The goal of this procedure is to show how polarization based total internal reflection fluorescence microscopy is implemented for the study of membrane remodeling during regulated exocytosis. This is accomplished by first ensuring optical elements, namely quarter wave plate and polarizing cubes are correctly positioned to generate discrete p and s excitation polarizations. The second step is to verify that the p and s beams are coline pass through the optical axis of the microscope and illuminate the same portion of the viewing field when in total internal reflection fluorescence or TIRF.
Next, the cells to be imaged are stained with the carbo cyanide die, die D and stimulated with a depolarizing stimuli to trigger exocytosis. The final step is to capture images of fusing dense core vesicles while in TIRF. Ultimately, P-T-I-R-F is used to acquire images in real time to monitor the membrane topological changes that result from chromin cell exocytosis.
The main advantage of this technique over existing methods like pyrometry and membrane capacitance, is that ptro F reports a membrane curvature and by extension fusion port dilation directly. The steepest part of the learning curve with this technique is first learning to align the p and s excitation polarizations, so they're coline travel through the optical axis of the microscope and illuminate the same part of the imaging field. A second issue that often arises concerns cell staining, namely how long to expose cells to dd and what constitutes a well stained cell versus one that is poorly stained.
Demonstrating the procedure will be TA square row, a graduate student from our laboratory. To begin power on all microscope components, lasers and computers direct a beam from the 488 nanometer laser to the back focal plane of the 1.49 numerical aperture lens. If the laser beam is focused on the back focal plane, it will emerge, collated, and appear as a small, well-defined spot on the ceiling directly above the objective.
Then adjust the X galvanometer mirror so that the laser beam is moved off axis and emerges from the objective at progressively steeper angles to the objective normal. Next, verify that total internal reflection or TIR is achieved. Add 10 microliters of fluorescent microspheres to one milliliter volume of physiological saline solution and a glass bottom dish only fluorescence from microspheres on the bottom of the dish closest to the TIR interface should be detected.
Detection of floating microspheres indicates that the angle between the incident light and objective normal is insufficient. For polarization based imaging, use the aligned 488 nanometer beam as a guide and adjust the position of the raw 561 nanometer laser beam so it is traveling along an identical optical path. The 561 nanometer laser will be used for imaging of the carbo cyanide dye.
A diverging lens and mirrors are downstream of the 561 nanometer laser. Using the adjustment knobs on the mirrors, adjust the beam so that it is co-align with the 488 nanometer beam and the spot is in focus on the ceiling. When the galvanometer mirrors are in the zero position, a polarization cube reflects the vertical component of the electric field and passes the horizontal component.
It is placed downstream of the elliptically polarized beam on a small translating stage. To facilitate subsequent alignment of polarization, beam paths use a second polarizing cube and mirrors to recombine the beams. A shutter is placed between the first polarization cube and the vertical component mirror.
A second shutter is placed between the horizontal component mirror and second polarization cube. These shutters are controlled by the imaging software enabling the user to rapidly select between beam polarizations. Use a polarizing filter to verify the electric field orientation in each beam path.
A polarizing filter will only allow transmission in line with the filter's axis. Verify that the vertical and horizontal components of the laser beam are aligned with each other and with the 488 nanometer beam, make adjustments as necessary. The three beams should all be focused on the back focal plane and emerge collated from the objective to the same spot on the ceiling.
This spot can be marked by an X to facilitate future alignment. Next place a drop of immersion oil on the objective. Place the dish containing fluorescent microspheres.
On the objective, enter TIR by moving the X galvanometer mirror in the mirror moving software in TIR. The vertical component of the beam is the S polarization and the horizontal component of the beam is now the P polarization center, A quarter wave plate or qw in the beam path. Immediately downstream of the laser aperture view each polarized evanescent field with a rod domine sample, which is predicted to be randomly oriented.
Rotate the qw plate to match the average pixel intensities. Isolate healthy chromin cells from the cat adrenal gland as described in the text protocol count cells using a hemo cytometer and prepare for transfection transfect cells with DNA encoding the protein of interest using the electroporation system according to the manufacturer's recommendations. The settings which provide the best balance between transfection efficiency and cell survival rate.
For this preparation of bovine chromin cells are 1, 100 volts, 40 milliseconds, and one pulse following transfection plate cells gently on poly de lycine and collagen treated dishes in one milliliter of warmed electro parading media. Place the dishes in a 37 degrees Celsius incubator. Add one milliliter of two x antibiotic media to each dish After six hours, change the media to normal media the next day.
Chromin cells are typically imaged 48 hours to five days after electroporation with the imaging system on start acquisition software. Verify that lasers are aligned. Check evanescent field profile using microspheres.
Prepare the global and local profusion system. Clean solution reservoirs with filtered deionized water and fill with basal and stimulating physiological saline solutions. Before imaging chromin cells verify that the P polarization and S polarization excitation illuminate the same region of the viewing field and that the illumination intensities are roughly equivalent as detailed in the text protocol.
Now proceed to staining bovine chroma cells with DD.Rinse culture media from the dish containing the chromin cells and replace with two milliliters of basal physiological saline solution. Add 10 microliters of 10 millimolar DD directly to the dish containing the cells. Gently agitate the dish for two to 10 seconds before removing the solution.
Wash the dish three to four times with basal physiological saline solution to remove any residual DD, the cells are now stained and ready to use. Add a drop of immersion oil to the objective. Place the dish containing Dai D stained chromin cells on top of the objective.
Position the local perfusion needle so that it is roughly 100 microns away from the cell. Focus on the cell membrane and activate the auto-focus hardware immediately before cell stimulation image acquisition. Begin image acquisition perfuse cells for 10 seconds with a basal solution, and then for 60 seconds with a depolarizing 56 millimolar potassium chloride solution.
Acquire images while rapidly shuttering between 561 nanometer P polarization and 561 nanometer s polarization to monitor changes in p and s emissions of DD and 488 nanometer excitation. To image the transfected vesicle probe exuberant membrane labeling of chromin cells is achieved after a brief incubation with Dai D.In this example, the cell membrane is stained well. A healthy adherent cell will exhibit distinct differences in p and s emissions.
The P emission image shows a brighter cell border with respect to the rest of the cell. The S emission image shows roughly uniform fluorescence across the cell footprint, calculated pixel to pixel P over S and p plus two s. Images are sensitive to membrane curvature and to dye concentration respectively.
The CHRO and cell shown has also been transfected with synap OIN one florin to label secretory vesicles. The CHRO and cell is stimulated with potassium chloride to depolarize the cell membrane and trigger exocytosis. A number of rightly fluorescent spots suddenly become evident as dense core vesicles fuse.
A white box is drawn around one fusion event To analyze this fusion event frame by frame changes in sit one florin po VS and p plus two s image intensities were examined. Fluorescence fluorescent intensity of CIT one quickly diminishes as the protein diffuses away from the fusion site. The indentation representing the fused vesicle plasma membrane complex diminishes at a relatively slower rate.
This illustration depicts one interpretation of these measurements. The rapid and localized membrane deformation are a result of stimulus evoked calcium influx membrane depolarization causes a significant increase in G Camp 5G fluorescence signifying an increase in sub plasmal calcium levels. A 30 by 20 pixel region of the cell is selected and frame by frame changes in G camp 5G PS and p plus two s.
Pixel intensities are shown in the images and graphs. Times zero designates the frame before a change in PS is evident. The white arrowheads indicate that the membrane deformation is accompanied by a decrease in p plus two S emission.
The cytosolic G CAMP 5G protein is excluded from the area by the fused dense core vesicle. Note also the sudden decrease in G camp 5G intensity at times zero. The long-lived increase in POS and decrease in p plus two s suggests a fusion pore that dilates slowly as illustrated.
Here After watching this video, you should have a good understanding of how to build a PTU F system and image cells to observe membrane topological changes while in turf after its development. This technique paved the way for researchers in the field of cell biology to explore changes in membrane shape during exocytosis endocytosis cytokinesis and cell motility in living cells.
