Universal Quenching of Standard Organic Fluorophores by Water and Alcohols through Resonance Energy Transfer

Abstract

Although biological imaging is mostly performed in aqueous media, it is often overlooked that water is a known fluorescence quencher. In this contribution, we show that H2O quenches the fluorescence of over 40 common organic fluorophores recommended for biological labelling by up to threefold. We find that the quenching efficiency is higher with red-emitting fluorophores and follows an energy gap law. The fluorescence quenching finds its origin in high-energy vibrations of the solvent (OH groups), as methanol and other linear alcohols are also found to quench the fluorescence which is restored in deuterated solvents. Our observations are consistent with a mechanism by which the electronic excitation of the fluorophore is resonantly transferred to overtones of vibrational stretching modes of the solvent through space and not through bond. H2O and methanol indeed weekly absorb through vibrational combination bands in the red part of the visible spectrum where the fluorescence quenching is highest. Our findings open the door to the rational design of brighter fluorescent probes and improved methods for single-molecule and super-resolution imaging, which we will briefly elude to.