Mice were injected intravenously with 10 MBq 111In-labeled PSMA ligands (0.3 nmol, molar activity 33.3 MBq/nmol) in PBS/0.5% BSA. Two hours post injection, mice were euthanized by CO2/O2-asphyxation and images were acquired with the IVIS fluorescence imaging system (Xenogen VivoVision IVIS Lumina II, Caliper Life Sciences), using an acquisition time of 10 s. Subsequently, µSPECT/CT images were acquired (U-SPECT II, MILabs) with a 1.0 mm diameter pinhole mouse collimator tube 26. Mice were scanned for 30 min followed by a CT scan (spatial resolution 160 μm, 65 kV, 615 μA) for anatomical reference. µSPECT/CT scans were reconstructed with MILabs reconstruction software, using an ordered-subset expectation maximization algorithm, energy windows 154 - 188 keV and 220-270 keV, 3 iterations, 16 subsets, voxel size of 0.4 mm. SPECT/CT scans were analyzed and maximum intensity projections (MIPs) were created using the Inveon Research Workplace software version 4.1 (Siemens Preclinical Solutions). NIRF images were analyzed using Living Image software version 4.2 (Caliper Life Sciences). Tissues of interest were dissected, weighed and measured for radioactivity in a gamma-counter as described above. In the fluorescence-guided surgery feasibility study, pre-operative imaging was performed to visualize tumor tissue. After white light resection, NIRF imaging was repeated to assess if any remaining tumor was present.

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