The overall goal of this imaging protocol is to describe the quantification of intracellular pH, using a genetically-encoded pH indicator and demonstrate how this method can be used to assess basolateral proton transport in an insect renal structure model, the malpighian tubule. This method can help answer key questions in the cellular transport field such as, how specific transporter proteins contribute to epithelial proton movement in intracellular pH regulation. The main advantage of this technique is that cellular transport can be quickly and easily assessed across multiple functionally distinct zones of intact epithelial.
Though this method can provide insight into pH regulation and insect epithelial, it can also be applied to other systems such as, the mammalian nephron and epithelial cells in culture. To begin this procedure, position two sets of soldering helping hands clamps on the imaging microscope stage, with one clamp on each side. Next, insert the respective bent glass capillaries and the inflow line, and the vacuum connected, outflow line.
Then, mount then in the helping hands and align them with the imaging stage of the microscope. Spread vacuum grease onto the ceiling tape and press the adhesive profusion well divider onto the tape to coat the bottom with grease. Peel off the adhesive perfusion well divider and place it grease side down, on top of a poly l lysine coated slide.
Following that, remove the perfusion well divider to leave individual specific wells traced in hydrophobic grease. After that, place 200 microliters of room temperature IPBS or insect phosphate buffer saline in the greased and circle well on the poly l lysine coated slide. Then, place the slide under the steroscope, pour ice cold Schneider's medium into the dissecting dish and transfer a single anesthetized female fly to it.
Hold the fly by the thorax with a set of forceps and use the other to gently grip the posterior abdomen. Pull open the posterior end of the fly in short deliberate motions. Once the hind guard is visible, grip the distal end and free the gut and antes from the underlying tracheales, by pulling the hind guard away from the body through repetitive brief tugs.
Then, pinch off the anterior antes of the ureter with fine forceps, once the second set of antes is free from the abdomen. Pick up the free anterior antes with the pole glass rod, by sliding the rod under the ureter, such that the tubules fall to either side. Following that, lift the antes straight up, out of the solution, after that, turn the glass rod such that the antes and ureter are adhered to the underside of the rod.
Lower the ureter straight down onto the slide, then, affix the ureter and seal the distal ends of the antes by further pressing the ureter onto the glass slide. Use the fine end of the glass rod to gently sweep each tubule across the slide surface. Graze the rod against the slide to avoid crushing the tubule and slide the rod over the top of the tubule, moving from distal to proximal, to attach the full length of each tubule to the surface of the poly l lysine coated slide.
Success of this technique depends entirely on accurate identification and careful handling of the anterior malpighian tubules. Damaged tubules will produce inconsistent results. Next, place the adhesive profusion well divider back on the slide to form a small fluid filled well over the mounted tubule.
After that, place the specimen on the microscope stage. Position the inflow and outflow capillaries over the inlet and outlet opening of the profusion well, respectively. In this procedure, turn on the microscope, light source and imaging system, then, open the imaging software.
Look through the eyepieces and manually adjust the focus until the lumen of the antes is clearly visible under the transmitted light. Then, click the acquisition tab and select, 2x2 in the dimming pull down menu, in the acquisition load section. Insert a 5%neutral density filter into the light path, to reduce illumination light and minimize photo-bleaching.
Click the GFP channel in the channels menu, then click live to observe the fluorescent signal via the camera. After that, adjust the time slider to set the exposure time, such that the brightest pixel values in the intensity histogram are approximately 40%of the maximum value. Then click stop, to stop the illumination.
Repeat the procedures in the RFP channel and confirm the presence of the dilated initial segment of the anterior ante and the absence of cytosolid m cherry aggregates, which indicates tissue damage or over-expression. Afterwards, enable a timelapse imaging protocol by clicking the time series checkbox. Adjust the duration in the pull down menu, in the time series section to 10 minutes, and the interval slider to zero, to set the total capture time with the maximum image acquisition.
Then, check both the GFP and RFP boxes in the channel section. Open the IPBS line of the profusion system by activating the appropriate valve controller and start the imaging protocol by clicking, start experiment. After one minute, switch to ammonium chloride post solution for 20 seconds, by opening the appropriate valve and closing the IPBS line.
Then, return to IPBS by closing the ammonium chloride line and reopening the IPBS valve. To perform a two point calibration, remove the well divider by peeling it away from the underlying slide and remove the profusion capillaries in clamps from the imaging well. Following that, apply 200 microliters of calibration IPBS buffer to pH 7.4.
Then, remove the solution from the imaging well and replace it with another 200 microliters of calibration solution. Repeat this process four times to ensure complete solution exchange. Next, incubate the preparation and calibration solution for 30 minutes before imaging.
Repeat the imaging protocol using the same parameters determined previously, with the modification of only one minute of image capture. Then add 200 microliters of calibration IPBS buffer to pH nine. Remove the solution from the imaging well and replace it with another 200 microliters of calibration solution.
Subsequently, incubate the preparation in the second calibration solution for 10 minutes before imaging and repeat the imaging protocol. After that, review the captured image stacked in the image analysis software to confirm that no pixels in either channel are saturated, by clicking MeanROI and that no values reported in the intensity histogram, reached the maximum detectable value, while scrolling through the image stack with the frame slider. Next, analyze the image stack by plotting fluorescent intensity and fluorescence ratio is a function of time, here, we see the expected fluorescent response to the ammonium chloride pulse in the pH sensitive and pH insensitive channels of the indicator.
The ratio of these signals reveal the intracellular pH transience. To perform this analysis, first click meanROI and select the free form tool. Hold the left click on the mouse, to trace a 50 micrometer MT and right click to finish drawing the ROI.
Then, repeat that in an area adjacent to the MT, to define background ROI. Next, click mean intensity under measurements, create a table of intensity values by clicking export, data table, create. Click the configuration clock wheel icon and deselect all parameters, except time and mean intensity.
Right click the tab for the newly created data table, select save as, and export the data as a csv file. Shown here, is a wide field image, a super-elliptic fluorin fluorescence in principle cells of the anterior ante and stellate cells of the anterior ante. Note that stellate cells are bar shaped in the initial segment, variable in the transitional segment and display distinct cellular projections in the main segment.
This graph shows the calibrated intercellular pH changes in response to 20 seconds, 40 millimolar ammonium chloride pulse in regions of interest. Dashed curves denote single exponential fits applied to the acid recovery phase, following ammonium chloride withdrawal. From which, the decayed constant values are derived.
This graph shows the acid extrusion rate plotted as a function of intracellular pH and this graph shows the acid flux plotted as a function of intracellular pH, derived from the exponential fits from the previous figure. Once mastered, the malpighian tubule extraction can be done within 10 minutes, if it is performed properly. While attempting this procedure, it's important to remember the success depends entirely on the quality of dissection and accurate calibration of the pH indicator.
This preparation can be combined with other genetically encoded biosensors such as, fluorescent calcium and halide indicators to study solute movement and intracellular signaling in epithelial cells. After watching this video, you should have a good understanding of how to prepare healthy drosophila malpighian tubules, image a genetically encoded pH indicator and quantify proton movement in epithelial cells. Don't forget that working with nitroizene can be hazardous and damage preparations and thus, precautions should be taken while performing this procedure, to ensure that it does not contaminate any equipment that is to be reused.
