Multiple orthogonal pairs can be rapidly unmixed. Established reporters examined include firefly luciferase (Fluc), NanoLuc (Nluc), and Gaussia luciferase (Gluc). The corresponding luciferins for each reporter are shown. Gradients of the luciferases were plated as shown. The corresponding substrates ([d-luc] = 100 μM, [FRZ] = 1:40 of commercial stock, [CTZ] = 100 μM) were administered, beginning with the dimmest luciferin. Images were acquired after each addition. The raw data were stacked, unmixed, and false colored.