A system configured to acquire and process image data, comprising: an imaging subsystem, a storage medium comprising program instructions which are executable by a processor for: storing data acquired for multiple images of particles, wherein each of the multiple images corresponds to a different wavelength band; selecting a reference image from one of the multiple images; and manipulating the coordinates of at least one of the multiple images that is not the reference image such that spots corresponding to the particles within each of the multiple images align with spots in the reference image, characterized in that the imaging subsystem is configured to: - image, at different wavelength bands, particles disposed within the imaging subsystem; - and acquire data corresponding to emissions of imaged particles; wherein the program instructions for manipulating the coordinate parameters comprise program instructions for: - adjusting coordinates of at least one image by a predetermined offset; - computing an aggregate error or mean squared difference among locations of spots within the different images; - repeating the steps of adjusting the coordinates of the at least one image and computing the aggregate error or mean squared difference of spot locations with different predetermined offsets for a predetermined number of iterations; - comparing the computed values of aggregate error or mean squared difference of the spot locations for each of the different adjusted coordinates of the at least one image; and - saving offset values for each image having adjusted coordinates corresponding to the set of images having the smallest aggregate error or mean squared difference of spot locations. an imaging subsystem, a storage medium comprising program instructions which are executable by a processor for: storing data acquired for multiple images of particles, wherein each of the multiple images corresponds to a different wavelength band; selecting a reference image from one of the multiple images; and manipulating the coordinates of at least one of the multiple images that is not the reference image such that spots corresponding to the particles within each of the multiple images align with spots in the reference image, characterized in that the imaging subsystem is configured to: - image, at different wavelength bands, particles disposed within the imaging subsystem; - and acquire data corresponding to emissions of imaged particles; - image, at different wavelength bands, particles disposed within the imaging subsystem; - and acquire data corresponding to emissions of imaged particles; wherein the program instructions for manipulating the coordinate parameters comprise program instructions for: - adjusting coordinates of at least one image by a predetermined offset; - computing an aggregate error or mean squared difference among locations of spots within the different images; - repeating the steps of adjusting the coordinates of the at least one image and computing the aggregate error or mean squared difference of spot locations with different predetermined offsets for a predetermined number of iterations; - comparing the computed values of aggregate error or mean squared difference of the spot locations for each of the different adjusted coordinates of the at least one image; and - saving offset values for each image having adjusted coordinates corresponding to the set of images having the smallest aggregate error or mean squared difference of spot locations. - adjusting coordinates of at least one image by a predetermined offset; - computing an aggregate error or mean squared difference among locations of spots within the different images; - repeating the steps of adjusting the coordinates of the at least one image and computing the aggregate error or mean squared difference of spot locations with different predetermined offsets for a predetermined number of iterations; - comparing the computed values of aggregate error or mean squared difference of the spot locations for each of the different adjusted coordinates of the at least one image; and - saving offset values for each image having adjusted coordinates corresponding to the set of images having the smallest aggregate error or mean squared difference of spot locations.