To prevent the perfusate from diffusing into the microgrooves, we designed a 3-inlet perfusion channel. In this design, the 2 outer inlets are filled with normal media, resulting in 3 fluid streams entering the perfusion channel. The 2 outer streams serve as virtual fluid barriers preventing diffusion of the perfusate into the microgrooves (Figure 4A). Fluorescence and DIC images of the perfusion are shown in Figure 4B. Another advantage of the 3-inlet design is that the width of the perfusion spot can be reduced considerably, owing to the streams of normal media that flank the perfusate on either side. For example, within microgrooves near the entrance of the perfusion, we were able to restrict the width of the perfusate to a minimum of 10 μm (Figure 4B-C). The z profile of a perfusion spot is shown in Figure 4C. Importantly, perfusion within a neuron-filled chamber shows no evidence of diffusion into the microgrooves. This demonstrates that the microfluidic local perfusion (μLP) chamber incorporating a 3-inlet design prevents diffusion of the perfusate into the microgrooves and can be used to apply pharmacological agents to focal synaptic regions present within the channel.
