Confocal microscopy approach for imaging cells transduced with LeGO vectors encoding 5FPs. NIH3T3 cells transduced with LeGO vectors encoding single FPs were used to define imaging parameters. (A) Spectral (xyλ) imaging was used to record reference emission spectra for each FP, illustrated in normalized histograms pseudo-colored with cyan (Cerulean), green (EGFP), yellow (Venus), magenta (tdTomato), and red (mCherry). The mCherry spectrum red shifts (12 nm at peak) when excited by 594 nm (solid red line) compared with 561 nm (dashed red line) wavelength. (B) Five channels were set to image sequentially: Cerulean (458 emission [em] 468-482), EGFP (488 em 496-514), Venus (514 em 523-558), tdTomato (561 em 579-597), and mCherry (594 em 618-670). (C) Imaging of NIH3T3 cells transduced with individual FP vectors. Each FP was visible only in the cells transduced with the corresponding vector imaged in the appropriate channel, and absent from the others (no crosstalk). (D-E) Populations of NIH3T3 cells transduced independently with the 5 LeGO vectors and then mixed (Mix, D) or NIH3T3 cells transduced with all 5 vectors concurrently (Co, E), plated and imaged 4-5 days later. (D) The 5 individual FPs are shown pseudo-colored cyan (Cerulean), green (EGFP), yellow (Venus), magenta (tdTomato), and red (mCherry). (E) Clusters of progeny cells inheriting a specific color within a diverse palette of colors based on combinations of 1-5 FPs at variable intensities in each originally transduced cell. Progeny inherit the specific color, allowing tracking of lineage relationships (related images; supplemental Videos 1 and 2). Scale bar represents 100 μm (D-E).