AbstractCerenkov luminescence imaging (CLI) is a promising approach to image-guided surgery and pathological sampling. It could offer additional advantages when combined to whole-body isotope tomographies. We aimed to obtain evidence of its applicability in lymphoma patho-diagnostics, thus we decided to investigate the radiodiagnostic potential of combined PET or SPECT/CLI in an experimental, novel spontaneous high-grade B-cell lymphoma mouse model (Bc.DLFL1). We monitored the lymphoma dissemination at early stage, and at clinically relevant stages such as advanced stage and terminal stage with in vivo 2-deoxy-2-[¹⁸F]fluoro-d-glucose (FDG) positron emission tomography (PET)/magnetic resonance imaging (MRI) and ⁶⁷Ga-citrate single photon emission computed tomography (SPECT)/MRI. In vivo imaging was combined with ex vivo high resolution CLI. The use of CLI with ¹⁸F-Fluorine (F-18) and ⁶⁷Ga-Gallium isotopes in the selection of infiltrated lymph nodes for tumor staging and pathology was thus tested. At advanced stage, FDG PET/MRI plus ex vivo CLI allowed accurate detection of FDG accumulation in lymphoma-infiltrated tissues. At terminal stage we detected tumorous lymph nodes with SPECT/MRI and we could report in vivo detection of the Cerenkov light emission of ⁶⁷Ga. CLI with ⁶⁷Ga-citrate revealed lymphoma accumulation in distant lymph node locations, unnoticeable with only MRI. Flow cytometry and immunohistochemistry confirmed these imaging results. Our study promotes the combined use of PET and CLI in preclinical studies and clinical practice. Heterogeneous FDG distribution in lymph nodes, detected at sampling surgery, has implications for tissue pathology processing and it could direct therapy. The results with ⁶⁷Ga also point to the opportunities to further apply suitable SPECT radiopharmaceuticals for CLI.