Intravitale Microscopy for Imaging Subcellulaire Structuren in Levende muizen die fluorescerende eiwitten

The overall goal of this procedure is to visualize subcellular structures in murine salivary glands. This is accomplished by first surgically exposing the salivary glands without damaging them. In the second step, the anesthetized animal is immobilized on the microscope stage.

Next, the salivary glands are carefully positioned so as not to compromise their function in the final step. The mouse is stabilized with a custom holder to minimize any motion artifacts due to the animal's heartbeat or respiration. Ultimately, the dynamics of the subcellular structures can be imaged by intra vital microscopy.

Prior to anesthesia, weigh the mice to determine the proper dose of anesthetic. Next, induce pre anesthesia by isof fluorine inhalation. Once the animals are unconscious, confirm sedation every 15 minutes by pop pinch and keep the mice under heat lamp for the duration of the surgery.

Monitor the body temperature of the animals with a rectal probe thermometer. Now shave the neck of the mouse with a pair of electric clippers, and then use an ethanol soaked gauze sponge to wipe down the ventral side of the neck, drying the excess alcohol with the kim wipe. Next, use a pair of dull curved tweezers to lift up a small piece of skin at the lower side end of the massiter muscles and make a small incision.

Insert the scissors into the incision, pointing the tips upwards towards the underside of the skin and opening the blades several times until the skin has separated from the underlying tissue. Then starting near the base of the salivary glands where the nerve duct and blood vessels connect the gland to the rest of the body. Use the scissors to cut a three millimeter wide, 10 millimeter long strip of loosened skin.

After cleaning the wound area, the resultant opening will be oval in shape and measure roughly eight by 12 millimeters. Both salivary glands will be visible. Use a syringe to apply optical coupling gel into the middle of the cut, and then use fresh pieces of sterile gauze to wipe the gel towards the outside of the opening until all the blood and loose hair is removed.

Now, use a pair of number seven tweezers to find the tip of the right salivary gland, grabbing the connective tissue a few millimeters above the top of the gland. Then tear the connective tissue around the gland without injuring the parenchyma. Once the gland is exposed, gently separate the connective tissue from the gland, washing away any bleeding with saline.

Make sure that all the connective tissues are separated and the gland is fully exposed. Prior to bringing the animal to the microscope, place a thick piece of gauze and a heat pack onto the microscope stage, covering the top of the heat pack with a thin piece of gauze as well. Place a cover slip over the center of the stage.

Then position the mouse on its right side on top of the gauze, securing the animal with a piece of masking tape on the ventral side of the front right paw and a silk suture hooked around the incisors. Create some tension between the thread and the paw and attach them onto the stage. With more masking tape, the salivary gland should rest naturally.

In the middle of the cover slip. Use plastic wedges coupled to the microscope stage to further stabilize the mouse's head and thorax. Next place a small piece of lenss cleaning tissue over the gland.

Extend the gland slightly and place a customized holder over the gland. Then tightly, couple the stabilizer to the stage with a metal clamp and masking tape. Cover the animal with gauze and a heat pad and monitor the exposed glands temperature with a flexible thermal coupled probe equipped thermometer Immediately after stabilizing the animal, use epi fluorescence illumination to focus on the surface of the gland.

Assess the blood flow in the capillaries and the overall morphology of the tissue. To excite the GFP gene, use a 488 nanometer laser. Set the peak power to 0.5 milliwatts to avoid photobleaching for the tandem tomato protein.

Use a 561 nanometer laser, setting the peak power to one milliwatt. Now assess the stability of the tissue by pre scanning the selected area. If the preparation is not stable, adjust the animal accordingly to find the proper focal plane.

In the pre mode. Gradually move the objective towards the cover slip until the aser structures, secretory granules and plasma membrane become clearly visible. Then to image the dynamics of the secretory granules, use a scan speed of 0.5 to two seconds per frame and 256 by 256 pixels with a 0.2 micrometer per pixel spatial scale.

In order to optimally visualize the granules and the plasma membrane, set the optical thickness between 0.9 to 1.2 micrometers. Next, record an image of the field of view to be used as a reference point. Finally, stimulate exocytosis by subcutaneously injecting 0.1 milligram per kilogram of freshly prepared isoproterenol and saline.

One minute after the injection. Fusion of the secretory granules with the plasma membrane will be detected by an increase in the GFP fluorescence around the granules and the diffusion of the tandem tomato peptide into their limiting membranes. In the GFPM tomato mouse, the asinine appear as clearly distinct structures that express cytosolic GFP and membrane targeted tandem tomato peptide as indicated by the broken lines.

In individual asinine aser cells are delineated by the tandem tomato peptide as indicated by the arrows. GFP is also detected in the nuclei that are clearly visible inside the aser cells. Cytosolic GFP indicated by the arrowhead is excluded from the secretory granules that appear as dark circular vesicles of approximately one to 1.5 micrometers in diameter.

To visualize exophytic events, a representative area of the plasma membrane that is enriched in secretory granules as indicated by the asterisk is shown after the injection of the isoproterenol. An increase in the levels of GFP fluorescence denoted by the green arrows around some of the secretory granules that are in close proximity to the plasma membrane can be observed. In addition, after fusion with the plasma membrane, the tandem tomato peptide diffuses into the limiting membranes of the secretory granules as highlighted by the red arrows.

After 30 to 40 seconds, the secretary granules gradually collapse and their limiting membranes are integrated into the apical plasma membrane. The approach presented here represents a major breakthrough in cell biology as it enables imaging of the dynamics of a specific membrane trafficking process in living mice.