Bioluminescence imaging: progress and applications

Abstract

Application of bioluminescence imaging has increased tremendously in the past decade and has significantly contributed to core conceptual advances in biomedical research. This technology provides valuable means for monitoring of different biological processes in immunology, oncology, virology and neuroscience. In this review, we discuss current trends in bioluminescence and its application in different fields with an emphasis on cancer research.

Figure 1

Bioluminescence reaction of Fluc, Rluc and Gluc. The luciferin chemical structure is shown as well as the chemical reaction and the peak light emission (λ_max) for each luciferase.

Figure 2. Imaging of protein-protein interaction. (a–b) Two-hybrid system

(a) Two vectors driving the expression of GAL4-protein X and VP16-protein Y fusions are co-expressed. Once protein X interacts with protein Y, the GAL4-X-Y-VP16 complex will subsequently bind to the GAL4 responsive elements in a third vector to drive the expression of luciferase, thereby re-activating its bioluminescence reaction. (b) Transgenic mice expressing the transgene in (B) were transfected in vivo with different plasmids: pGal4BDp53 combined with pVP16-TAg or pGal4BDp53 combined with off-target pVP16-CP, or each plasmid separately. A plasmid expressing Rluc was used for normalization. Mice were imaged 24 hours after transfection for Fluc expression (d-luciferin substrate) and Rluc expression (coelenterazine substrate). BD, binding domain; CP, polyomavirus coat. Adapted with permission from Pichler et al., 2008. (c–d) Split reporter system or PCA. (c) Schematic overview of the Rluc-PCA system to study the interaction of the PKA subunits. Low levels of cAMP cause the regulatory (R) and catalytic (C) subunits to form a heterodimer and induce Rluc activity. Under high cAMP levels, both subunits are dissociated and the Rluc activity is decreased. (d) BLI of PKA subunits-interaction in vitro. HEK293T human fibroblast cells were transfected with full-length Rluc (upper panel) or with Rluc-PCA (middle and lower panel). Images were detected using a CCD camera after addition of coelenterazine. Forskolin which increases the cAMP levels was used to treat cells co-expressing PKA C and R subunits. This cAMP induction disrupted the Rluc activity. DAPI was used for nuclear staining. Scale bar, 5 µm. Adapted with permission from Stefan et al., 2007.

Figure 3. Imaging of circulating hematopoietic and neuronal precursor cells

(a) Human T cells expressing extGluc and CAR accumulate in subcutaneous tumors. BLI of mice with subcutaneous CD19+ tumors at day 1 and 2 after systematic injection with human T-cells co-expressing CAR targeting CD19+ cells (19z1) and extGLuc or non-specific CAR (Pz1) and extGLuc. Adapted with permission from Santos et al., 2009. (b) Monitoring of neuronal precursor cells (NPC) using the Gluc-blood reporter. Mice were injected with NPC cells expressing Gluc or PBS (control). Gluc activity was monitored in 5 µl blood over time after the addition of coelenterazine using a luminometer. Adapted with permission from Wurdinger et al., 2007.

Figure 4. Imaging of hydrogen peroxide production

(a) The PCL-1 sensor. H₂O₂ mediates the release of luciferin thereby generating bioluminescence. (b–e) In vivo BLI of H₂O₂. Mice ubiquitously expressing Fluc were injected with PCL-1 followed by injection with different doses of H₂O₂. A dose-dependent increase in BLI expressed as total photon flux was observed few minutes after injection of H₂O₂ (b–c). Injection of the antioxidant N-acetylcysteine (NAC) resulted in descrease in the BLI signal further confirming the specificity on PCL-1 as an in vivo reporter of H₂O₂ (d–e). Adapted with permission from Van de Bittner et al, 2010.