GFP-tagged photoreceptor proteins, like the ion channel TRPL GFP, allow us to study protein transport in neurons using noninvasive techniques. This method can also be used to monitor photoreceptor degeneration. Thus, the molecular basis of hereditary diseases resulting in blindness in humans can be studied in the Drosophila eye.
Cellular localization of GFP-tagged proteins can be assessed by observing the fluorescence in the deep pseudopupil or by water-immersion microscopy. Both methods allow fast determination of the localization of visual proteins and observing structural defects of rhabdomeres due to the degeneration of photoreceptor cells. To obtain high-quality images, the most important aspect is the orientation of the fly eye.
While performing this technique, one should focus on ommatidia located slightly towards the eye's periphery. I will be demonstrating the procedure of DPP imaging, while the two techniques using water-immersion microscopy will be demonstrated by my colleague, Dr.Krystina Wagner, and Matthias Zeger, a PhD student in our group. To begin deep pseudopupil, or DPP, imaging, anesthetize one-to three-day-old flies, and position one of them in the center of the microscope objective on its side so that either the left or right eye is facing the objective precisely radially.
Increase the magnification to fit the entire eye, and center the central ommatidia of the eye. If possible, decrease the depth of field of the microscope by adjusting the double-iris diaphragm to a shallow setting. Next, switch on the microscope UV lamp at maximum intensity.
Then, select the fluorescence filter set of the microscope according to the fluorescence protein expressed in the eyes, and set the light path toward the microscope-mounted camera. Using the live imaging feature within the software, adjust the image brightness to a setting that detects only specific signals from the eye by raising the exposure time and gain value. Readjust the microscope focus into the eye to generate the superimposed image of the DPP.
Then, take a snapshot of the fluorescent DPP. To prepare a fly in a lethal variation, adhere a piece of Plasticine onto an object slide and another piece into the center of a Petri dish. Fill the Petri dish with ice-chilled distilled water and some ice flakes.
Then, put one ice-anesthetized fly under a stereo microscope on top of the Plasticine-coated object slide. Turn the fly on its back, and pierce an insect pin through the center of the thorax. Fix the pin horizontally on the Plasticine-coated object slide, and orient either the left or the right eye of the fly upward.
Then, carefully fix the object slide with its Plasticine-free side facing down in the Petri dish, preventing rotation of the fly head. Ensure that the fly eye is covered with water. Alternatively, to prepare a fly in a non-lethal variation, transfer the ice-anesthetized fly head first 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 tif 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 tif 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 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 two to six, 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 categories 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. Alternatively, to quantify eye morphology by eGFP fluorescence in water-immersion micrographs, choose three adjacent ommatidia in a representative region of the image that is in focus. Evaluate the 18 rhabdomeres of the selection individually according to their eGFP intensity, edge sharpness, and contrast with respect to the surrounding background signal.
Finally, score the clearly visible rhabdomeres with a value of two, weekly visible rhabdomeres with a value of one, and absent rhabdomeres with a value of zero to generate a degeneration index. In transgenic Drosophila flies expressing an eGFP TRPL fusion protein, rhabdomeral fluorescence disappears in light due to translocation of the eGFP TRPL. This allows for performing a genetic screen to identify mutants defective in the internalization of the eGFP TRPL.
In control flies, eGFP TRPL translocates out of the rhabdomeres into the cell body after 16 hours of illumination, resulting in a significant decrease of rhabdomeral fluorescence compared to the dark-adapted state. The second dark incubation raises the rhabdomeral fluorescence again towards the initial value. However, in the TRPL translocation defective mutant vps35MH20, the fluorescence pattern does not change drastically after 16 hours of illumination and a subsequent dark adaptation for 24 hours.
This quantification method can detect a statistically highly significant recycling defect. White-eyed flies allow the detection of fluorescence signals from both rhabdomere and cell body. In contrast, in red-eyed flies, fluorescence signals are only detectable in rhabdomeres but not in the cell body.
Regarding the quantification of retinal degeneration, eGFP TRP fluorescence can be assessed over the course of several weeks. When kept in a 12-hour light, 12-hour dark cycle for two weeks, the degeneration index declined in mutant flies but not in control flies. In this protocol, one needs to differentiate between pigmented and non-pigmented eyes.
Since pigmentation affects the fluorescent signal, quantitative water-immersion microscopy has been optimized for both cases individually. DPP imaging and water-immersion microscopy are high-throughput methods with limited resolution. To investigate subcellular localization, we recommend immunofluorescence microscopy on tissue sections.
For a detailed analysis of degenerative phenotypes, electron microscopy can be performed.
