Five colour variants of bright luminescent protein for real-time multicolour bioimaging

Luminescence imaging has gained attention as a promising bio-imaging modality in situations where fluorescence imaging cannot be applied. However, wider application to multicolour and dynamic imaging is limited by the lack of bright luminescent proteins with emissions across the visible spectrum. Here we report five new spectral variants of the bright luminescent protein, enhanced Nano-lantern (eNL), made by concatenation of the brightest luciferase, NanoLuc, with various colour hues of fluorescent proteins. eNLs allow five-colour live-cell imaging, as well as detection of single protein complexes and even single molecules. We also develop an eNL-based Ca²⁺ indicator with a 500% signal change, which can image spontaneous Ca²⁺ dynamics in cardiomyocyte and neural cell models. These eNL probes facilitate not only multicolour imaging in living cells but also sensitive imaging of a wide repertoire of proteins, even at very low expression levels.