented eyes.