Tools like firefly and Renilla luciferase are commonly used in diverse assays. However, autocatalytic fluorescent proteins such as GFP still dominate in most imaging applications, especially those involving microscopy. Fluorescent proteins have the advantages of being independent of cofactor for fluorescence and having emission spectra that are narrow enough for straightforward multicolor imaging.

A diverse palette of bioluminescent tools will enable gentle imaging, especially in animals. Credit: Marina Corral Spence/Springer Nature

However, luciferases offer complementary advantages. Because these probes are not excited by light, bioluminescence imaging has minimal phototoxicity and succeeds at greater depths within tissue than imaging with fluorescent proteins. Bioluminescence is also uniquely suited to imaging in freely moving organisms. For these reasons, there has been a push in recent years to develop improved luciferases and luciferins.

One advance has come in the form of ‘nanolanterns’, which fuse luciferases with fluorescent proteins for bioluminescent resonance energy transfer to combine the power of luciferases with the spectral benefits and brightness of fluorescent proteins (Nat. Commun. 3, 1262; 2012). Other groups have engineered improved luciferases that have higher substrate turnover, are brighter, or are color-shifted relative to their original versions. Alongside these developments, luciferins have been developed for optimal brightness, solubility, affinity, and color.

Perhaps the most useful of these combinations emit light in the near-infrared range, where tissues are most transparent (Nat. Commun. 7, 11856, 2016; Nat. Methods 14, 971–974, 2017). A stunning example of the power of these tools showed that an all-engineered, red-shifted luciferase–luciferin pair could be used to visualize individual tumor cells in the lungs of mice and to detect small numbers of neurons in freely behaving marmosets (Science 359, 935–939; 2018).

Another push to increase the versatility of luciferases depends on making them fully genetically encoded. This requires identification and expression of the enzymes involved in the synthesis of their luciferins—a challenging prospect because many aspects of these synthetic pathways are poorly understood. Fully genetically encoded bacterial luciferase systems have been developed (Proc. Natl Acad. Sci. USA 115, 962–967; 2018), and headway made in this direction for other luciferases will greatly enable biological studies. Continued development of luciferases and their substrates will transform these tools into nearly ideal probes, especially for gentle imaging in living mammals.