Although biological imaging is mostly performed in aqueous media, it is hardly ever considered that water acts as a classic fluorescence quencher for organic fluorophores. By investigating the fluorescence properties...

We report design, synthesis, and evaluation of fluorescent flipper probes for single-molecule super-resolution imaging of membrane tension in living cells. Reversible switching from bright-state ketones to dark-state hydrates, hemiacetals, and hemithioacetals is demonstrated for twisted and planarized mechanophores in solution and membranes. Broadband femtosecond fluorescence up-conversion spectroscopy evinces ultrafast chalcogen-bonding cascade switching in the excited state in solution. According to fluorescence lifetime imaging microscopy, the new flippers image membrane tension in live cells with record red shifts and photostability. Single-molecule localization microscopy with the new tension probes resolves membranes well below the diffraction limit. The imaging of physical forces in living systems is a central challenge in current biology. 1 To contribute solutions, we have introduced small-molecule chemistry tools to image membrane tension in living cells. 2Based on the concept of planarizable push-pull probes, 3,4 the so-called flipper probes allowed us to image the change of plasma 5 and organellar membrane tension in living cells. 6 Flipper probes are twisted dithienothiophene (DTT) push-pull dimers (Figure 1a). Their mechanical planarization in the ground state

HydroFlippers respond to membrane compression and hydration in the same fluorescence lifetime imaging microscopy histogram: the responses do not correlate.

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 H 2 O. The amount of H 2 O in their direct surrounding was modulated in homogeneous H 2 O−D 2 O 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 H 2 O 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.