Abstract: An automated method for identifying the precise location of each sub-array and dot of a micro-array is provided. The sub-arrays of the micro-array are located using a template sub-array. The estimated location of each sub-array is estimated according to the expected physical dimension of the sub-array. A search of each sub-array is conducted for the dots of each sub-array. A constraining shape mask is constructed, and a segmentation method is applied to detect the location of each dot within the collection of pixels. The constraining shape mask is applied to each detected dot, limiting the boundary of and precisely identifying the location of each dot.