Stable ultrabright nanoprobes for two-photon excitation microscopy based on octupolar merocyanine-loaded nanovesicles

Two-photon microscopy is a powerful technique for deep-tissue and in vivo fluorescence imaging, yet its full potential is often constrained by the low two-photon absorption efficiency of conventional fluorophores. In this work, for the first time non-liposomal, unilamellar quatsome nanovesicles were loaded with octupolar fluorophores. Molecular engineering of centrosymmetric merocyanines led to dyes emitting in the 600-700 nm range with a fluorescence quantum yield ≈ 0.16 in polar solvents. Centrosymmetric architecture made it possible to perform heretofore unachievable systematic investigation of the impact of hydrophobic and hydrophilic (triethylene glycol) moieties on the stability of nanocarriers. It was discovered that two long alkyl chains are prerequisites to achieving stable incorporation of dyes within nanovesicles. Through systematic formulation screening, we identified a lead nanoprobe exhibiting high brightness, in the order of 10⁷ M^-1 cm^-1, combined with strong and broad nonlinear optical properties leading to large two-photon brightness, in the order of 10⁴ GM, in a highly stable nanoparticle. The performance of these very bright nanoprobes in two-photon microscopy was assessed in ex vivo permeation studies using porcine tissues, demonstrating their potential for bioimaging applications. This work underscores the synergy between molecular design and nanocarrier engineering in advancing next-generation fluorescent probes for nonlinear optical imaging.