Amira 5

Animals that were scanned on both PET and x-ray CT systems were placed on a custom-built platform in a rigid body fixed position (using 0.1-mm polyethylene wrapping) (20 (link)). The bed was placed into an integrated heated air, anesthesia bed (MultiCell, Mediso). The bed was fixed in place on the microPET gantry and imaged as above. The bed was then moved for CT imaging using the NanoSPECT/CT (Bioscan). General acquisition parameters were 55 kVp with a pitch of 1 and 240 projections in a spiral scan mode. The entire animal was scanned using a multiple-field-of-view procedure (with an approximate field of view of 4 cm × 4 cm × 4 cm per bed position), commonly requiring three bed positions per scan. Total scan time was about 10 min. A Shepp-Logan filter was used during the reconstruction process to produce image matrices with isotropic volumes of 221 μm.
PET data were reconstructed using a 3D-filtered back projection maximum a priori algorithm using a ramp filter with a cutoff frequency equal to the Nyquist frequency into a 128 × 128 × 95 matrix (50 (link)). Data were exported in raw format, and the rigid body (three degrees of freedom) coregistration between PET and CT data (and MRI, if applicable) was performed in Amira 5.3.3 (FEI). Amira and FIJI were used to produce most of the figures in the manuscript.

Data visualization was accomplished with a combination of the open source FIJI (NIH)^{41 (link)}, ASIPro (Concorde Microsystems) and Amira 5.3.3 (FEI). FIJI was used to generate whole body 3D images of the MR acquisitions. Fused and nuclear medicine tomographic images were generated using Amira. These images are 3D volume rendering of PET or SPECT data overlaid onto surface-rendered CT data (or CT and MR data). To generate these images, a weighted average of the intensities along projections through the PET is computed and this in turn is blended with the CT-rendered image. The three dimensionally rendered MR data used an alpha transfer function (mapping transparency to intensity) included as Supplementary Fig. 9. To show the PET data are represented with a semiquantitative blue (low)–yellow (high) colour scale; the SPECT data uses a green (low)–blue (high) colour scale.

PET imaging experiments were conducted on a microPET Focus 120 scanner (Concorde Microsystems). Mice were anesthetized by inhalation of 1–2% isoflurane/oxygen gas mixture in order to record PET images (21 (link)). PET images were acquired at 120h after injection. The PET-bed was then moved for CT imaging using a NanoSPECT/CT (Bioscan). Data were exported in raw format, and the rigid body (three degrees of freedom) co-registration between PET and CT data was performed in Amira 5.3.3 (FEI). Details on PET and CT acquisition and analysis are provided in the supplementary methods.

Mice were brought to the plane of anesthesia and maintained under isofluorane in air for agent administration and imaging. Mice were evaluated using an image guided small animal micro-irradiator (XRad225Cx, Precision X-Ray, Inc., North Brantford, CT). This device utilizes a dual focal spot X-ray tube at 45 kVp with a flat-panel amorphous silicon imager mounted on a C-arm gantry. All subjects were imaged with an isotropic resolution of 100 μm, using a volume that encompassed the entire abdomen. Animals imaged with contrast were given approximately 3 mL of 75 mg Iodine/mL (iohexol; Omnipaque, GE Healthcare) between 5 and 15 minutes prior to scan. Data visualization was accomplished with a combination of the open source FIJI (National Institutes of Health) (19 (link)) and Amira 5.3.3 (FEI, Hilsboro, OR) as previously described (20 (link)). Semi-automated generation of organ volumes was accomplished with the Amira region-grow tool.