Fluorescent serum (i.v. tetramethylrhodamine isothiocyanate (TAMRA) dextran, 150 kDa in physiological saline, 5% wt/vol) was visualized using an Olympus BX 51WI upright microscope and water-immersion LUMPlan FL/IR 20X/0.50 W objective. Excitation was provided by a PrairieView Ultima multiphoton microscopy laser scan unit powered by a Millennia Prime 10 W diode laser source pumping a Tsunami Ti: Sapphire laser (Spectra-Physics, Mountain View, CA, USA) tuned to 750 nm center wavelength. Band-pass-filtered epifluorescence (570-600 nm for TAMRA and 425-475 nm for NADH) was collected by photomultiplier tubes of the Prairie View Ultima system. Images (512 X 512 pixels, 0.15 um/pixel in the x- and y-axes) or line scans were acquired using Prairie View software. Red blood cell flow velocity was measured in microvessels ranging from 3-50 μm diameter up to 500 μm below the surface of the parietal cortex, as we described previously [6 (link)]. Tissue hypoxia was assessed by measurement of NADH autofluorescence. In offline analyses using NIH ImageJ software, the three-dimensional anatomy of the vasculature in areas of interest was reconstructed from two-dimensional (planar) scans of the fluorescence intensity obtained at successive focal depths in the cortex (XYZ stack).