20 1.0 na water immersion objective

2% w/v fluorescein isothiocyanate-dextran (FITC-dextran, MW 70,000, 50 μl, Sigma-Aldrich) was administered into the femoral vein to label the blood plasma. In vivo imaging of blood vessel diameter and pericyte location was performed using a commercial two-photon microscope (SP5, Leica, Wetzlar, Germany), a MaiTai HP Ti:Sapphire laser (Millennia Pro; Spectra Physics, Santa Clara, CA, mean output power 10mW), and a 20× 1.0 N.A. water-immersion objective (Leica). Tissue was excited at 900 nm wavelength, and the emitted light was filtered to collect red and green light from DsRed (pericytes) and FITC-dextran (vessel lumens). Penetrating arterioles were identified unequivocally in vivo in two ways, firstly by tracing their connections back to pial arterioles with obvious smooth muscle around them, and secondly by observing the direction of flow of the red blood cells from the arterioles into the parenchymal capillaries. Z-stack images were taken of the area of interest. XY-time series were taken to image pericytes and blood vessels during stimulation, with a frame size 512 × 300 pixels (170 msec/frame; pixel size was 93-201 nm depending on the magnification used, with a mean value of 155 nm; pixel dwell time was 1.1 μsec).