For analysis of flow properties, Alexa Fluor 488 (Invitrogen) was used to visualize perfusion. XZ line scans were taken every 3 min transecting both the local perfusion and a microgroove. Fluorescence intensity was measured from region of interests (ROIs) (50 μm length, 5 μm high) within the microgroove and local perfusion channel close to the glass surface where neurons and their processes attach. Any detectable fluorescence in the microgroove proximal to the postsynaptic cell bodies would indicate diffusion or leakage of perfusate. Mean pixel values were taken from 16-bit images. Fast XT line scans were taken at a rate of 0.00316 seconds per line.
