Validation of method for enhanced production of red-shifted bioluminescent photons in vivo

Bioluminescence Imaging (BLI) is an increasingly useful and applicable technique that allows for the non-invasive observation of biological events in intact living organisms, ranging from single cells to small rodents. Though the photon production occurs within the host, significant exposure times can be necessary due to the low photon flux compared to fluorescence imaging. The optical absorption spectrum of haemoglobin strongly overlaps most bioluminescent emission spectra, greatly attenuating the total detectable photons in animal models. We have developed and validated a technique that is able to red-shift the bioluminescent photons to the more desirable optical region of > 650 nm, a region of minimal absorbance by hemoglobin. This red-shift occurs by using bioluminescence as an internal light source capable of exciting a fluorophore, such as a fluorescent protein or a quantum dot, that emits in the red. Interestingly, in the absence of an absorber, this excitation can occur over substantial distances (microns to centimeters), far exceeding distances associated to, and thereby precluding, resonance energy transfer phenomena. We show this novel technique yields a substantial increase in the number of red photons for in vitro and ex vivo conditions, suggesting eventually utility for in vivo studies on, for example, intact living mice.