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We present a practical approach for co-registration of bioluminescence tomography (BLT), computed tomography (CT), and magnetic resonance (MR) images. To this end, we developed a customized animal shuttle composed of non-fluorescent, MR-compatible Delrin plastic that fits a commercially available MR surface coil. Mouse embryonic stem cells (mESCs) were transfected with the luciferase gene and labeled with superparamagnetic iron oxide (SPIO) nanoparticles. Cells were stereotaxically implanted in mouse brain and imaged weekly for 4 weeks with BLI (IVIS Spectrum CT scanner) and MRI (11.7T horizontal bore scanner). Without the use of software co-registration, in vitro phantom studies yielded root-mean-square errors (RMSE) of 7.6×10 -3 , 0.93 mm, and 0.78 mm along the medial-lateral (ML), dorsal-ventral (DV), and anterior-posterior (AP) axes, respectively. Rotation errors were negligible. Software co-registration by translation along the DV and AP axes resulted in consistent agreement between the CT and MR images, without the need for rotation or warping. In vivo co-registered BLT/MRI mouse brain data sets demonstrated a single, diffuse region of BLI photon signal and MRI hypointensity. Over time, the transplanted cells formed tumors as validated by histopathology. Disagreement between BLT and MRI tumor location was greatest along the DV axis (1.4±0.2 mm) compared to the ML (0.5±0.3 mm) and AP axis (0.6 mm) due to the uncertainty of the depth of origin of the BLT signal. Combining the high spatial anatomical information of MRI with the cell viability/proliferation data from BLT should facilitate pre-clinical evaluation of novel therapeutic candidate stem cells.

Co-Registration of Bioluminescence Tomography, Computed Tomography, and Magnetic Resonance Imaging for Multimodal In Vivo Stem Cell Tracking