Evaluation of the Light Emission Kinetics in Luciferin/Luciferase-Based In Vivo Bioluminescence Imaging for Guidance in the Development of Small Animal Imaging Study Design

Bioluminescence imaging (BLI) is gaining acceptance as a small animal imaging modality useful for visualizing cellular and molecular activity in vivo, and especially for evaluating tumor development and efficacies of treatments. Various studies have validated the technique for a number of purposes, including the quantification of tumor burden; however, many basic questions have not been investigated whose answers may ultimately impact the conclusions drawn from the results. Primarily, consideration of the impact of BLI emission kinetics has not been rigorously addressed. This study provides information on the effects of different routes of luciferin substrate injection on the BLI kinetic profile, including time to peak emission, magnitude of peak emission, and emission decay characteristics. This study also presents for the first time the use of subcutaneous (s.c.) luciferin injection and the use of s.c. luciferin injection followed by continuous s.c. infusion (s.c.i.) for establishment of stable BLI light emission. Further, results are presented of the kinetic profile changes associated with 1) inhaled and injected anesthesia; and 2) ambient air heating on mouse core temperature. The study demonstrated substantial differences in the peak light emission with i.v. providing the highest, with s.c., s.c.i. and i.p yielding 30% or less of the light emission of the i.v. route. The correlations between tumor burden and BLI light emission were moderately strong (R>0.75) for each administration route, but at varying times following injection, providing information for establishment of optimal image start times. Surprisingly, ambient cooling of the animal while under anesthesia yielded peak light emissions of up to 100% higher than those obtained when ambient air heating was used to maintain mouse core temperature. Finally, guidelines are presented to aid investigators in development of BLI study design to give due consideration to luciferin administration routes, anesthesia protocol, and animal temperature maintenance.