Assay apparatuses, methods and reagents

Apparatuses, systems, method, reagents, and kits for conducting assays as well as process for their preparation are described. They are particularly well suited for conducting automated analysis in a multi-well plate assay format. A method is provided for focusing an optical sensor to a spaced apart platform comprising the steps of: providing at least a higher, middle and lower patterned surface, wherein the middle patterned surface and the platform are aligned to each other and wherein a first distance between the higher and middle patterned surfaces and a second distance between the middle surface and lower patterned surface are substantially equal; obtaining a first difference in contrast values between the higher and middle patterned surfaces with the optical sensor; obtaining a second difference in contrast values between the middle and lower patterned surfaces with the optical sensor; and comparing the first and second differences in contrast values.