Molecular engineering of individual dye-based nanoparticle photostability for ultrabright two-photon fluorescence

Dye-based fluorescent organic nanoparticles (dFONs) represent a promising class of bioimaging probes combining high brightness with molecular tunability. While their fluorescence performance is well established for one-photon excitation, their single-particle properties under two-photon excitation remain to be evaluated. Here, we perform a comprehensive optical characterization of two distinct dFONs designed to exhibit the same two-photon brightness but with different photostabilities. Saturation measurements of individual nanoparticles allowed for an estimation of their two-photon absorption cross sections, which were found to be consistent with ensemble values when taking into account a local-field correction. Time-resolved experiments further revealed that nanoparticles with the highest absorption cross section photobleach significantly faster, confirming the trade-off between absorption efficiency and photostability. These results demonstrate that the photophysical behavior of dFONs can be rationally engineered at the molecular level and provide design principles for the development of optimized organic nanoparticles for nonlinear fluorescence microscopy and bioimaging applications.