Ultrabright Föster Resonance Energy Transfer Nanovesicles: The Role of Dye Diffusion

The development of contrast agents based on fluorescent nanoparticles with high brightness and stability is a key factor to improve the resolution and signal-to-noise ratio of current fluorescence imaging techniques. However, the design of bright fluorescent nanoparticles remains challenging due to fluorescence self-quenching at high concentrations. Developing bright nanoparticles showing FRET emission adds several advantages to the system, including an amplified Stokes shift, the possibility of ratiometric measurements, and of verifying the nanoparticle stability. Herein, we have developed Förster resonance energy transfer (FRET)-based nanovesicles at different dye loadings and investigated them through complementary experimental techniques, including conventional fluorescence spectroscopy and super-resolution microscopy supported by molecular dynamics calculations. We show that the optical properties can be modulated by dye loading at the nanoscopic level due to the dye's molecular diffusion in fluid-like membranes. This work shows the first proof of a FRET pair dye's dynamism in liquid-like membranes, resulting in optimized nanoprobes that are 120-fold brighter than QDot 605 and exhibit >80% FRET efficiency with vesicle-to-vesicle variations that are mostly below 10%.