Ultima multiphoton laser scan unit

An Olympus BX51WI upright microscope and a water-immersion LUMPlan FL/IR 20×/0.50 W 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, USA). Blood plasma was labeled by i.v. 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 and NADH autofluorescence at 425–475 nm. Imaging data processing and analysis were carried out using the NIH ImageJ.

For long-term in-vivo imaging of the mouse cortex, we used an optical clearing skull window using two clearing solutions without performing a craniotomy [8 (link)]. For skin imaging, the ear was shaved and treated with a skin-clearing solution [9 (link)] before each imaging. The number of perfused capillaries, microcirculation, and tissue oxygen supply were visualized using Olympus BX 51WI upright microscope and water-immersion XLUMPlan FI 20x/0.95W objective as previously described [4 (link)]. Excitation 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). 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 and ear skin. NADH autofluorescence measurement was used to evaluate mitochondrial activity (metabolic status) and tissue oxygenation [10 (link)]. In offline analyses using NIH ImageJ software, 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).

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)].