Murine models are a valuable tool in medical research, commonly imaged using MR or bioluminescent techniques. Although MR has excellent soft tissue contrast and resolution, the cost of imaging can be high. Bioluminescence imaging has excellent specificity, but suffers poor resolution and sensitivity due to the high absorption and scatter of light in biological tissue. Cryo-Fluorescence Tomography (CFT), an imaging modality based on serial slicing and off-the-block fluorescence imaging, is examined for its utility as a complimentary assay in preclinical oncology studies. CFT is incorporates into existing workflows and allows for registration and evaluation with in vivo imaging techniques.
Molecular fluorescence data is shown as a 3D maximum intensity projection with a multi-planar slice view of corresponding white light data. In this way, molecular fluorescence can be analyzed in the accessible context of white light images.
An immunocompromised mouse is intracranially inoculated with GL26-luc2 cells in the right hemisphere of the brain. After a waiting period of two weeks, the subject is imaged with a T1-weighted MR sequence with Gadolinium contrast enhancement. A bioluminescence image is also acquired. Finally, the subject is intravenously administered 100uL of 0.1mM indocyanine green (ICG) and 100uL of 0.2mM of Angiosense680Ex. At the 24-hour time point, the subject is sacrificed. The brain is then removed and embedded in Optimum Cutting Temperature material. The specimen is then imaged using CFT with a section thickness of 25um. Three images are collected for each section: a white light image and two fluorescence images. One fluorescence image is taken for Angiosense detection, and one is taken for ICG detection. Using VivoQuant, the resulting image stacks are aligned, corrected for biological optical effects, and reconstructed to recover a 3D distribution of Angiosense and ICG, as well as 3D white light information.
Traditional BLI offers a highly sensitive imaging technique used in a variety of oncology models. However the modality is limited in its ability to accurately give information on 3D structure. It is common practice to incorporate MRI into Murine GBM studies. Using CFT as an additional data point the researcher can also evaluate how the fluorescent reporter or the white light visualization correlate structural information from the MRI.
Using CFT as a complementary imaging platform adds additional information that bridges the gap between in vivo imaging and traditional histopathology. Both Angiosense and ICG are nonspecific tracers that have shown good correlation with contrast enhanced MRI in this model.
The fluorescence provides an enormous amount of specificity in this study, as it is only the tumor site that is appreciably fluorescent in the imaging domain. However, the imaging technique does not sacrifice structural information for specificity, as there is also a collection of white light images for anatomical landmarking. Thus, imaging with CFT provides valuable but complimentary information in preclinical oncology studies.
Figure 3 description: Comparison graphs between AngioSense and ICG were plotted. Correlation between AngioSense and ICG were greater than .90. Comparison between the contrast enhanced MRI and the Angiosense was performed to show a 0.80 and 0.72 Correlation between animal 1 and 2, respectively.
The CFT process also allows for the collection for tissue samples for traditional histology analysis. Future research will incorporate models that use molecular targeted probes as well as the use of transgenic models that express fluorophores.