The ratiometric characteristic of BRET approaches allows for drug screening and mechanism/target validation directly in living subjects. The BRET6 system also compares favorably with other systems for imaging PPIs. For example, a split luciferases system (split CBR), with emission maxima at 615 nm, has been recently reported (36). This system could also enable PPIs imaging in small animal models; however, the reassembled CBR luciferase exhibits a relatively low light output, which may be insufficient for applications requiring sensitive detection of PPIs in deep tissues. A direct comparison between the red light-emitting BRET system and the red light-emitting split luciferase system remains to be assessed. Nonetheless, our red light-emitting BRET systems are optimized for deep-tissue imaging and represent an unprecedented development of RET technologies to monitor PPIs directly in disease models in living subjects.