Compositions and methods for in vivo evaluation of bioluminescence

Abstract

Compositions and methods are described for normalizing a bioluminescent signal in a live animal.

1 . A composition comprising a bioluminescent substrate and a tracking dye. 
     
     
         2 . The composition of  claim 1 , wherein the bioluminescent substrate comprises luciferin. 
     
     
         3 . The composition of  claim 1 , wherein the tracking dye comprises a fluorescent dye. 
     
     
         4 . The composition of  claim 2 , wherein the tracking dye comprises a fluorescent dye. 
     
     
         5 . A method of normalizing a bioluminescent signal detected in a live animal comprising a bioluminescent protein, the method comprising:
 administering a composition according to  claim 4  to a plurality of live animals;   measuring the bioluminescent signal generated by reaction of the bioluminescent substrate and bioluminescent protein from each animal;   measuring the signal of the tracking dye from each animal; and   normalizing the bioluminescent signal to the signal of the tracking dye in each animal.   
     
     
         6 . The method of  claim 5 , wherein the signal of the tracking dye is determined as a fluorescent normalization factor (FLI) percentage using the following equation:
   (fluorescence ( FLI ) signal−average fluorescence ( FLI ) signal)/average  FLI  signal.  Equation (1):
   
     
     
         7 . The method of  claim 6 , wherein the step of normalization comprises the steps of
 (a) determining a bioluminescence (BLI) normalization value using the following equation:
     BLI  Normalization Value= BLI×FLI  Normalization Factor; and  Equation (2)
 
   (b) determining a normalized bioluminescent signal using the following equation:
   Normalization  BLI=BLI−BLI  Normalization Value  Equation (3)
 
   
     
     
         8 . The method of  claim 5 , wherein the method is preformed using a computer program. 
     
     
         9 . A method of validating bloodstream injection of a bioluminescent substrate into a living animal, the method comprising the steps of
 administering a composition according to  claim 4  to an animal;   localizing the signal emitted by the tracking dye, wherein if the tracking dye signal is localized at the site of injection, the bioluminescent substrate was not distributed into the bloodstream.   
     
     
         10 . A kit comprising a composition according to  claim 1 . 
     
     
         11 . The kit of  claim 10 , further comprising instructions selected from the group consisting of instructions for reconstitution of the composition, instructions for administration of the composition, instructions for imaging of live animals, instructions for normalization, instructions for validation and combinations thereof.