Abstract: Reporter genes are genetically encoded proteins that are used to indicate the transcriptional activity of a gene or promoter. One example of a reporter gene is the luciferase from the North American firefly, Photinus pyralis . Firefly luciferase (Fluc) catalyzes the reaction of luciferin with oxygen to produce a photon of light. Fluc has been used in a variety of applications including gene delivery, signal transduction, and tumor growth analysis. While luciferase is often utilized in mammalian cells, little is known about its kinetic activity inside live cells. In Chapter 2, we analyzed the kinetics of Fluc inside living cells and developed a model to quantitatively predict the bioluminescence signal. We found the K_M to be 1 mM, a value much higher than the 10 μM reported in vitro, and found the half-life of the luciferase to be 2 hours. While most of the studies involving luciferase use it to generate information about populations, luciferase also has the ability to kinetically monitor individual cells using an ultra-sensitive digital camera. In Chapter 3, we design and test an ultra-sensitive microscopic imaging system and find we are able to quantitatively distinguish different behaviors of individual mammalian cells. Specifically, we monitored the degradation of the Fluc signal using and found a population half-life of 2.5 hours; however, individual cells exhibited a diversity of half-lives ranging between 2 and 7.5 hours. One of the limitations of using a light-based signal in vivo is the absorption of photons by tissue and hemoglobin. The lowest absorption coefficient for hemoglobin occurs in the red region of the visible spectrum, while Fluc has an emission maximum at 560 nm. A red-shifted Fluc would enhance the sensitivity of an in vivo assay and also create the possibility of using various colors to monitor multiple events. In Chapter 4, we describe the evolution, expression, and imaging of several red-emitting Flucs (λ_max=600 nm) in living mammalian cells.