Timing of imaging after d-luciferin injection affects the longitudinal assessment of tumor growth using in vivo bioluminescence imaging

The peak signal or the signal at a predetermined, fixed time point after D-luciferin injection may be used for the quantitative analysis of in vivo bioluminescence imaging. We repeatedly performed sequential bioluminescence imaging after subcutaneous injection of D-luciferin in mice bearing subcutaneous tumors. The peak time in each measurement became shorter early after cell inoculation, presumably due to gradual establishment of intratumoral vasculature, and reached a plateau of about 10 min on day 10. Although the correlation between the signal at a fixed time point and the peak signal was high, the signal at 5 or 10 min normalized for the peak signal was lower for earlier days, which caused overestimation of tumor growth. The time course of the signals after D-luciferin injection may vary with time after cell inoculation, and this variation should be considered when determining the imaging protocol for quantitative bioluminescence tumor monitoring.