Semisynthetic, multicolor probes for bioluminescence imaging.

Bioluminescence imaging is a sensitive and noninvasive technique for monitoring molecular and cellular processes in living systems. NanoLuc is one of the brightest and most widely used luciferase enzymes, however, the native blue emission of NanoLuc limits its applications due to poor sample penetration. NanoBiT, a split luciferase reporter derived from NanoLuc, consists of two subunits: LgBiT and HiBiT. Herein, we take advantage of bioluminescence resonance energy transfer (BRET) to red-shift the emission of NanoBiT by chemically labeling the N-terminus of HiBiT with fluorescent probes. Upon association of HiBiT and LgBiT, the appended fluorophores emit red-shifted light. When LgBiT is genetically encoded on the surface of mammalian cells, each probe produces unique emission profiles that can be distinguished by spectral phasor analysis microscopy. Moreover, by conjugating HiBiT peptides to fluorescent quantum dots, we extended BRET emission into the near-infrared. This modular system allows easy synthesis, rapid color switching, and tunable emission across the visible to near-infrared spectrum, providing a versatile platform for multicolor bioluminescence imaging across biological scales.