High aspect-ratio microstructures were hot-embossed in polymer substrates with a molding tool fabricated using lithography/electroplating/forming (LIGA). The resulting devices were used for the electrophoretic separation of oligonucleotides labeled with near-infrared (near-IR) dyes. Near-IR time-resolved fluorescence was used as an identification method for the labeling dyes. The detection apparatus consisted of a pulsed laser diode operating at 680 nm, a single-photon avalanche diode, an integrated microscope, and a PC-board incorporating time-correlated single photon counting electronics. Investigation of the optical quality and amount of autofluorescence generated from different polymer substrates was carried out in the near-IR region for determining compatibility with time-resolved fluorescence. Our results revealed that of several poly(methylmethacrylate) (PMMA) substrates, brand Plexiglas' offered minimal replication errors in the embossed features using appropriate embossing conditions with low background fluorescence contributions to the observed decay. Near-IR dye-labeled oligonucleotides were separated to determine the applicability of fluorescence lifetime discrimination between Cy5.5 (tau(f) = 930 ps) and IRD700 (tau(f) = 851 ps) labeling dyes during the microchip separation. These dyes were used to label T-fragments (thymine) of an M13mp18 ssDNA template. The DNA ladders were electrophoresed at 130 V/cm in a 4% linear polyacrylamide gel (LPA) gel matrix in a 9.5 cm long serpentine channel heated to 50degreesC. The electropherogram revealed that the lifetimes could be accurately read well beyond 450 bases, although single-base pair resolution in the electropherogram was difficult to achieve due to potential solute-wall interactions in the polymer microdevice or the electroosmotic flow (EOF) properties of the device. The relative standard deviations secured for individual bands in the electropherogram were similar to those obtained using capillary gel electrophoresis, in spite of the lower load volume.