Bioluminescence imaging of human cultured cells transfected with an expanded color palette of bioluminescent proteins (eNLEX). (A) HeLa cells expressing each color of eNLEX. Scale bar: 20 μm. (B) A mixture of cells of each color. Scale bar: 100 μm. (C) Time course observation (hours: minutes). Scale bar: 100 μm. All images were taken with a color CMOS camera. [Image: Nagai et al., Science Advances (2025)]
In the tiny world of living cells, bioluminescent proteins don’t need external light for excitation, unlike their fluorescent cousins. This quality gives bioluminescence the upper hand in experiments requiring precise imaging and tracking of biological processes. But this advantage comes with costs: a limited number of colors and problems with detecting these colors simultaneously.
Now, researchers in Japan have broadened the spectrum of available bioluminescence colors by fusing a type of enzyme called a luciferase with differing amounts of two fluorescent proteins (Sci. Adv., doi: 10.1126/sciadv.adp4750 ). The team captured a single image of 20 different bioluminescence color samples with an ordinary smartphone camera, and the scientists also made time-lapse observations of biological reactions inside mammalian cells.
Fluorescence versus bioluminescence
In fluorescence imaging, researchers typically use six to eight fluorescent molecules as optical markers in cell populations. Since the process requires one or more external light sources, though, scientists often must use computational image processing to sort out the tangle of wavelengths. Bioluminescence imaging eliminates problems with background light as well as potential toxic reactions from cells exposed to too much light. However, before the current study, scientists had not been able to distinguish among more than 10 bioluminescence colors simultaneously in experiments with cells and organelles.
Mitsuru Hattori, Takeharu Nagai and their colleagues at Osaka University realized that complementary metal-oxide semiconductor (CMOS) sensors, such as those found in smartphone cameras, already pick up a vast range of colors. Thus, if the glowing proteins needed for bioluminescence imaging could be tweaked to produce more distinct colors, existing imagers could tell them apart.
Testing the concept
Top: The method for changing bioluminescence color. Bottom: Image of 20 bioluminescence colors, captured by a smartphone camera. [Image: Nagai et al., Science Advances (2025)]
Previous studies had used bioluminescence resonance energy transfer (BRET), an energy exchange involving a fluorescent protein acceptor and a donor enzyme from the luciferase family. The Osaka team fused a particularly bright luciferase with two fluorescent proteins instead of just one to produce dual-acceptor BRET. By choosing the proteins carefully, the researchers could produce multiple color variants that could be imaged together—no need to change optical filters.
To see how dual-acceptor BRET would work outside the test tube, the scientists prepared samples of HeLa cells—from a famous cultured human cell line—containing the 20 different combinations of donors and acceptors. They captured images of the variously colored cells with a CMOS camera attached to a microscope. Finally, Hattori and colleagues transferred the bioluminescence molecules into mouse cells and imaged the resulting colorful patterns just under the animal’s skin.