Evaluating Brightness and Spectral Properties of Click Beetle and Firefly Luciferases Using Luciferin Analogues: Identification of Preferred Pairings of Luciferase and Substrate for In Vivo Bioluminescence Imaging

Abstract

Purpose: Currently, 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.

Procedures: Transduced 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.

Results: Our 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.

Conclusion: 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 BLI applications.

Keywords: Bioluminescence; Emission spectrum; In vivo imaging; Luciferase; Luciferin.

Fig. 1.

(a) Bioluminescence profiles for Luc2, CBR2, CBG99, and Akaluc luciferases combined with four different luciferin analogues in live cells. (b-f) Photon flux (ph/s) in HEK 293T cells expressing individual luciferases upon addition of substrates (0.1 mM) was quantified using an exposure time of 30 s. Statistical analysis (N = 3) was performed using one-way ANOVA followed by Tukey’s T test (*p < 0.01 for Luc2/d-LH2 compared with all combinations with the exception of Akaluc/Cycluc1 which was not significantly different).

Fig. 2.

In vivo (BALB/C) emission spectra for different combinations of luciferase (Luc2, CBG99, CBR2, or Akaluc; expressed in HEK393T cells implanted subcutaneously in the flanks) and luciferin or luciferin analogue. (a) D-LH2 (150 mg/kg), (b) NH₂-NpLH2 (220 mg/kg), (c) CycLuc1 (7.6 mg/kg), and (d) Akalumine-HCl (50 mg/kg); substrates were injected intraperitoneally). Spectral data was acquired 15–20 min after injection.

Fig. 3.

Superficial bioluminescence imaging of BALB/C mice in which 1 × 10⁵ HEK293T cells transduced with Luc2 and Akaluc or CBR2 and CBG99 were implanted subcutaneously into the left and right flanks of mice, respectively, and treated (intraperitoneally) with (a) D-LH2 (150 mg/kg), (b) NH₂-NpLH2 (220 mg/kg), (c) CycLuc1 (7.6 mg/Kg), and (d) Akalumine-HCl (50 mg/kg). Imaging data was collected using open filters and with an exposure time of 30 s. Average luminescence is reported as photons/s/cm²/sr.

Fig. 4.

Quantification of photon flux (ph/s) measured in vivo for all combinations of luciferase and substrate (d-LH2 (a), Akalumine-HCl (b), CycLuc1 (c), and NH₂-NpLH2 (d). Combined data is also presented in logarithmic scale (e). Statistical analysis of data was performed using one-way ANOVA followed by Tukey’s post-test (**p < 0.0019; ***p < 0.001; ****p < 0.0001).