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Rhodamine derivatives and analogues have been widely used for different super-resolution imaging techniques, including photoactivated localization microscopy (PALM). Among them, rhodamine spirolactams exhibit great superiority for PALM imaging due to a desirable bright-dark contrast during the photochromic switching process. Although considerable attention has been paid to the chemical modifications on rhodamine spirolactams, the on-time of photochromic switching, one of the key characteristics for PALM imaging, has never been optimized in previous developments. In this study, we proposed that simply installing a carboxyl group close to the lactam site could impose an intramolecular acidic environment, stabilize the photoactivated zwitterionic structure and thus effectively increase the on-time. Based on this idea, we have synthesized a new rhodamine spirolactam, Rh-Gly, that demonstrated considerably longer on-time than the other tested analogues, as well as an enhancement of single molecule brightness, an improvement on signal-to-noise ratio and an enlargement of total collected photons of single molecule before photobleach. Finally, super-resolution images of live cell mitochondria stained with Rh-Gly have been obtained with a good temporal resolution of 10 s, as well as a satisfactory localization precision of ~25 nm. Through self-labeling protein tags, Rh-Gly modified with HaloTag ligand enabled super-resolution imaging of histone H2B proteins in live HeLa cells; through immunostaining antibodies labeled with an isothiocyanate-substituted Rh-Gly, super-resolution imaging of microtubules was achieved in fixed cells. Therefore, our simple and effective strategy provides novel insight for developing further enhanced rhodamine spirolactams recommendable for PALM imaging.
This article was published in the following journal.
Name: Journal of the American Chemical Society
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