The upright laser scanning microscope (BX61WI, Olympus) was attached to a Ti:sapphire femtosecond pulsed laser system (80 MHz repetition rate, pulse width, Spectra Physics, Santa Clara, California) and the software (Fluoview 1000) was used for two-photon fluorescence imaging. air, water-immersion [NA, 1.05; working distance (WD), 2 mm, Olympus], and water-immersion objectives (NA 0.80, WD; 3.3 mm, Olympus) were selectively chosen for fluorescence imaging in vivo. To excite OGB-1 and SR 101 simultaneously, 800-nm irradiation was used, and emission light was detected with 515/50 and 605/55 filters, respectively. In addition, we visualized pial arteries and veins under widefield fluorescence and identified penetrating arteries or collecting veins (10 to 35 mm diameter) by following the direction of flow from the pial surface.22 Capillaries were identified by their diameter (). The average laser power for imaging was . Arterioles, capillaries, and veins were discriminated by vessel diameter and blood flow direction. Series stacks of images (step-size: 1 μm) were acquired from the cortical surface to depths below by vertically translating the objective of the two-photon intravital microscopy system.