Figure 2.

Bioluminescence resonance energy transfer performance in deep tissue imaging experiments. (A) Upper panel represents mouse images comparing improvements in the signal output from lungs. Mammalian cells engineered for equivalent over-expression of donor alone (RLuc8 or RLuc8.6) or BRET proteins (mOrange-RLuc8 or TurboFP-RLuc8.6) as marked, were compared. Note the photon output values in the reference color scale bars. Highest signal output from same number of cells placed within lungs was noted with TurboFP-RLuc8.6 BRET protein imaged with Clz substrate. (B) Schematic illustration of the most successful BRET format tested for monitoring the rapamycin induced FRB-FKBP12 association. (C) Representative bioluminescence images of nude mice with accumulated mammalian cells in the lungs which stably over-express FRB and FKBP12 interacting partners fused to RLuc8.6 and TurboFP respectively. Cells (3 × 10⁶ in 150 μL PBS) were injected through the tail vein, resulting in significant trapping in the lungs. One group of mice (n = 8) was injected 2 h before cell injection with 40 μg rapamycin dissolved in 20 μL DMSO and further diluted in 130 μL PBS administered through the tail vein. A second group of mice (n = 8) was injected with DMSO (20 in 130 μL PBS). Two hours after cell injection, the mice were injected i.v. with Clz substrate and sequentially imaged using open/donor/acceptor filters. Substrate-only control mice (n = 4) were used for background subtraction. The figure is partially represented with permission from PNAS (7).