Autonomous multicolor bioluminescence imaging in bacteria, mammalian, and plant hosts

Bioluminescence imaging has become a valuable tool in biological research, offering several advantages over fluorescence-based techniques, including the absence of phototoxicity and photobleaching, along with a higher signal-to-noise ratio. Common bioluminescence imaging methods often require the addition of an external chemical substrate (luciferin), which can result in a decrease in luminescence intensity over time and limit prolonged observations. Since the bacterial bioluminescence system is genetically encoded for luciferase-luciferin production, it enables autonomous bioluminescence (auto-bioluminescence) imaging. However, its application to multiple reporters is restricted due to a limited range of color variants. Here, we report five-color auto-bioluminescence system named Nano-lanternX (NLX), which can be expressed in bacterial, mammalian, and plant hosts, thereby enabling auto-bioluminescence in various living organisms. Utilizing spectral unmixing, we achieved the successful observation of multicolor auto-bioluminescence, enabling detailed single-cell imaging across both bacterial and mammalian cells. We have also expanded the applications of the NLX system, such as multiplexed auto-bioluminescence imaging for gene expression, protein localization, and dynamics of biomolecules within living mammalian cells.