A new rainbow of colour for bioluminescence imaging

d-luciferin and several luciferin 

analogues synthesized that show 

varying colours of emitted light.

Luciferins are a class of molecules that are oxidized by the luciferase enzymes (for example firefly luciferase), producing oxyluciferin and energy which is released in the form of a photon of light (termed bioluminescence).[1]  Bioluminescence is a very sensitive imaging technique, making it one of the most popular methods for visualizing biological processes in vivo, especially in cancer biology research.[1]  Bioluminescencent imaging is preferred over the fluorescent counterpart because no external light source is required,[2] and the lack of endogenous bioluminescent reactions in mammalian tissue allows for near background-free imaging conditions.[3]  Unfortunately, luciferin-based bioluminescence imaging has been limited to monitoring one cell type or feature at a time, as nearly all the enzymes act on the same substrate (D-luciferin).  Additionally, light of wavelengths below 600 nm is absorbed and scattered by cells, which restricts the application of this technique to only superficial tissue depths.[1]

A range of luciferin analogues have been synthesized which show excellent bioluminescence properties and great potential in cell and tissue imaging. This series of luciferin analogues which absorb at different wavelengths has raised the possibility of multicomponent imaging using multiple colours.  Additionally, several of the analogues display red-shifted emission (>600nm) which give their signal better tissue penetration properties.

Researchers led by Stephen Miller from the University of Massachussets Medical School synthesized four alkylaminoluciferin substrates, which showed red-shifted and more intense light emissions than D-luciferin.[4]  They have also engineered several luciferase mutants that yield improved sustained light emission with aminoluciferins in both lysed and live mammalian cells.[5]

More recently, the Stanford University lab of William Moerner developed an analogue with a selenium atom in place of the native sulfur atom at position 1.  The resulting selenoluciferin emits 55% of its light above 600 nm.[6]

Soon after, Jennifer Prescher and co-workers at the University of California developed two further types of luciferin analogues, replacing the sulfur in either of the two heteroaromatic rings with nitrogen.[7]  One compound specifically shows the highest blue-shift of any luciferins.

Depending on the substitution pattern, the luciferin-emitted light can span a broad range, from deep in the red (>600 nm) up to bright blue (around 460 nm).  This is dependent on the identity and nature of the atoms that are substituted – for example, the more strongly electron-donating nature of the alkylamino group was hypothesized to red-shift the spectral properties.  The polar effect of the selenium atom was also predicted to red-shift the emission maximum; both assumptions turned out to be correct.  

While a palette of luciferin colours has now been developed, many of the analogues are still not ideal substrates.  The alkylaminoluciferins show a significant reduction in light output compared to D-luciferin, consistent with product inhibition and hence lower rate of enzymatic turnover.[4]  The selenocysteine analogue also has reduced light output, partly as a result of lower quantum yield.[6]  Some analogues synthesized displayed very limited or even no bioluminescence, making them of little use for imaging studies.[7]  Further tweaking of their structure will be required before luciferin-based multicomponent imaging is possible.

Background

Luciferases and Fluorescent Proteins: Principles and Advances in Biotechnology and Bioimaging 2007, V. R. Viviani, Y. Ohmiya (eds). Transworld Research Network, 2007.

References

[1] Y.-Q. Sun, J. Liu, P. Wang, J. Zhang, W. Guo. d-Luciferin analogues: a multicolour toolbox for bioluminescence imaging.  Angew. Chem. Int. Ed. 2012, 51(34), 8428-8430

[2] M. Baker. A broader palette for luciferase.  Nat. Methods. 2012, 9(3), 225.

[3] D. M. Close, T. Xu, G. S. Sayler, S. Ripp.  In vivo bioluminescent imaging (BLI): noninvasive visualization and interrogation of biological processes in living animals.  Sensors 2011, 11(1), 180-206.

[4] G. R. Reddy, W. C. Thompson, S. C. Miller.  Robust light emission from cyclic alkylaminoluciferin substrates for firefly luciferase.  J. Am. Chem. Soc. 2010, 132(39), 13586-13587.

[5] K. R. Harwood, D. M. Mofford, G. R. Reddy, S. C. Miller.  Identification of mutant firefly luciferases that efficiently utilize aminoluciferins.  Chem. Biol. 2011, 18(12), 1649-1657.

[6] N. R. Conley, A. Dragulescu-Andrasi, J. Rao, W. E. Moerner.  A selenium analogue of firefly d-luciferin with red-shifted bioluminescence emission.  Angew. Chem. Int. Ed. 2012, 51(14), 3350-3353.

[7] D. C. McCutcheon, M. A. Paley, R. C. Steinhardt, J. A. Prescher.  Expedient synthesis of electronically modified luciferins for bioluminescence imaging.  J. Am. Chem. Soc. 2012, 134(18), 7604-7607.