Fluorogenic dimers as bright switchable probes for enhanced super-resolution imaging of cell membranes
Résumé
Super-resolution fluorescence imaging based on single-molecule localization microscopy (SMLM) enables visualizing cellular structures with nanometric precision. However, its spatial and temporal resolution largely relies on the brightness of ON/OFF switchable fluorescent dyes. Moreover, in cell plasma membranes the single-molecule localization is hampered by the fast lateral diffusion of membrane probes. Here, to address these two fundamental problems, we propose a concept of ON/OFF switchable probes for SMLM (points accumulation for imaging in nanoscale topography, PAINT) based on fluorogenic dimers of bright cyanine dyes. In these probes, the two cyanine units connected with a linker were modified at their extremities with low-affinity membrane anchors. Being self-quenched in water due to intramolecular dye H-aggregation, they displayed light up on reversible binding to lipid membranes. Charged group in the linker further decreased the probe affinity to the lipid membranes, thus accelerating its dynamic reversible ON/OFF switching. The concept was validated on cyanines 3 and 5. SMLM of live cells revealed that the new probes provided higher brightness and ~10-fold slower diffusion at the cell surface, compared to reference probes Nile Red and DiD, which boosted axial localization precision of biomembrane imaging >3-fold down to 31 nm. The new probe allowed unprecedented observation of nanoscale fibrous protrusions on plasma membranes of live cells with 40-s time resolution, revealing their fast dynamics. Thus, going beyond the brightness limit of single switchable dyes by cooperative de-quenching in fluorogenic dimers and slowing down probe diffusion in biomembranes open the route to significant enhancement of superresolution fluorescence microscopy of live cells.
Domaines
Chimie
Fichier principal
MS-Dimer-PAINT-final.pdf (1.29 Mo)
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SI-Dimer-PAINT-final.pdf (2.09 Mo)
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