Before the surgery, animals were anesthetized with isoflurane (3% in oxygen for induction, and 1.5–2% for surgery to maintain a breathing frequency around 1 Hz). Body temperature was kept at 37.5 °C with a feed-back controlled blanket (Harvard Apparatus), and eyes were covered with eye ointment. Glycopyrrolate (0.01mg/kg body weight), dexamethasone (0.2mg/kg body weight), and ketoprofen (5mg/kg body weight) were administrated intramuscularly. Dexamethasone and ketoprofen were also administrated in two consecutive days following the surgery. The anesthetized animal was fixed on stereotaxic, and hair was removed from scalp with scissors and Nair. The scalp was further sterilized by alcohol wipes, and then cut open and removed to expose both parietal plates as well as the bregma and lambda. Sterile saline was applied to the skull immediately after the exposure, and it is critical to keep the entire bone surface covered by saline to insulate from air. Fascia and connective tissue on the skull were gently removed with forceps and sterile wet cotton tips to avoid any internal bleeding inside the brain. At this point, the whole skull was transparent, with blood vessels underneath visible with sharp edges. The saline covering the skull was then wiped completely dry with cotton tips, and the following actions were taken quickly before the bone turns opaque. Ultra-violet curable glue (Loctite 4305) was applied to the skull surface within 2 seconds afterwards. A sterile and dry round coverslip of 5-mm diameter (#1 thickness, Electron Microscopy Sciences) was placed on the skull, centered at 2.5 mm lateral, and 2 mm caudal from the bregma point. The coverslip was pressed closely against the skull surface by forceps to minimize the amount of glue between the coverslip and the skull. The glue was left to cure by itself for about 5 minutes without any ultra-violet light, during which time the skull transparency tends to increase visually. Afterwards, an ultra-violet light source (385–515nm, Bluephase Style 20i, Ivoclar vivadent) was used to completely cure the glue, with roughly 1s on and 1s off for 3s. The coverslip is necessary to keep the glue layer as thin as possible (down to ~10 μm at the thinnest part on the skull), and to form a flat interface to reduce aberration. The exposed part of the skull surrounding the coverslip was further covered with dental cement. Supplementary Fig. 1 shows an example of successful preparation. For awake imaging, a head-bar for head fixation during imaging was glued to the exposed parts of the skull surrounding the coverslip by metabond glue.