Two-Photon Microscopy for In Vivo Imaging of Mouse Brain Microvasculature

Begin with an anesthetized mouse with a headplate secured over a surgically prepared thin-skull cortical window.

Position the mouse supine. Remove the medial thigh skin to expose the femoral vein.

Inject a fluorescent dye conjugate into the vein to label blood plasma, enabling blood vessel visualization.

Suture the incision.

Flip the mouse and secure the headplate in a harness to stabilize the mouse.

Transfer the setup to a two-photon microscope stage.

Add saline over the cortical window. Lower the objective lens until it contacts the saline.

Using bright-field illumination, locate the imaging region.

Switch to two-photon imaging.

The microscope’s laser focuses low-energy near-infrared light into the brain.

At the laser’s focal point, two photons combine their energy to excite the dye, making the blood vessels fluoresce.

Identify a capillary bed—a network of small blood vessels connecting arteries to veins— and magnify it.

Acquire images to analyze the brain’s microvasculature.

In this procedure, remove the skin on the medial left thigh of the mouse. Next, locate the femoral vein. Using a 1-milliliter syringe and a 30-gauge needle, draw up 130 microliters of the fluorescent dye solution. Inject 100 microliters of the dye slowly into the femoral vein.

After removing the needle, apply steady, gentle pressure to the injection site to stop any bleeding, and allow the dye to circulate for five minutes. Then, close the surgical site with sutures. Carefully flip the mouse onto its stomach and place it into the mouse head plate harness. For in vivo two-photon imaging, move the surgical apparatus to the two-photon microscope, and make sure to maintain the animal's anesthesia level.

Next, place a small amount of 0.9% saline into the head plate reservoir, and lower the microscope objective so that it comes into contact with the saline. Then, locate the area of interest, using the bright field viewing objective. Afterward, begin two-photon imaging. Locate a capillary bed on the view screen, and magnify this area using the optical zoom 2. Acquire images of the capillaries using the two-photon imaging software.