Interpreting optical signatures to assess monomer aggregation

Abstract Intracellular in situ self-assembly of small molecular building blocks offers a promising route to functional nanoarchitectures, yet its characterization is complicated by disorder, inhomogeneous broadening, and light scattering. Optical spectroscopy is widely employed to probe aggregation, but steady-state spectral signatures are often weak or entirely absent. Here, we present a physically grounded optical spectroscopy framework to identify and interpret aggregation in soft, noncovalently assembled nanoarchitectures. We analyze how excitonic coupling, rotational disorder, and oscillator strength redistribution shape absorption and emission, explaining why steady-state measurements alone are frequently insufficient. We show that time-resolved observables, including excited-state lifetime shortening, polarization memory loss, and intensity-dependent deactivation, provide robust fingerprints of intermolecular coupling and exciton transport. We further emphasize the need to explicitly account for the complex dielectric function in pump–probe experiments conducted in highly scattering environments. Overall, we define experimentally accessible, materials-relevant criteria that distinguish aggregation, excitonic coupling, and energy transport in disordered environments. Graphical abstract