Described herein are methods for co-expression analysis of multiple markers in a tissue sample comprising: computing a heat map of marker expression for each of a plurality of single marker channel images, wherein each of the plurality of single marker channel images comprise a single marker; identifying one or more candidate regions of interest in each heat map of marker expression; computing overlay masks comprising the identified one or more candidate regions of interest from each heat map of marker expression; determining one or more co-localized regions of interest from the overlay masks; mapping the one or more co-localized regions of interest to a same coordinate position in each of the plurality of single marker channel images; and estimating a number of cells in at least one of the determined one or more co-localized regions of interest in each of the plurality of single marker channel images.