Afterglow or persistent luminescence eliminates the need for light excitation 
and thus circumvents the issue of autofluorescence, holding promise for 
molecular imaging. However, current persistent luminescence agents are rare and 
limited to inorganic nanoparticles. This study reports the design principle, 
synthesis, and proof-of-concept application of organic semiconducting 
nanoparticles (OSNs) with ultralong phosphorescence for in vivo afterglow 
imaging. The design principle leverages the formation of aggregates through a 
top-down nanoparticle formulation to greatly stabilize the triplet excited 
states of a phosphorescent molecule. This prolongs the particle luminesce to the 
timescale that can be detected by the commercial whole-animal imaging system 
after removal of external light source. Such ultralong phosphorescent of OSNs is 
inert to oxygen and can be repeatedly activated, permitting imaging of lymph 
nodes in living mice with a high signal-to-noise ratio. This study not only 
introduces the first category of water-soluble ultralong phosphorescence organic 
nanoparticles but also reveals a universal design principle to prolong the 
lifetime of phosphorescent molecules to the level that can be effective for 
molecular imaging.