Water immersion objective

For chronic transcranial time-lapse imaging, the thin skull procedure was used^{52 (link)}. P20–22 NG2cre:ZEG and NG2cre:ZEG:PLPDsRed mice were anesthetized by intraperitoneal injection of ketamine and xylazine, and the scalp was shaved and sterilized. A region of the skull approximately 1 mm in diameter was thinned with a high speed drill and a microsurgical blade to a thickness of 20–30µm. Images were acquired using a two-photon microscope (Prairie Technologies) with a mode locked MaiTai tunable near infrared laser (Spectra Physics) and a 20x water immersion objective with a numerical aperture of 1.0 (Leica). In some cases 70,000 MW Texas Red dextran (Life Technologies) was intravenously injected to visualize the cortical vasculature. The following wavelengths were used for two-photon fluorescence excitation: 900nm for GFP alone, 975nm for GFP and DsRed, and 1040nm for DsRed alone. Z stacks of the same cortical region over a depth of 200–320μm with a 4μm step size were captured through the thinned skull on consecutive days at intervals depicted in the text. Images were analyzed using ImageJ.