Bioluminescence is the active luminescence light producing event encountered among diverse group of living organisms in nature, more commonly in deep sea organisms. The event is a result of high‐quantum yield enzyme–substrate catalysis reaction resulting in the production of light devoid of any heat. The chemical reactions behind bioluminescence light emission can be broadly categorised as adenosine triphosphate (ATP)‐dependent and ATP‐independent mechanisms. Bioluminescence identified from firefly beetle is an example of ATP‐dependent photo‐production, whereas in jellyfish and Renilla sp. bioluminescence is an ATP‐independent process. In recent years several new biotechnology applications have used bioluminescence demonstrating their utility as highly sensitive assays for measuring cellular functions in vivo. Often genetic modifications in the luciferase structure or chemical modifications of their substrate have added new functionalities making these assays even more robust. Finally, noninvasive bioluminescence imaging has also evolved as an attractive tool for interrogating human cellular biology in rodent models.

Key Concepts:

  • Bioluminescence is light producing event caused by luciferase enzyme mediated substrate catalysis.

  • Bioluminescence can be co‐factor (e.g. ATP and Ca2+) dependent or independent process.

  • Evolutionary relationship among the structure of various luciferase proteins or their organic chemical substrates are hard to establish, with little or no commonality.

  • Luciferase proteins obtained from diverse organisms are used as bioluminescent reporters because they enable monitoring of intracellular events with high sensitivity, and without destroying the cells or tissues.

  • In biotechnology, mutations can be performed on luciferase gene sequence obtained from nature to make it suitable for use in higher order organisms and/or attribute better characters such as brightness, change spectral properties or protein stability.

  • Bioluminescence is extensively used in basic laboratory experiments for biotechnology and biomedical research, particularly in the format of various assays such as BRET and other diagnostic methods.

  • Noninvasive small animal imaging using luciferase reporter is gaining ground to provide simultaneous qualitative and quantitative measurements of disease interrogation in vivo.

Keywords: luciferase; luciferin; coelenterazine; firefly; Renilla; aequorin; bioluminescence imaging

Figure 1.

The range of colour of bioluminescent systems is represented by these two terrestrial species, exposed by their own bioluminescence. (a) The Australasian glowworm, Arachnocampa (Dipteran), approximately 5 mm in length. (b) The South American railroad worm, Phrixothrix (Coleopteran), approximately 20 mm in length. Reproduced with permission of Vivian and Bechara (), Figure 2D. (c) With external illumination the jellyfish, Aequorea, is approximately 20 cm in diameter. (d) The light organs of Aequorea are distributed in a circle around the edge of the umbrella. Exposed by its own bioluminescence. Photograph by permission of Dr. John Blinks, Friday Harbor Laboratories, University of Washington. © Dr. John Blinks.

Figure 2.

(a) Overall chemical steps in firefly bioluminescence. These reactions occur on FLuc. The asterisk indicates that the molecule is in its first electronic (fluorescent) state. (b) Many marine bioluminescence systems proceed by this overall chemistry, including the crustacean Vargula and the jellyfish Aequorea. For Clz the R substituents are phenyl or benzyl groups. (c) Reduced FMN reacts with oxygen on bacterial luciferase to produce a metastable peroxyflavin on the luciferase. Reaction with an aliphatic aldehyde generates the bioluminescence.

Figure 3.

Live A375 human melanoma cells over‐expressing FLuc and 293T human embryonic kidney cells over‐expressing RLuc was imaged using Olympus LV200 luminescence microscope after addition of respective substrates to the culture media. The image was captured over 300 s without using any filter. The cells were maintained live inside this luminescence microscope while imaging.

Figure 4.

(a) Overlay image of luminescence spectral profile measured from live mammalian cells over‐expressing RLuc8 (X), mOrange‐RLuc8 BRET fusion protein (Z). Wells marked as Y are blank wells. Cells over‐expressing these proteins were exposed to live cell substrate and emission profiles were imaged using IVIS spectrum imaging system loaded with 20 nm band pass filters between 460 and 720 nm. (b) The chart below represents values of light signal quantitated from wells marked as X and Z. Note the spectral gain in signal in the BRET fusion expressing cells at 560±10 nm filter, characteristic wavelength where the acceptor monomeric orange (mOrange) fluorescent protein emits. RLuc8 is a genetically engineered mutant RLuc selected for BRET partnering.

Figure 5.

Noninvasive serial imaging of luciferase signal time kinetics in representative mice with either tumour xenograft (upper panel) or metastatic tumours in the lungs (lower panel) by implanting with HT1080 human fibrosarcoma cells over‐expressing RLuc reporter. These mice were injected with 30 µg of ‘live cell’ Clz substrates and scanned repeatedly over time as marked. Serial images were scaled to single pseudocolor scale bar representing average radiance (Photon sec−1 cm−2 sr−1).



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Further Readings

Lubov B (2007) Bioluminescence for food and environmental microbiological safety. Tutorial Texts Series, vol. TT74, 75 pp. Bellingham, Washington, USA: SPIE. ISBN 978‐0‐8194‐6643‐3.

Shimomura O (2012) Bioluminescence: Chemical Principles and Methods, pp. 468. Singapore: World Scientific Publishing Co. Pte. Ltd. ISBN‐13:978–9814366083.

Stanley PE and Kricka LJ (2002) Bioluminescence & Chemiluminescence: Progress & Current Applications, pp. 530 Singapore: World Scientific Publishing Co. Pte.Ltd. ISBN 981‐238‐156‐2.

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De, Abhijit(Jul 2014) Bioluminescence. In: eLS. John Wiley & Sons Ltd, Chichester. [doi: 10.1002/9780470015902.a0001412.pub2]