**3.1 Background on bioluminescence imaging**

Developed over the last decade, bioluminescence imaging is a technology that enables visualization of viral gene expression in live tissues and animals (Tang et al, 2008). With little surprise, BLI has become a powerful technique for studying VZV pathogenesis.

Bioluminescence is the production of light by living organisms, resulting from a chemical reaction in which chemical energy is converted to light energy (Hastings, 1983; Kurfurst et al., 1983). BLI systems generate bioluminescence using two compounds – luciferase and its substrate luciferin. Luciferase is a class of enzymes commonly exploited as a reporter gene for transcriptional regulation studies (Doyle et al., 2004). Most extensively employed is the luciferase of the North American firefly (*Photinus pyralis*), which can be expressed in mammalian cells by inserting the gene under the control of a promoter. Because firefly luciferase generates a bioluminescence wavelength that can efficiently penetrate tissues, it serves as an excellent indicator for *in vivo* studies (Tang et al, 2008).

There are many advantages to employing BLI over other bio-imaging techniques; one of the key factors is its use of luciferin. Not only can luciferin permeate all tissues *in vivo* (including cell membranes and the blood-brain barrier) (Contag et al., 1997; Rehemtulla et al., 2002), the substrate can also be administered numerous times to the same animal and provides great accuracy, due to its low toxicity and high sensitivity, respectively (Contag et al., 1997; Rehemtulla et al., 2000). When exposed to the appropriate luciferin substrate, luciferase will catalyze an oxidation reaction to produce light visible to the human eye. The light's intensity depends on the amount of luciferase present, which can be determined by quantifying the relative amounts bioluminescence emitted *in vivo* via computer-based analysis. For this reason, engineering viral BACs to express luciferase can be especially valuable for monitoring the activities of the promoter that mediates gene regulation, detecting sites of viral infections, and quantifying viral replication in living cultures and animals (Zhang et al., 2007; Zhang et al., 2008; Dulal et al., 2009; Zhang et al., 2010).
