Transforming Rhodamine Dyes for (d)STORM Super-Resolution Microscopy via 1,3-Disubstituted Imidazolium Substitution

We introduce a strategy to optimize the photoswitching behavior of rhodamines for (d)STORM super-resolution microscopy. By replacing the benzene ring in the rhodamine core with a permanently charged 1,3-disubstituted imidazolium, the resultant dyes are markedly sensitized toward photoswitching, and exhibit outstanding (d)STORM performance with fast on-off switching, long-lasting blinking, and bright single-molecule emission. We thus attain excellent (d)STORM images under green excitation that are on par with the ideal red-excited dyes, including for difficult structures such as the mammalian actin cytoskeleton, and demonstrate high-quality two-color three-dimensional (d)-STORM.

Automated summary

We propose a strategy to optimize the photoswitching behavior of rhodamines for (d)STORM super-resolution microscopy. By replacing the benzene ring with a permanently charged 1,3-disubstituted imidazolium, the resulting dyes show enhanced photoswitching capability and exhibit excellent performance in (d)STORM with fast on-off switching, long-lasting blinking, and bright single-molecule emission. Our method achieves high-quality (d)STORM images under green excitation that are comparable to those obtained using ideal red-excited dyes, even for challenging structures like the mammalian actin cytoskeleton. We also demonstrate high-quality two-color three-dimensional (d)-STORM.