The TRIO Platform imaging system enables secure head fixation for in vivo, optical imaging of the mouse brain through a cranial window or an implanted microlens. It has a nose cone with tube fittings to connect to a gas anesthesia system. The base plate of the system has channels for circulation of warm water to maintain body heat. It has fittings to connect to a water heater/pump system. The TRIO Platform can be used on almost any upright microscope. We recommend using a multiphoton microscope for deep brain imaging.