Temporal Variations of Skin Pigmentation in C57Bl/6 Mice Affect Optical Bioluminescence Quantitation

ABSTRACT PURPOSE: Depilation-induced skin pigmentation in C57Bl/6 mice is a known occurrence, and presents a unique problem for quantitative optical imaging of small animals, especially for bioluminescence. The work reported here quantitatively investigated the optical attenuation of bioluminescent light due to melanin pigmentation in the skin of transgenic C57B1/6 mice, modified such that luciferase expression is under the transcription control of a physiologically and pharmacologically inducible gene. PROCEDURE: Both in vivo and ex vivo experiments were performed to track bioluminescence signal attenuation through different stages of the mouse hair growth cycle. Simultaneous reflectance measurements were collected in vivo to estimate melanin levels. RESULTS: Biological variability of skin pigmentation was found to dramatically affect collected bioluminescent signal emerging through the skin of the mice. When compared to signal through skin with no pigmentation, the signal through highly-pigmented skin was attenuated an average of 90%. Correlation of reflectance signals to bioluminescence signal loss forms the basis of the proposed correction method. We observed, however, that variability in tissue composition, which results in inconsistent reflectance spectra, limits the accuracy of the correction method but can be improved by incorporating more complex analysis. CONCLUSION: Skin pigmentation is a significant variable in bioluminescent imaging, and should be considered in experimental design and implementation for longitudinal studies, and especially when sensitivity to small signal changes, or differences among animals, is required