E2-Orange is useful as a second or third color. (A) Shown is a dot plot generated by flow cytometry of S. cerevisiae cells expressing E2-Orange, EGFP, or no FP. The three strains were grown separately, then pooled at equivalent cell concentrations and analyzed. Cells were excited using a 488-nm laser, and orange and green fluorescence signals were detected using PE and FITC filter sets, respectively. On the right are the same data with E2-Orange-expressing cells boxed in orange and EGFP-expressing cells boxed in green. (B) A three-color yeast strain was generated with GFP-labeled Golgi, E2-Orange-labeled cytosol, and mCherry-labeled mitochondria. Cells were imaged using confocal microscopy. E2-Orange and GFP were immediately resolvable, and E2-Orange and mCherry were resolvable after linear unmixing, yielding clear three color images as shown in the overlay.