Single-Molecule Localization Microscopy and Tracking with a Fluorescent Mechanosensitive Probe.

A milestone in optical imaging of mechanical forces in cells has been the development of the family of flipper fluorescent probes able to report membrane tension noninvasively in living cells through their fluorescence lifetime. The specifically designed Flipper-CF3 probe with an engineered inherent blinking mechanism was recently introduced for super-resolution fluorescence microscopy of lipid ordered membranes but was too dim to be detected in lipid disordered membranes at the single-molecule level (García-Calvo, J. J. Am. Chem. Soc. 2020, 142(28), 12034-12038). We show here that the original and commercially available probe Flipper-TR is compatible with single-molecule based super-resolution imaging and resolves both liquid ordered and liquid disordered membranes of giant unilamellar vesicles below the diffraction limit. Single probe molecules were additionally tracked in lipid bilayers, enabling to distinguish membranes of varying composition from the diffusion coefficient of the probe. Differences in brightness between Flipper-CF3 and Flipper-TR originate in their steady-state absorption and fluorescence properties. The general compatibility of the Flipper-TR scaffold with single-molecule detection is further shown in super-resolution experiments with targetable Flipper-TR derivatives.

HydroFlipper membrane tension probes: imaging membrane hydration and mechanical compression simultaneously in living cells.

HydroFlippers are introduced as the first fluorescent membrane tension probes that report simultaneously on membrane compression and hydration. The probe design is centered around a sensing cycle that couples the mechanical planarization of twisted push-pull fluorophores with the dynamic covalent hydration of their exocyclic acceptor. In FLIM images of living cells, tension-induced deplanarization is reported as a decrease in fluorescence lifetime of the dehydrated mechanophore. Membrane hydration is reported as the ratio of the photon counts associated to the hydrated and dehydrated mechanophores in reconvoluted lifetime frequency histograms. Trends for tension-induced decompression and hydration of cellular membranes of interest (MOIs) covering plasma membrane, lysosomes, mitochondria, ER, and Golgi are found not to be the same. Tension-induced changes in mechanical compression are rather independent of the nature of the MOI, while the responsiveness to changes in hydration are highly dependent on the intrinsic order of the MOI. These results confirm the mechanical planarization of push-pull probes in the ground state as most robust mechanism to routinely image membrane tension in living cells, while the availability of simultaneous information on membrane hydration will open new perspectives in mechanobiology.

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.

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.

Universal quenching of common fluorescent probes by water and alcohols.

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 of 42 common organic fluorophores recommended for biological labelling, we demonstrate that H2O reduces their fluorescence quantum yield and lifetime by up to threefold and uncover the underlying fluorescence quenching mechanism. We show that the quenching efficiency is significantly larger for red-emitting probes 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 emission, whereas it 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 and combination transitions of high-frequency vibrational stretching modes of the solvent through space and not through hydrogen bonds. Insight into this solvent-assisted quenching mechanism opens the door to the rational design of brighter fluorescent probes by offering a justification for protecting organic fluorophores from the solvent via encapsulation.