This article details a method for imaging microglial dynamics in transgenic mice using two-photon microscopy. The procedure involves surgical implantation of a cranial window and subsequent imaging of fluorescently labeled cells and blood vessels.
Start with an anesthetized transgenic mouse with a circular glass imaging window and a head plate implanted on its skull.
The microglial or immune cells and neurons are labeled with fluorescent proteins.
Next, secure the mouse onto the two-photon microscope stage.
Then, inject a fluorescent dye intraperitoneally to stain the blood vessels red.
Clean the glass imaging window, apply a saline drop, and lower the microscope objective.
Next, set the laser light to capture an image of the blood vessels. Record the branching pattern and coordinates to locate and image the same region later.
Now, set the laser to excite the fluorescent proteins, causing the microglia and neurons to fluoresce.
Using the recorded branching pattern, locate the same region and regularly capture images of cells.
The vascular network and neurons remain stable, while microglial position changes over time reflect immune dynamics in response to neuronal activity.
Drill a circular opening approximately 4 millimeters into the skull using a dental drill bit. During drilling, regularly moisten the skull with sterile saline and cotton swabs to cool the brain. Clear off bone debris and soften the skull bone.
When finished, carefully remove this portion of the skull with pointed forceps. After the skull is removed, carefully place a small cover glass moistened with saline in the craniotomy. Dry off excess saline using a sterile wipe. Using a pointed applicator, apply cyanoacrylate glue around the window and allow it to attach to the brain and skull.
Apply the primer glue to the rest of the skull and cure it with a curing light for 20 to 40 seconds. Create a well around the window with the final glue and cure it with a curing light for 20 to 40 seconds. Glue a small head plate onto the skull on the contralateral hemisphere of the craniotomy with the primer glue, and then, with the final glue, curing both for 20 to 40 seconds.
After the mouse wakes up, inject one subcutaneous dose of buprenorphine SR as post-operative analgesia, which will be sufficient for 72 hours. Provide the mouse with extra soft food to facilitate a healthy recovery. Imaging can be performed as early as two weeks after the implantation surgery. Use screws to mount the head plate on the two photon microscope stage, which should be maintained at 35 degrees Celsius with a heating plate.
Inject the mouse subcutaneously with 100 microliters of blood vessel dye, such as Rhodamine B. Gently clean the surface of the cranial window with a cotton swab dabbed in 70% ethanol. Put a few drops of water or saline on the window and lower the objective lens into the solution. Ensure that the laser is turned off as the objective is lowering.
Hand draw a course map to denote the major blood vessel landmarks, while looking through the eyepiece. Use this drawing to identify the specific regions during two photon imaging. Collect images of fluorescent cells and vessels using two photon imaging. See the text manuscript for imaging parameters. Take careful notes with appropriate coordinates to ensure that the precise regions can be revisited for subsequent imaging.
Collect several fields of view in this initial imaging session.
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