1. A method of registering an analytical image of a microsphere array comprising: a) providing a microsphere array, wherein said microsphere array comprises: i) a substrate with a surface comprising discrete sites; and ii) a population of microspheres comprising at least a first and a second subpopulation each comprising a bioactive agent, wherein said population of microspheres is distributed on said surface such that each of said discrete sites contain no more than 1 microsphere; b) decoding said microsphere array so that the location of each bioactive agent is elucidated; c) computing a registration grid based on known locations of said bioactive agents from information obtained from step (b); d) contacting said array with a sample comprising target analytes; e) acquiring a hybridization intensity image of said microsphere array, wherein said hybridization intensity image comprises a first bright bead type to serve as a fiducial; f) overlaying the registration grid onto said hybridization intensity image; g) determining an average intensity for the first bright bead type; and h) positioning the registration grid such that the average intensity of the first bright bead type is maximal, thereby registering the analytical image of the microsphere array.