Fluorescence microscopy and spectroscopy are nowadays standard tools to address biological questions. Small organic fluorescent probes are frequently chemically linked to biomolecules of interest to enable their detection in an aqueous environment. Although many fluorophores approved for biological applications are rather insensitive to their environment, i.e they display little solvatochromism and small Stokes shifts, water is nonetheless known to quench their emission. The fluorescence may efficiently be restored in D₂O, which leads to a better contrast and an improved localization precision in single-molecule based super-resolution imaging experiments [1, 2].

In this contribution, we explore the quenching of 40 organic fluorophores by water and alcohols and explain why solvent-assisted quenching is stronger for red-emitting fluorophores. The efficiency of the quenching can indeed be related to the spectral overlap between the emission of fluorophores with the absorption of  water in the 700-800 nm region originating from overtone and combination bands of the vibrational modes of OH groups, thus enabling through-space resonant energy transfer [3]. Inspired by those observations, we further selected a handful of red-emitting fluorophores to explore the sensitivity of their fluorescence properties to water more quantitatively. We demonstrate that oxazine fluorophores are particularly well suited to act as nanoscopic water sensors both in homogeneous solvent mixtures and in complex environments such as in reverse micelles, in host-guest complexes, and on protein surfaces [4]. We directly relate the fluorescence lifetime of the probe to the average quantity of water in contact with the fluorophore by defining a residual quenching fraction, which brings us one step closer to our goal of using red-emitting fluorophores to count water molecules.

[1]  S. F. Lee, Q. Vérolet, A. Fürstenberg, Angew. Chem. Int. Ed. 2013, 52, 8948-8951.
[2]  K. Klehs, C. Spahn, U. Endesfelder, S. F. Lee, A. Fürstenberg, M. Heilemann, ChemPhysChem 2014, 15, 637-641.
[3]  J. Maillard, K. Klehs, C. Rumble, E. Vauthey, M. Heilemann, A. Fürstenberg, Chem. Sci. 2021, 12, 1352–1362.
[4]  J. Maillard, C. Rumble, A. Fürstenberg, submitted.