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Abstract
PurposeCurrently, a variety of red and green beetle luciferase variants are available for bioluminescence imaging (BLI). In addition, new luciferin analogues providing longer wavelength luminescence have been developed that show promise for improved deep tissue imaging. However, a detailed assessment of these analogues (e.g., Akalumine-HCl, CycLuc1, and amino naphthyl luciferin (NH2-NpLH2)) combined with state of the art luciferases has not been performed. The aim of this study was to evaluate for the first time the in vivo brightness and spectral characteristics of firefly (Luc2), click beetle green (CBG99), click beetle red 2 (CBR2), and Akaluc luciferases when paired with different d-luciferin (d-LH2) analogues in vivo.ProceduresTransduced human embryonic kidney (HEK 293T) cells expressing individual luciferases were analyzed both in vitro and in mice (via subcutaneous injection). Following introduction of the luciferins to cells or animals, the resulting bioluminescence signal and photon emission spectrum were acquired using a sensitive charge-coupled device (CCD) camera equipped with a series of band pass filters and spectral unmixing software.ResultsOur in vivo analysis resulted in four primary findings: (1) the best substrate for Luc2, CBG99, and CBR2 in terms of signal strength was d-luciferin; (2) the spectra for Luc2 and CBR2 were shifted to a longer wavelength when Akalumine-HCl was the substrate; (3) CBR2 gave the brightest signal with the near-infrared substrate, NH2-NpLH2; and (4) Akaluc was brighter when paired with either CycLuc1 or Akalumine-HCl when paired with d-LH2.ConclusionWe believe that the experimental results described here should provide valuable guidance to end users for choosing the correct luciferin/luciferase pairs for a variety of BLI applications.
Highlights
Bioluminescence imaging (BLI) is a well-known, noninvasive technique employed during preclinical studies to track cells and monitor biological processes in living animals [1,2,3]
We believe that the experimental results described here should provide valuable guidance to end users for choosing the correct luciferin/luciferase pairs for a variety of bioluminescence imaging (BLI) applications
Akalumine-HCl has a spectral peak in the near infrared (NIR) (677 nm) and enhanced emission with Luc2 when administered at low concentration [10]
Summary
Bioluminescence imaging (BLI) is a well-known, noninvasive technique employed during preclinical studies to track cells and monitor biological processes in living animals [1,2,3]. Cycluc has been shown to enhance emission of codon optimized firefly luciferase (Luc2), especially in the brain. This system provides slightly red-shifted emission resulting in deeper light penetration and less scattering of the bioluminescence signal [8, 9]. Amino naphthyl luciferin (NH2-NpLH2) represents another new substrate with potential for deeper tissue BLI [12]. This substrate was shown to emit in the NIR with a peak of 740 nm when reacting with an engineered version of click-beetle luciferase (CBR2). CBR2 can utilize D-LH2 and this combination was shown to improve imaging in black fur mice compared with Luc2/ D-LH2
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