Two-photon Dye-Based Fluorogenic Organic Nanoparticles as Intracellular Thiols Sensors

Optical bioimaging is an ever-growing field that benefits both from the fast progress of optical instrumentation and modalities, and from the development of light-emitting probes. The efficacy of molecular fluorescent dyes is crucial, yet hindered by limited brightness and hydrophilicity. Addressing these challenges, self-stabilized fluorogenic organic nanoparticles only made of pure dyes (dFONs) are introduced in this work. Comprising thiol-sensitive fluorogenic chromophores, these dFONs exhibit enhanced brightness exclusively in the presence of biological thiols, notably glutathione, overcoming the need for water-solubilizing moieties. Importantly, these nanoparticles demonstrate large fluorescence and one- and two-photon brightness, enabling sensitive bioimaging of intracellular thiols at micromolar concentrations. Notably, only the pristine fluorogenic nanoparticles can penetrate the cells and does not require to wash the cells before imaging, emphasizing their unique role as dye carriers, fluorogenic probes and ease of use. This work highlights the transformative potential of dFONs in advancing optical bioimaging, paving the way for the use of dFONs not just as tracers, but also now as biosensors and ultimately in the future as biomarkers. The study introduces self-stabilized fluorogenic organic nanoparticles composed entirely of thiol-sensitive dyes, which fluoresce exclusively in the presence of biological thiols, notably glutathione. These sensors exhibit significant one- and two-photon brightness, allowing sensitive bioimaging of intracellular thiols at micromolar levels. Uniquely, only the pristine nanoparticles can penetrate cells, serving as efficient dye carriers and wash-free fluorogenic probe. image