Compositions and methods for in vivo evaluation of bioluminescence
Inventors
Oldfield, Stephen • Ansaldi, Daniel • Singh, Rajendra • Zhang, Ning
Assignees
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Publication Number
US-9486542-B2
Publication Date
2016-11-08
Expiration Date
Abstract
Compositions and methods are described for normalizing a bioluminescent signal in a live animal.
Core Innovation
The invention relates to an in vivo bioluminescence imaging approach in live non-human mammalian animals. The approach normalizes bioluminescence signals produced by an exogenous transgene encoding a bioluminescent protein by co-administering a bioluminescent substrate together with a fluorescent tracking dye that emits light of different wavelengths than the bioluminescent substrate.
The bioluminescent substrate is transformed to an excited state in the presence of an oxygenase, oxygen, and ATP, producing bioluminescence. The fluorescent tracking dye is not a protein and is configured to co-distribute with the bioluminescent substrate so that fluorescence measured in a reference ROI provides a quantified indicator of systemic distribution of the substrate.
Bioluminescent signal from a target ROI is normalized using fluorescence from the tracking dye, and predefined deviation thresholds can be applied to flag aberrant substrate injections. The document also describes validating correct bloodstream distribution of substrate using localization of the tracking dye, and provides examples using multicolor fluorescent dye emission including green, orange, red, violet, and near-IR, together with fluorescence quantification alongside bioluminescence imaging.
Claims Coverage
The partial content provides one independent claim. Across the dependent claims, the fluorescent tracking dye’s emission wavelength and optional composition components such as luciferin and buffer are further specified.
Live non-human mammalian animal with exogenous bioluminescent transgene and wavelength-differentiated substrate/tracking dye
A live non-human mammalian animal comprising an exogenous transgene encoding a bioluminescent protein and a composition comprising a bioluminescent substrate and a fluorescent tracking dye, wherein the bioluminescent substrate and the fluorescent tracking dye emit light of different wavelengths, the bioluminescent substrate is transformed to an excited state in the presence of an oxygenase, oxygen, and ATP, and the fluorescent tracking dye is not a protein.
The claim scope centers on a live non-human mammalian animal with an exogenous transgene encoding a bioluminescent protein, where a bioluminescent substrate and a non-protein fluorescent tracking dye emit at different wavelengths and the substrate is excited via an oxygenase/oxygen/ATP mechanism, enabling normalization based on dye distribution.
Stated Advantages
Enables normalization of bioluminescent signals in live animals using a fluorescent tracking dye that co-distributes with the substrate.
Allows identification and flagging of aberrant substrate injections using fluorescence-based quantification and bioluminescence normalization with deviation thresholds.
Provides validation of correct bloodstream distribution of the bioluminescent substrate using localization of the fluorescent tracking dye.
Documented Applications
In vivo bioluminescence imaging in live non-human mammalian animals with fluorescence imaging to normalize substrate-dependent bioluminescent signals.
Detection and identification of an outlier animal with poor substrate injection by using reduced dye distribution and fluorescence normalization calculations.
Validation of correct bloodstream substrate injection through fluorescence localization of the tracking dye.
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