Although animal models of leukemia often utilize survival time as the primary therapeutic end point, bioluminescence imaging (BLI) is increasingly being used to provide quantitative and more rapid assessment of drug efficacy in pre-clinical oncology research [
1‐
5]. BLI of firefly luciferase activity provides a cost-effective and extremely sensitive method for imaging fundamental biological processes
in vitro and
in vivo[
6‐
8]
. In vivo BLI is an excellent method to gain a dynamic, longitudinal profile of engraftment [
9]. Luciferase oxidizes luciferin in the presence of adenosine tri-phosphate (ATP) and oxygen to form an electronically excited oxy-luciferin species. Visible light is emitted following the relaxation of excited oxy-luciferin to its ground state [
10,
11]. Because this light can be transmitted through mammalian tissues, it is possible to use bioluminescence for non-invasive and quantitative monitoring of leukemia burden. However, the establishment of clinically relevant animal models that include sensitive detection of early cancer growth and leukemia burden remains an ongoing challenge in translational oncology research [
12]. Therefore, the difficulty in molecular imaging is in the development of effective imaging strategies with reporter systems that reveal cellular and molecular processes consistently throughout an entire study period [
13‐
16]. Nevertheless, there are limitations associated with this approach. Using firefly luciferase as a reporter system requires exogenous luciferin addition and is currently not practical for large animal models. The rapid consumption of D-luciferin can potentially lead to an unstable signal [
17]. Further mammalian tissue is known to be a turbid medium that both scatters and absorbs photons. This is mostly due to changes in refractive index at cell membranes and internal organelles, and can lead to a scattered and attenuated bioluminescence signal, which has influence on investigations especially in deeper tissue [
18]. Bioluminescence imaging using firefly luciferase
in vitro and
in vivo is also often performed with potentially unstable luciferase-expressing polyclonal cell populations. In this study we investigated the limitations, advantages and disadvantages of bioluminescence imaging using a firefly luciferase system with monoclonal and polyclonal human leukemia cell populations
in vitro and in a xenograft mouse model.