Ex vivo IVIS fluorescent imaging

The amount of fluorescent light emitted from the GFP transplants was quantified in the isolated fixed spinal cords using ex vivo imaging with the IVIS Spectrum. Spinal cords were placed on a Petri-dish with a black backing and inserted into the IVIS Spectrum. The number of photons emitted by the GFP⁺ SCs within the spinal cord was determined using the following IVIS settings: excitation/emission = 500/540; exposure = 0.5 s; binning = 4, F-stop = 2; field of view = B. A photographic image of the spinal cord overlaid with the photon intensity count was generated. The ROI of GFP fluorescence was then auto-sized and the average radiant efficiency [(p/s/cm²/sr)/(μW/cm²)] determined for each case. To account for variability in light intensity between images, the same settings were used to show visible photons on all the images (min = 3.25 × 10⁸; max = 10 × 10⁹).