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).