Colour palette expands for bioluminescent cell imaging

Imaging live cells has long been a crucial technique for understanding cellular behaviour — but while bioluminescent proteins offer several advantages, the limited availability of colour variants has made it difficult to observe multiple targets simultaneously. Now, researchers from SANKEN (The Institute of Scientific and Industrial Research) at Osaka University have found a way to expand the colour palette of bioluminescent protein to 20 distinct colours, enabling advanced simultaneous multicolour imaging.

Understanding how cells function is essential for progress in biological sciences, medicine and drug discovery. Optical labelling techniques allow scientists to observe cell behaviour, track cell fate, and identify cells with specific traits. While fluorescent proteins are widely used for these purposes, bioluminescent proteins are gaining popularity due to certain advantages.

Bioluminescence — the natural emission of light by living organisms — is powered by a chemical reaction catalysed by an enzyme, typically a luciferase, acting on a bioluminescent substrate. Unlike fluorescent proteins, bioluminescent proteins do not require external light for excitation, avoiding issues like phototoxicity and background light. However, their use has been limited by the small number of available colours, and having distinct and easily distinguishable colours is vital to tracking multiple targets simultaneously.

Previously, a five-colour series of bioluminescent labels was created by coupling one of the brightest luciferases, NanoLuc, with a fluorescent protein. This technique leverages the transfer of excited-state energy from the substrate to the fluorescent protein, altering the bioluminescence colour; but while it is effective, the five-colour palette was insufficient for more complex imaging needs. The Osaka researchers have now addressed this challenge by expanding the bioluminescent colour palette to 20, with their results published in the journal Science Advances.

“Instead of fusing NanoLuc with single fluorescent protein, we fused it with two,” said lead author Mitsuru Hattori. “This approach allowed us to access a much broader range of bioluminescence colours by fine-tuning the combinations of fluorescent proteins.”

The researchers achieved an impressive milestone with their new bioluminescent protein labels. They captured a single-shot image of a mixture of cells expressing all 20 bioluminescent proteins, used the labels to visualise distinct subcellular components, and even demonstrated their capability in live mice. Additionally, they successfully conducted time-lapse observations of cell behaviour over several hours, simultaneously tracking seven distinct labels.

“What’s truly exciting is that we could detect all 20 colours simultaneously without any time lag, using a standard smartphone camera,” said senior author Takeharu Nagai. “This innovation makes it significantly easier and more cost-effective to monitor multiple targets or track individual cells within a population.”

The newly developed bioluminescent colours have the potential to revolutionise cell fate tracking, offering insights concerning how cells develop into specific cell types and identifying cells with unique responses to drugs. The team’s breakthrough in bioluminescent imaging thus opens new doors for advancements in biological research, drug discovery and medical science.

Image caption: Bioluminescence imaging of human cultured cells transfected with the expanded colour palette (scale bar = 100 μm). Image credit: 2025 Nagai et al, ‘Creating coveted bioluminescence colors for simultaneous multi-color bioimaging’, Science Advances