Prime 10 w

An Olympus BX51WI upright microscope and water-immersion LUMPlan FL/IR 20×/0.50W objective were used. Excitation (740 nm) was provided by a Prairie View Ultima multiphoton laser scan unit powered by a Millennia Prime 10 W diode laser source pumping a Tsunami Ti: sapphire laser (Spectra-Physics, Mountain View, CA). Blood plasma was labeled by i.v. injection of tetramethylrhodamine isothiocyanate dextran (155 kDa) in physiological saline (5% wt/vol). All microvessels in an imaging volume (500×500×300 µm) were scanned at each study point, measuring the diameter and blood flow velocity in each vessel (3–20 µm Ø). Tetramethylrhodamine fluorescence was band pass filtered at 560–600 nm, NADH autofluorescence was band pass filtered at 425–475 nm. Imaging data processing and analysis were done using Fiji image processing package [11 (link)].

Using in-vivo 2PLSM through a cranial window over the rat parietal cortex we measured microvascular red blood cell flow (RBC) velocity and diameters (tetramethylrhodamine dextran), NADH autofluorescence (tissue oxygenation) and blood brain barrier (BBB) integrity by tetramethylrhodamine dextran extravasation. For 2PLSM, an Olympus BX51WI upright microscope and water-immersion LUMPlan FL/IR 20x/0.50W objective was used. Excitation (740 nm) was provided by a Prairie View Ultima multiphoton laser scan unit powered by a Millennia Prime 10 W diode laser source pumping a Tsunami Ti: sapphire laser (Spectra-Physics, Mountain View, CA). Blood plasma was labeled by i.v. injection of tetramethylrhodamine isothiocyanate dextran (155 kDa) in physiological saline (5% wt/vol). All microvessels in an imaging volume (500X500X300 μm) were scanned at each CPP, measuring the diameter and blood flow velocity in each vessel (3-20 μm Ø). Cortical Doppler flux (probe Ø = 0.8 mm), rectal and cranial temperatures, ICP, arterial pressure and arterial blood gases were monitored.

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).