Fluorescence Imaging of Neuro-Immune Dynamics Using a Skull Implant

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, c