Two-photon imaging was performed using a Thorlabs two-photon setup attached to a Mai Tai HP Ti:Sapphire laser (Spectra-Physics). A 20× objective (0.9 numerical apperture; Olympus) was used. Intravascular fluorescein isothiocyanate–dextran (FITC-dextran; 2.5% in saline) was injected to the tail of the mouse and excited at 820 nm. To prepare the sulfite puffing solution, 0.01 or 1 M sodium sulfite was dissolved in 10 mM Hepes (with a drop of HCl to adjust pH to 7.3) containing 100 μM Alexa Fluor 594 and loaded into a glass micropipette with a tip diameter of 2 to 3 μm. The puffing micropipette was then carefully loaded into a capillary bed in the cerebral cortex, and the sulfite solution was puffed at 10 psi (~20 ms) controlled by a Picospritzer III. Capillary RBC velocity was captured with line scans (scan rate, ~1 kHz) placed along the length of the capillary. RBC velocities (Δxt, mm/s) were calculated from parallel-to-flow line-scan images using the contrast between FITC-dextran–labeled plasma and unlabeled RBCs, using a modified version of the LS-PIV algorithm in MATLAB.

Local tissue PO2 measurement. Local tissue O2 tension was recorded with a calibrated modified Clark-type polarographic O2 microelectrode (OX-10, Unisense A/S, Aarhus, Denmark) inserted in close proximity to the puffing pipette (~50 μm). The signal was amplified and analog-to-digital converted by a high-impedance picoammeter (OXY-Meter, Unisense A/S). Data were recorded in pCLAMP 9.0 and analyzed using Clampfit 10.4 and MATLAB.

Parameter estimation. A zero-phase running average filter (window size, ~1 s) was used to smooth raw velocity data after outliers (raw data points of >3 SDs from mean of trace) were removed. Onset of the evoked response was estimated by fitting a line to the slope between 20 and 80% to the peak of the response and calculating the time of the line’s x intercept. Five to 10 s immediately before puffing stimulation was considered baseline, and responses occurring within 20 s after the start of stimulation were analyzed.

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