Advances in bioluminescence imaging: new probes from old recipes
Introduction
Bioluminescent enzymes (luciferases) are among the most sensitive probes for imaging in thick tissues and whole organisms [1]. Luciferases catalyze light emission via the oxidation of small molecule substrates (luciferins). Since no external light is required, the background emission is virtually zero, enabling sensitive imaging in vivo. Bioluminescence has long been used to track cells, gene expression, and other biological features in tissues and whole organisms [2]. The emitted light is inherently weak, though, compared to conventional fluorescent tools. For this reason, luciferases are typically used in conjunction with fluorescent reporters. The bioluminescent enzymes survey processes on the macro scale and in heterogeneous environments. The fluorescent probes capture events at the micro scale or ex vivo — environments where excitation light is more efficiently delivered.
Historically, the most popular bioluminescent reporter for imaging in vivo has been firefly luciferase (Fluc). This enzyme emits the largest percentage of tissue-penetrant light with its cognate luciferin (d-luciferin, Figure 1a) [3]. Other luciferases, including Renilla luciferase (Rluc) and Gaussia luciferase (Gluc) have also found broad utility in biological research [4]. These enzymes oxidize coelenterazine and emit blue light in the process. Rluc and Gluc require no additional cofactors (other than oxygen), making them well suited for extracellular work. Compared to their fluorescent protein counterparts, though, luciferases have been less frequently employed in bioimaging studies. Fewer bioluminescent probes have been developed and even fewer have been optimized for application in vivo. There is a constant demand for more bioluminescent colors, improved enzymes, and more biocompatible substrates.
Advances in protein engineering and chemical syntheses are addressing voids in the bioluminescent toolbox. The past few years, in particular, have seen an uptick in the number of sensitive and substrate-selective luciferases available for use. Much of the progress mirrors trends in fluorescent protein development, including identifying mechanistically distinct probes in nature and subsequently evolving for new function [5]. Systematic efforts to engineer fluorescent probes for altered colors of emission, photo-switching capabilities, and other features ultimately enabled new studies in biology. This iterative cycle of tool development and biological discovery is similarly driving the field of bioluminescence. Below we highlight recent efforts to discover and evolve new bioluminescent tools, and showcase their application to biological sensing.
Section snippets
Discovering new luciferases and luciferins
Thousands of luminescent species exist in the natural world, but only a fraction of the associated luciferases and luciferins have been characterized in detail [4, 6]. Even fewer have been coopted for use in heterologous systems [1]. Continued efforts to mine new luciferase and luciferin architectures from natural sources are expanding the number of available tools. For example, the luciferase gene from Photinus scintillans was recently cloned [7]. P. scintillans emits predominantly orange
Generating a palette of bioluminescent probes
The discovery and characterization of native bioluminescent systems, while important, have often not kept pace with the demand for user-friendly imaging tools. Thus, efforts to engineer bioluminescent probes with desirable properties have been critical to fill voids in the imaging toolbox. Many of the approaches have mirrored those in fluorescent protein development: mutagenesis and screening for desired properties such as thermostability, turnover, and color. Some of the most impactful
Engineering orthogonal luciferase-luciferin pairs
Discriminating among wavelengths in vivo is challenging, as the perceived color changes with depth. Multi-component bioluminescence imaging has thus been most often achieved using substrate-resolved luciferases versus spectrally resolved pairs. For example, Fluc and Rluc oxidize completely different luciferins and can therefore be readily distinguished in two-component assays [31]. The Fluc/Rluc combination has further inspired the expansion of orthogonal bioluminescent tools. Unique patterns
Monitoring new facets of biology
Advances in luciferase engineering have ushered in a flurry of new sensors for metabolites and enzyme activities [44, 45, 46, 47]. Many of these probes have parallels to classic fluorescent sensors, but are more tailored for in vivo work. A notable example is CalfluxVTN, a BRET-based calcium sensor comprising Nluc and Venus fluorescent protein (Figure 4a). In the absence of Ca2+, Nluc emission is observed. Upon Ca2+ binding, the sensor undergoes a conformational change and BRET is observed.
Conclusions and future directions
Many advances in bioluminescent probe technology have mirrored trends in fluorescent probe development. Dozens of luciferases have been evolved for new functions via iterative mutagenesis and screening. Collections of robust and structurally distinct luciferins have also been synthesized. A variety of unique bioluminescent mechanisms have further been uncovered in the natural world, providing platforms from which to craft new tools. The continued discovery and development of bioluminescence
References and recommended reading
Papers of particular interest, published within the period of review, have been highlighted as
• of special interest
•• of outstanding interest
Acknowledgements
This work was supported by a grant from the National Institutes of Health (R01-GM107630 to J.A.P.). B.S.Z. was supported by a GAANN Fellowship and Z.Y. was supported by the BEST IGERT program (National Science FoundationDGE-1144901). We thank members of the Prescher lab for helpful discussions.
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These authors contributed equally.