8.1. Bioluminescence imaging
Many tissues contain molecules with strong autofluorescence; therefore, the use of fluorescence excitation with visible light in such samples is complicated. Bioluminescence imaging allows background-free image acquisition since no excitation light is required (Figure 2m). Bioluminescence is produced by the chemical transformation of a substrate by the luciferase enzyme. The absence of a background signal allows for very long exposure times (minutes) to detect even very weak luminescence intensities. However, this is also a major drawback of bioluminescence imaging because such long exposure times are often required for image acquisition, limiting its applicability for the study of dynamic processes. A particularly promising approach is bioluminescence complementation, which is based on the reconstruction of a complete functional luciferase by binding its two split pieces together (Remy & Michnick, 2006). Bioluminescence complementation has been used in live mice to detect the interactions of β-arrestin2 with β2AR (Takakura et al., 2012) and sphingosine-1-phosphate receptor 1 (Kono et al., 2017) and visualise β2AR activation by ligands in breast cancer cells (Alcobia et al., 2018). Bioluminescence has been used to report Gs activation in live mice using the cAMP response element (Dressler et al., 2014). Bioluminescence imaging is also compatible with high throughput screening reporting GPCR interactions with β-arrestins in multi-well plates (Hattori & Ozawa, 2015).