A miniature head-mounted two-photon microscope: High-resolution brain imaging in freely moving animals

Two-photon microscopy has enabled anatomical and functional fluorescence im aging in the intact brain of rats. Here, we extend two-photon imaging from anesthetized, head-stabilized to awake, freely moving animals by using a mi niaturized head-mounted microscope. Excitation light is conducted to the mi croscope in a single-mode optical fiber, and images are scanned using vibra tions of the fiber tip. Microscope performance was first characterized in t he neocortex of anesthetized rats. We readily obtained images of vasculatur e filled with fluorescently labeled blood and of layer 2/3 pyramidal neuron s filled with a calcium indicator. Capillary blood flow and dendritic calci um transients were measured with high time resolution using line scans. In awake, freely moving rats, stable imaging was possible except during sudden head movements.