Single-Particle Photophysics Reveals Pathway-Dependent Packing in Kinetically Trapped Hydrophobic Aggregates

Abstract Hydrophobic self-assembly frequently produces kinetically trapped, nonequilibrium aggregates whose internal molecular packing is difficult to control and characterize. Here, using a donor–acceptor aggregation-induced-emission probe, we show that the onset concentration of assembly (below 200 nM) controls packing density and thereby the photophysical properties of the metastable states via different kinetic routes at room temperature. High-sensitivity ensemble photoluminescence spectroscopy and single-particle fluorescence lifetime imaging reveal pathway-dependent emission peak positions and lifetimes that both remain stable over several days. Fluorescence correlation spectroscopy shows that size distributions are largely decoupled from these photophysical changes, while all-atom molecular dynamics (MD) simulations confirm that higher onset concentrations produce denser packing, accounting for the observed redshift and longer lifetime. These results establish molecular packing density as a key determinant of the photophysics of kinetically trapped hydrophobic aggregates and provide a general room-temperature route to interrogate otherwise inaccessible nonequilibrium intermediates.