Confocal microscopy imaging of primary BM HSPCs cotransduced with 5FP lentiviral vectors. (A) A schematic overview of the transplantation of LeGO-transduced murine HSPCs. Donor Lin⁻ HSPCs were harvested, transduced in vitro with all 5FP vectors concurrently (Co) as shown in the schematic, or individually and then mixed (Mix), and transplanted into lethally irradiated recipient mice or continued in culture for flow cytometric, CFU, and imaging analyses. Tissues from transplanted recipients were removed at specific time points for imaging. (B) Lin⁻ cells co-transduced with the 5 LeGO vectors and imaged 4 days after transduction, showing a merged tiled-3D image of highly diverse color marking of individual cells, resulting from the variety of vector combinations transducing each cell. (C) Higher magnification view of the boxed area from the image shown in panel B; line intensity profiles were drawn manually through the center of 8 cells, generating 5-channel intensity histograms demonstrating unique spectral “colorprints” to unequivocally distinguish each transduced cell. (D) Cells from panel B were segmented as spot objects using Imaris software, and the MFI/spot in each of the 5 channels was quantitated and displayed as stacked columns, either as (i) absolute values (AU arbitrary units) or as (ii) percent relative contributions of the 5 FP channels. This graphically and quantitatively demonstrates the huge diversity of spectral identity for individual Lin⁻ cells. Original raw data for the first 100 spots are shown in supplemental Table 1. Scale bar represents 100 μm.