Pmod software version 3.5

SPECT imaging was conducted 40 min after the NIR-PIT. Tumor-bearing mice from each group (¹¹¹In-DTPA, n = 7; ^99mTc-HSA-D, n = 8) were intravenously injected with ¹¹¹In-DTPA (7.4 MBq) and ^99mTc-HSA-D (7.4 MBq), respectively. Dynamic SPECT data acquisition was conducted for 60 min, using a VECTor/CT SPECT/CT Pre-Clinical Imaging system with a multi-pinhole collimator (MILabs, Utrecht, the Netherlands) under 1.5% isoflurane anesthesia. SPECT images were reconstructed using a pixel-based ordered-subsets expectation-maximization algorithm with eight subsets and two iterations on a 0.8-mm voxel grid without attenuation correction. Computed tomography scans were acquired with an X-ray source set at 60 kVp and 615 μA after the SPECT scan, and images were reconstructed using a filtered backprojection algorithm for the cone beam. Merged images were obtained using PMOD software (version 3.5; PMOD Technologies, Zurich, Switzerland). The VOI was drawn over tumors and radioprobe uptake was quantified as the % ID/cc, and AUCs covered up to each time point were also calculated appropriately.

Also, semiquantitative analysis of FDG-PET was conducted. The images have been anonymized before analysis. PMOD software (version 3.5, PMOD Technologies Limited, Zürich, Switzerland) was used for coregistration of all individual FDG-PET image volumes to an in-house FDG-PET template within the MNI space (Daerr et al., 2017 (link)). In analogy to the visual analysis, we measured the mean activity within bilateral parietal, bilateral temporal, and bilateral posterior cingulate cortex volumes of interest (VOIs) of the Hammers atlas (Hammers et al., 2003 (link)) and scaled the measured regional activities by a cerebellum reference region to generate standardized uptake value ratios (SUVr). Asymmetry was calculated by the Asymmetry-Index [AI = (left − right)/(left + right)*100] for hemispheres and subregions.

PMOD software Version 3.5 (PMOD Technologies Ltd.) was used for quantitative analysis of PET data. PET and structural MR images were co-registered. The binding potential (BP_ND) was calculated using the simplified reference model 2 (Wu and Carson, 2002 (link)), with the cerebellum as the reference region. Using Statistical Parametric Mapping 8 software (SPM8, Wellcome Department of Imaging Neuroscience), whole-head structural images were then normalized to the Montréal Neurological Institute (MNI) T1 image template, with the same parameters applied to the BP_ND image. BP_ND images were resampled to a voxel size of 2.0 × 2.0 × 2.0 mm, using SPM8. The between-condition comparison (love vs. control) of the BP_ND was performed on a voxel-by-voxel basis using t statistics, with the statistical threshold set at P < 0.001 at the voxel level and more than 20 voxels at the cluster level for the entire brain. The co-ordinate of each brain region was defined by using the Wake Forest University Pick-atlas (Maldjian et al., 2003 (link)).