(a) Measured pO₂ values in microvasculature at various depths, overlaid on the MIP image of the vasculature structure (grey). Digital processing was performed to remove images of the dura vessels. For easier identification, the edges of the major pial arterioles and venules were outlined in red and blue, respectively. Scale bar, 200 µm. (b) Composite image showing a projection of the imaged vasculature stack. Four pO₂ measurement locations in the capillary vessels at 240 µm depth are marked with red arrows. Five orange arrows point to the consecutive branches of the vascular tree, from pial arteriole (arrow at left) to the capillary level and further to the connection with ascending venule (arrow at upper right). Scale bar, 200 µm. (c) The pO₂ dependence with cortical depth in three mice averaged across all vessels excluding pial arterioles. Dotted black line shows average pO₂ dependence with cortical depth in three ascending venules, at positions outlined with the circles in (a). The error bars represent standard error at each depth. Composite image in the corner shows a side projection of the two circled ascending venules from the upper right corner in (a). These ascending venules are marked with black arrows in (c).

(a) Measured pO₂ values overlaid with the grey scale phosphorescence intensity image at 40 µm depth. Measurements were performed at locations of descending arteriole (left) and ascending venule (right). Measurement locations were also marked with the white rectangles in the inset (top left), which shows a MIP of 80 µm-thick FITC-labeled microvasculature stack. Artery (red) and vein (blue) are color-coded for easier identification. Scale bars, 100 µm. (b) Measured pO₂ values overlaid with the grey scale phosphorescence intensity image at 60 µm depth. Measurements were performed at the location of an ascending venule. Measurement location is marked with the white rectangle in the inset (bottom right), showing MIP of 80 µm-thick FITC-labeled microvasculature stack. Ascending venule (blue) and branches of the descending arteriole (red) are color-coded for easier identification. Scale bars, 50 µm.

(a) Experimental setup. PC₁ and PC₂ – computers, SH – shutter, EOM – electro-optic modulator, GS – galvanometer scanner. (b) Maximum intensity projection (MIP) along z direction of a 250 µm thick stack in the mouse cortex. The vasculature was labeled with FITC. (c) Phosphorescence intensity image of microvasculature obtained at 166 µm depth below the cortical surface. The color bar shows the average number of counts in each pixel collected during single phosphorescence decay. Scale bar, 100 µm. (d) Experimental measurement (dots) and corresponding single exponential fits (solid lines) of two phosphorescence decays from the diving arteriole (A, red) and ascending venule (V, blue), with positions marked with the arrows in (b) and (c).

(a) Volumetric image of the 200 µm-thick FITC-labeled microvasculature stack in the rat cortex. A large pial artery (red, marked with arrows in (a) and (b)) and two pial veins (blue) are color-coded for easier identification. (b) An integrated phosphorescence intensity image at 100 µm depth. Grey bar represents average number of counts from single phosphorescence decay at each pixel. Scale bar, 200 µm. (c) Measured pO₂ values during normoxia are overlaid with the grey-scale microvasculature structural image at 100 µm depth. (d)–(f) Temporal profiles of the measured pO₂ values at selected locations (b) during hypoxia. The period of the hypoxia is indicated by the grey bar.