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A simple method for fabricating nanoscale channels based on thermomechanical deformation of rigid polymer substrates is described. The process uses thermoplastic preforms containing microchannels with cross-sectional dimensions on the order of tens of microns, which are controllably deformed to produce sub-micron dimensions. Nanochannels with circular or elliptical cross-sections are defined in polycarbonate by varying the channel position and preform geometry prior to deformation. Arrays of parallel nanochannels with critical dimensions down to 400 nm are described. Using the fabrication method, a nanochannel network is fabricated for the detection of single protein molecules via confocal fluorescence microscopy, with channel dimensions matched to the confocal volume dimensions of the detection objective. Single molecule measurements of fluorescently-labeled bovine serum albumin at 15 nM and 150 nM concentrations are presented.