Red-Emitting Fluorophores as Local Water-Sensing Probes

Fluorescent probes are known for their ability to sense changes in their direct environment. We introduce here the idea that common red-emitting fluorophores recommended for biological labeling and typically used for simple visualization of biomolecules can also act as reporters of the water content in their first solvent sphere by a simple measurement of their fluorescence lifetime. Using fluorescence spectroscopy, we investigated the excited-state dynamics of seven commercially available fluorophores emitting between 650 and 800 nm that are efficiently quenched by H₂O. The amount of H₂O in their direct surrounding was modulated in homogeneous H₂O-D₂O mixtures or, in heterogeneous systems, by confining them into reverse micelles, by encapsulating them into host-guest complexes with cyclodextrins, or by attaching them to peptides and proteins. We found that their fluorescence properties can be rationalized in terms of the amount of H₂O in their direct surroundings, which provides a general mechanism for protein-induced fluorescence enhancements of red-emitting dyes and opens perspectives for directly counting water molecules in key biological environments or in polymers.