Abstract: Methods and apparatus for detecting polynucleotide hybridization in luminescence-based assays. The methods may include (1) contacting a sample polynucleotide with a reference polynucleotide at an assay site, where at least one of the polynucleotides is capable of emitting luminescence, (2) illuminating the assay site with light capable of stimulating such luminescence, (3) detecting light transmitted from the assay site, and (4) deriving information relating to the extent of hybridization between the sample and reference polynucleotides based on the detected light. The methods may further include (1) illuminating with and/or detecting polarized light, (2) deriving information relating to the sequence of the sample polynucleotide from the extent of hybridization, and (3) converting the light to a signal and distinguishing between a portion of the signal attributable to luminescence and a portion attributable to background.