100 μl/h) (Figure 3B,C). A uniform fluorescence was rapidly established within the channel; notably, there was no fluorescent signal apparent within the microgrooves. To determine the stability of the perfusion stream and to determine, conclusively, if there is any diffusion from the perfusion stream into the microgrooves, we monitored the fluorescence intensity of Alexa Fluor 488 within the chamber over 60 min. We used single-line z scans within a selected region of interest to determine the signal intensity within the perfusion channel and the neighboring microgroove. The regions of interest (ROI) were limited to 3 μm high (i.e., the height of the microgroove). As shown in Figure 3D, the signal within the perfusion channel was stable over an hour and there was no signal detected within the adjacent microgroove ROI over this same time period. We next examined the on/off kinetics to determine the minimum time required to switch perfusion solutions. We found that the perfusate can be added or removed within approximately 1 min (Figure 3E). These results show that we can locally and rapidly perfuse molecules within a neuron-free chamber.
