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.