This work reports the development of iterative indirect immunofluoresence imaging (4i), which allows 40-plex immunostaining of hundreds of thousands of single cells using conventional primary and secondary antibodies in high-throughput. Combined with cellular computer vision, this generates a wealth of quantitative information about biological samples from the millimeter to the nanometer scale. It also reports a novel pixel-based computational approach that allows the unsupervised comprehensive quantification of protein sub-compartmentalization within various multicellular, cellular, and pharmacological contexts. These approaches have enormous potential for biomedicine in both fundamental research, diagnostics and personalized medicine.