Imaging vasodynamics in the Awake mouse brain with two-photon microscopy

In vivo two-photon laser-scanning microscopy is widely used to study brain structure and function in mice. Recent studies using awake mice have revealed a rich dynamism in cerebrovascular flow and neural activity that is significantly masked by anesthesia. Imaging of awake animals is necessary to fully understand neurovascular coupling during naturalistic sensorimotor activity. We provide detailed instructions to rigorously quantify blood flow at the level of single cortical vessels in awake mice. This includes a description of surgical techniques to obtain optical access to the cortex, improved head-restraint devices to reduce motion artifacts, and robust algorithms to quantify red blood cell flow and vessel caliber. Finally, we provide examples on how these techniques are used to measure sensory-evoked cortical hemodynamics.