This protocol demonstrates how In Vivo Two-Photon microscopy can be used to do repetitive visualization of cellular dynamics, in the same brain location over extended time periods. The advantage of this technique is that, it makes it possible to image the longterm changes in cellular elements of brain, including the arrangement, morphology, and physical interactions between different cell type of the CNS. This technique can be especially useful in eliciting cell-cell interactions, cell dynamics, and morphological changes during brain plasticity, and neurodegeneration.
Begin by preparing a juvenile or young adult, four to 10 week old, a CX3CR1 GFP heterozygote mouse, that weighs 17 to 25 grams, for a cranial window implantation. After anesthetizing the mouse, use a hair trimmer to shave the hair on its head, between the ears, approximately, from the eye level, to the top of the neck region. Move the mouse to the stereotactic surgery station, nose cone, and stabilize its head using ear bars.
Maintain the mouse on the heating pad, throughout the procedure. Lubricate both eyes with oil ointment, then inject 100 microliters of 0.25%bupivacaine, and 100 microliters of four milligram per milliliter dexamethasone, subcutaneously at the incision site. Wait at least five minutes before proceeding.
Bleed the shaved head, with three alternating swabs of betadine, and 70%alcohol. Then make a midline scalp incision with a surgical blade or scissors. Extending from the back of the skull region, between the ears, to the frontal area between the eyes.
Cut the remaining skin to expose the skull. Bleed the connective tissue located between the scalp and the underlying skull with 3%hydrogen peroxide, and localize the brain area to be imaged with stereotactic coordinates. Drill a circular opening, approximately four millimeters into the skull, using a dental drill bit.
During drilling, regularly moistened 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, 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 postoperative analgesia, which will be sufficient for 72 hours. Revive the mouse with extra salt 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, and 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 a subsequent imaging. Draw like several fields of view in this initial imaging session, At the end of the imaging, take the mouse off the stage, allow it to wake up from anesthesia, and return it to its home cage. Use the obtained notes and images for re-imaging the areas during subsequent sessions.
This protocol was used to visualize Microglial Dynamics In Vivo, in Taiwan YFP-H line mice. Specific dendrites, served as stable landmarks for the fine mapping of brain regions. While dendrites are a stable, some microglia move daily.
Single transgenic CX3CR1/GFP mice, were used to follow microglia for up to eight weeks. Injections of rhodamine B, were used to label the vasculature, during each imaging session. While the vasculature remains stably fixed, the microglia are dynamic.
The CX3CR1/GFP mice, were used to follow microglia before and after severe seizures. The vascular bed structure was maintained, but the microglial cellular network, and position landscape was transient. Greater changes were observed within 24 to 48 hours of seizures.
Double transgenic CX3CR1/GFP and NG2DS-Red mice, were used to track microglia, and NG2 to positive cells In Vivo. Without labeling the vasculature, microglia as well as vessel associated parasites, and oligodendrocyte precursor cells, or OPCs, were identified. Parasites have elongated processes that follow along the vascular wall, and OPCs typically show larger cell bodies that reside in the brain parenchyma, away from the vasculature.
When rhodamine B was used, the brighter fluorescence of parasites could be distinguished from the fainter fluorescence of luminal rhodamine, despite similar excitation during imaging. Daily imaging showed that parasites were stably positioned, while OPCs and microglia were dynamic. It is important to make careful observation of the area being visualized, to draw a representative map that is as accurate as possible, and denote the steps taken to reach the other locations with respect to the starting point.
In subsequent imaging sessions, these steps, the hand drawn maps, and the images previously taken, will be critical in identifying the same locations repeatedly. This technique has allowed researchers to elucidate neuron-glial interactions, neuro-immune interactions, and neuro-cell dynamics of extended time periods in both health and pathology.
