Experimental procedures for two-photon fluorescence imaging in the cortex of anesthetized mice. (A) Schematic view of experimental setup. Stable fluorescence imaging is enabled by restraining the animal's head via a head plate and head holder. A blow-up of the head plate (yellow dashed box) is shown in (B). (B) Firm and repeatable head restraint is facilitated by implanting a lightweight head plate. Coverslip and metal ring are used to stabilize and seal open skull preparations. (C,D) Lateral view showing how skull, head plate, and head holder interface with each other. Scale bar, 2 mm. (C) Schematic view of a thinned skull preparation. A restricted area of the skull (typically <350 µm in diameter) is reduced to an ∼25- to 35-µm-thin layer. Cells and their processes can be visualized over short and long timescales up to a depth of ∼250 µm. (D) Open skull preparation. To reduce heartbeat- and breathing-induced motion artifacts as well as optical aberrations, the exposed brain is covered with agarose gel and a coverslip. This preparation is particularly useful for short-term investigation of deep cortical regions and large contiguous brain areas.