The physical morphology and chemical functionality of fluid microchannels formed in poly(methyl methacrylate) substrates were studied to increase the fundamental understanding of polymer microchannel surface properties for 'lab-on-a-chip' devices.  Microchannels were formed by a hot imprint method using a silicon template or by a laser ablation process (248nm KrF laser) operating at low to moderate fluence levels (up to 1180 mJ/cm2).  The carboxylate groups, which are responsible for the surface charges, were fluorescently labeled by reaction with an ethyl-dimethylaminopropyl-carbodiimide hydrochloride/amino-fluorescein solution.  Fluorescence microscopy was then used to locate and measure qualitatively the charge present on the microchannel walls.  Results suggest that surface charges are localized on the corners of trapezoidal channel formed by the hot imprint method and that the amount of charge present is significantly less compared with laser-ablated microchannels where charges appear to be distributed uniformly.  For substrates irradiated at fluences above the laser ablation threshold, it was found that one pass of the laser produced a surface with greater charge than channels made with multiple passes, that ablation under nitrogen resulted in more charge than ablation under oxygen, and that non-sonicated substrates had more charge than samples that were sonicated after ablation.  Finally, we have determined that the surface charge on the substrate can be modified by using the laser at fluence levels lower than those required to ablate the substrate.