Cell-permeable organic fluorescent probes for live-cell super-resolution imaging

Abstract

The recent development of super-resolution microscopies has allowed the diffraction-limited resolution to be surpassed and biological structures to be imaged with resolution as high as about 20 nm. These super-resolution imaging approaches are providing new insights into cell biology. Use of bright and anti-photobleaching organic fluorophores to specifically label biomolecules inside live cells will greatly benefit live-cell fluorescence microscopy, especially live-cell super-resolution fluorescence microscopy. Although many small-molecule organic fluorophores with excellent optical properties have been commercialized, necessary cell permeability, particularly after conjugation with a recognition unit, severely restricts the applications of these organic fluorophores in live-cell imaging. Here we develop general methods for delivering cell-impermeable organic fluorescent probes into living cells. By using our delivering methods, cell-impermeable fluorescent probes containing various cell-impermeable fluorescent dyes (e.g., Alexa Fluor dyes, Atto dyes, and Cy dyes,), not only were able to be delivered into living cells efficiently but also can specifically label various intracellular organelles. We anticipate that these fluorescent probes and our delivery strategies will be helpful tools for live-cell super-resolution imaging and substantially benefit characterizations of dynamic physical interactions between intracellular organelles in living cells.