The overall goal of this procedure is to measure the intracellular pH of follicle cells within live explanted Drosophila ovarian tissue. This method can help us answer key questions in stem cell and developmental biology such as, how changes to intracellular pH contribute to sulfate decisions. The main advantage of this technique is that it makes it possible to accurately quantify pH within living intact tissue.
To begin the experiment, prepare mounting chambers by applying a thin coating of vacuum grease to the flatter side of a 3D printed mounting chamber. Place the flatter side on a 22 x 40 millimeter cover slip to seal the cover slip to the mounting chamber. Next, dissect the ovaries from female adult flies in 500 microliters of dissection buffer.
Anesthetize three to four flies using CO2 gas and transfer the flies onto a fly pad. Use forceps to select a fly and place it into the dissection media. Pinch the thorax of the fly with one pair of forceps while tugging on the tip of its abdomen until its ovaries are exposed.
Set a timer for exactly 10 minutes. After detaching the ovaries from the other organs, tease apart the ovarioles using 22 and a half gauge syringe needles. Use one syringe needle to hold the cluster of ovarioles in place at its anterior end and stroke downwards along the length of single ovarioles to carefully separate the muscle sheath from the ovaries.
Incubate the dissected ovaries in dissection media for the remainder of the time on the timer. It is important the ovaries remain in the dissection media for exactly 10 minutes to provide enough time for the cells. Place a small drop of dissection media onto the glass cover slip inside the mounting chamber.
Transfer the separated ovarioles using forceps. Separate later stage egg chambers from germaria associated with earlier stage egg chambers and ensure that the dissected germaria are placed at the center of the dissection media drop. Add two small drops of nail polish to opposite sides of around 12 millimeter cover slip and let it air dry for about 10 seconds.
Place the round cover slip on the drop of dissection media containing separated germaria, so that the side with nail polish faces down and contacts the dissection media. Press down on the edges to flatten and secure the germaria in position. Fill mounting chamber with additional dissection media and move on to live imaging.
Image ovarioles in ny dissection buffer at pH 6.5. Adjust settings such that pixel intensities of the pHluorin and mCherry images are low, but not below the limits of detection of the camera. Next, image another set of ovarioles in nygerion dissection buffer at pH 7.5 and adjust the settings such that the pixel intensities of the pHluorin and mCherry images are high but not saturated.
Image ovaries and bi-carbonate dissection buffer and ensure that the pixel intensities of the pHluorin and mCherry images are not saturated with the chosen settings. If pixels are saturated, adjust the settings. After selecting appropriate live imaging settings, proceed to collecting images of at least five germaria in each of the conditions.
Subtract the background by opening unprocessed images in Fiji with each channel in a separate window. Use the rectangle tool to draw a region of interest or ROI in the pHluorin channel window and a part of the image without signal. Set the lower limit of the threshold so that pixels with intensity values below the background are excluded and set the upper limit of the threshold to maximum.
When the threshold is set in this way, the areas of the image without signal are mostly blue and the signal is clearly visible. If the threshold was set, navigate to the set measurements dialog box and make sure to check the limit to threshold box. Measure the mean fluorescence intensity in the ROI, go to process, then, math menu to subtract the measured background intensity from each channel and slice of the image using the subtract function.
Repeat the steps for the mCherry channel window but, use the restore selection function instead of drawing a new rectangle with the rectangle tool found in the edit selection menu, to add a rectangle with the same dimensions and position on the image. Choose one or more slices in which the cell of interest has highest fluorescence intensity in the mCherry channel and make sure that the concanavalin A staining in the far red channel, is in focus within the selected slice. Next, draw an ROI around each follicle stem cell or FSC and measure the mean fluorescence intensity of pHluorin and mCherry and all of the slices chosen for measurements.
Finally, divide the mean fluorescence intensity of the pHluorin channel by the mean fluorescence intensity of the mCherry channel, to calculate a ratio of pHluorin to mCherry fluorescence. Two germaria were imaged using quantitative fluorescence microscopy with UAS mCherry pHluorin and 10930 gal 4, stained with concanavalin A and ROI outlining of follicle stem cell or FSC or PFC and a follicle cell is shown in each image. The ratio of the fluorescence intensities in the GFP and mCherry channels is converted to pH I values, using a standard curve.
The pH I increases with differentiation in the early FSC lineage, from 6.8 in FSCs to 7.0 in prefollicle cells to 7.3 in follicle cells. The pH I values of each cell type can be represented as a pseudocolored micrograph that shows the differences in pHluorin to mCherry ratios, display these differences in color as defined by an LUT. When attempting this procedure, it is important to remember to have all the reagents ready before hand because many steps in this protocol are time sensitive.
After its development, this technique paved the way for researchers in the field of developmental biology, to explore the role of pH in sulfate decisions intracellular malano gaster.
