Multiplexed molecular imaging of freshly excised breast tissues with REMI, in which automated DMV staining of a 5-flavor mixture of NPs is employed. Each tissue specimen is stained with an equimolar mixture of HER2-NPs, EGFR-NPs, CD44-NPs, CD24-NPs and isotype-NPs (5 min), followed by a quick rinse in PBS (10 s) and raster-scanned imaging (< 3 min) to simultaneously quantify the expression of four biomarkers: EGFR, HER2, CD44 and CD24. a) Photograph of a human breast tumor and a normal tissue specimen from one patient. b) Ratiometric images of EGFR-NPs vs. isotype-NPs, HER2-NPs vs. isotype-NPs, CD44-NPs vs. isotype-NPs and CD24-NPs. c) Validation data: H&E and IHC for EGFR, HER2, CD44 and CD24. Unlabeled scale bars represent 200 μm. d) Cumulative results from multiple regions of interest from a total of 5 patient specimens: measured NP ratios on IHC-validated biomarker-negative and biomarker-positive tissue regions. Each data point in the plots is the average ratio from one region of interest.