Fluorescent Membrane Tension Probes for Super-Resolution Microscopy: Combining Mechanosensitive Cascade Switching with Dynamic-Covalent Ketone Chemistry

We report the 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.