Creating coveted bioluminescence colors for simultaneous myriad-color bioimaging

Abstract Bioluminescence, an optical marker that does not require excitation by light, allows researchers to simultaneously observe multiple targets, each exhibiting a different color. Notably, the colors of the bioluminescent proteins must sufficiently vary to enable simultaneous detection. Here, we aimed to introduce a method that can be used to expand the color variation by tuning dual-acceptor bioluminescence resonance energy transfer (BRET). Using this approach, we could visualize multiple targets with up to 20 colors through single-shot acquisition using a color CMOS camera. Overall, this method enables simple and simultaneous observation of numerous biological targets and phenomena. Competing Interest Statement The authors have declared no competing interest. Copyright The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.