**Abstract**

The firefly bioluminescence reaction has been exploited for *in vivo* optical imaging in life sciences. To develop highly sensitive bioluminescence imaging technology, many researchers have synthesized luciferin analogs and luciferase mutants. This chapter first discusses synthetic luciferin analogs and their structure–activity relationships at the luminescence wavelength of the firefly bioluminescence reaction. We then discuss the development of luciferin analogs that produce near-infrared (NIR) light. Since NIR light is highly permeable for biological tissues, NIR luciferin analogs might sensitively detect signals from deep biological tissues such as the brain and lungs. Finally, we introduce two NIR luciferin analogs (TokeOni and seMpai) and a newly developed bioluminescence imaging system (AkaBLI). TokeOni can detect single-cell signals in mouse tissue and luminescence signals from marmoset brain, whereas seMpai can detect breast cancer micro-metastasis. Both reagents are valid for *in vivo* bioluminescence imaging with high sensitivity.

**Keywords:** Firefly bioluminescence, Bioluminescence imaging, Structure–activity relationships, Multicolor, Near-infrared light

### **1. Introduction**

In Japan, watching the light of fireflies has been a summer tradition for over one thousand years. Modern fireflies are known to glow yellow-green, but in ancient times they emitted a dark green luminescence, as confirmed by recent molecular biology techniques [1]. The detailed mechanism of firefly bioluminescence is described in previous chapters. This chapter focuses on synthetic substrates of firefly luciferase, which are employed in firefly bioluminescence imaging (BLI).

In recent biological research, BLI technology has observed biological events *in vivo* [2–8]. For example, in cancer research, BLI has been applied to real-time monitoring of gene expression, cell numbers, and other biological events in transgenic mouse models [9–16]. Our group has developed firefly substrate analogs for use in these research fields.

The firefly bioluminescence reaction proceeds via the oxidation of *d*-luciferin (**1**, LH2, **Figure 1**) catalyzed by firefly luciferase (Fluc) in the presence of adenosine triphosphate (ATP), Mg2+ and O2 by a two-step reaction. In the first step, LH2 is adenylated with ATP, and is then oxidized by O2, forming excited-state oxyluciferin that relaxes to the ground state with yellow-green light emission (*λ*max = 560 nm) [17–19]. However, yellow-green light is not able to easily penetrate biological tissues [20], and is useful only for imaging shallow tissues such as subcutaneous tissues. To detect signals from deep tissues such as brain and lung [21], near-infrared (NIR)

**Figure 1.** *Structures of d-luciferin (1, LH2) and aminoluciferin (2).*

light should be used, as it is highly permeable to biological tissues [20] and is suitable for *in vivo* deep tissue imaging [21]. Recently, many synthetic luciferin analogs have been reported. Our group has synthesized various luciferin analogs and compared them with **1**. By studying the structure–activity relationships of these analogs and *Photinus pyralis* (*Ppy*) luciferase, we have developed luciferin analogs that produce wide-spectrum light (from blue to red), along with NIR luciferin analogs (AkaLumine, TokeOni, seMpai) for BLI. Our different analogs are described in this chapter.
