An additional advantage of microfluidic devices is the ability to create multiple distinct microenvironments. This is particularly relevant for the study of neurons which have long processes forming synapses in multiple microenvironments or compartments. Using the syringe pump-based flow within the perfusion channels and the virtual boundaries set up with the outer streams of buffer, we can also create distinct microenvironments for post- and pre-synaptic compartments as well as local synaptic regions. To illustrate this feature, we added three different wavelength fluorescent dyes (Alexa Fluor 488, Alexa Fluor 568, and Alexa Fluor 633) to the postsynaptic, perfusion channel and pre-synaptic compartments and imaged them after 30 min of perfusion, showing that the separation of the microenvironments is stable (Figure 4E). We expected that dye from the pre- and postsynaptic compartments would enter the microgrooves but that the buffer streams on either side of the perfusion stream would provide a barrier to maintain the isolation of the fluid microenvironments– this was indeed the case. In addition, the Alexa Fluor 488 dye was restricted only to the postsynaptic compartment. The Alexa Fluor
