Directed self-assembly of small molecules in living systems could enable a myriad of applications in biology and medicine, and it has been widely used to synthesize supramolecules and nano/ microstructures in solution and in living cells. However, controlling self-assembly of synthetic small molecules in living animals is challenging because of the complex and dynamic in vivo physiological environment. Here we employed an optimized first-order bioorthogonal cyclization reaction to control self-assembly of a fluorescent small molecule, and demonstrated its in vivo applicability by imaging of casapae-3/7 activity in human tumor xenograft mouse models of chemotherapy. The in situ assembled fluorescent nanoparticles have been successfully imaged in both apoptotic cells and tumor tissues using three-dimensional structured illumination microscopy. This strategy combines the advantages offered by small molecules with those of nanomaterials and should find widespread use for non-invasive imaging of enzyme activity in vivo. Controlling small molecules self-assembly into supramolecular complexes is pervasive among living things, building complex structures capable of high-order functions necessary for life1,2. In the laboratory, this principle is also widely used to synthesize supramolecules and nano/microstructures3–5. Recently, extensive efforts have been made to design small molecules with the propensity for controlled self-assembly in living cells6–10. In these examples, small molecules are shown to enter cells and undergo self-assembly after activation by subcellular targets. Introduction of this technology into whole mammalian organisms would offer myriads of applications in biology and medicine such as controlled *Correspondence and requests for materials should be addressed to J.R.
