AbstractBrown adipose tissue (BAT) is present in human adults and the current gold standard to visualize and quantify BAT is [¹⁸F]FDG PET-CT. However, this method fails to detect BAT under insulin-resistant conditions associated with ageing and weight gain, such as type 2 diabetes. The aim of this study was to develop a novel triglyceride-based tracer for BAT. For this purpose we designed a dual-modal fluorescent/PET fatty acid tracer based on commercially available BODIPY-FL-C₁₆, which can be esterified to its correspondent triglyceride, radiolabeled and incorporated into pre-synthesized chylomicron-like particles. BODIPY-FL-C₁₆ was coupled to 1,2-diolein with a subsequent radiolabeling step resulting in [¹⁸F]BODIPY-C₁₆-triglyceride that was incorporated into chylomicron-like particles. Various quality control steps using fluorescent and radioactive methods were conducted before BAT visualization was tested in mice. Triglyceride synthesis, radiolabeling and subsequent incorporation into chylomicron-like particles was carried out in decent yields. This radiotracer appeared able to visualize BAT in vivo, and the uptake of the radiotracer was stimulated by cold exposure. The here reported method can be used to incorporate radiolabeled triglycerides into pre-synthesized chylomicron-like particles. Our approach is feasible to visualize and quantify the uptake of triglyceride-derived fatty acids by BAT.