摘要： Non-invasive and precise stem cell tracking after transplantation in living subject is very important to monitor both stem cell destinations and their in vivo fate, which was closely related to their therapeutic efficacy. Herein, we developed bicyclo[6.1.0]nonyne (BCN)-conjugated glycol chitosan nanoparticles (BCN-NPs) as a delivery system of dual-modal stem cell imaging probes. Near-infrared fluorescent (NIRF) dye, Cy5.5, was chemically conjugated to the BCN-NPs and then oleic acid-coated superparamagnetic iron oxide nanoparticles (OA-Fe3O4 NPs) were encapsulated into BCN-NPs, in resulting Cy5.5-labeled and OA-Fe3O4 NP-encapsulated BCN-NPs (BCN-dual-NPs). For bioorthogonal labeling of human adipose-derived mesenchymal stem cells (hMSCs), firstly, hMSCs were treated with tetra-acetylated N-azidoacetyl-D-mannosamine (Ac4ManNAz) for generating azide (-N3) groups onto their surface via metabolic glycoengineering. Second, azide groups on the cell surface were successfully chemically labeled with BCN-dual-NPs via bioorthogonal click chemistry in vitro. This bioorthogonal labeling of hMSCs could greatly increase the cell labeling efficiency, safety, and imaging sensitivity, compared to only nanoparticle-derived labeling technology. The dual-modal imaging-guided precise tracking of bioorthogonally labeled hMSCs was tested in the photothrombotic stroke mouse model via intraparenchymal injection. Finally, BCN-dual-NPs-labeled hMSCs could be effectively tracked of their migration from implanted site to brain stroke lesion using NIRF/T2-weighted magnetic resonance (MR) dual-modal imaging for 14 days. Our observation would provide a potential application of bioorthogonally labeled stem cell imaging in regenerative medicine by providing safety and high labeling efficiency in vitro and in vivo.