One approach to super-resolution fluorescence imaging uses sequential activation and localization of individual fluorophores to achieve high spatial resolution. Essential to this technique is the choice of fluorescent probes; the properties of the probes, including photons per switching event, on-off duty cycle, photostability and number of switching cycles, largely dictate the quality of super-resolution images. Although many probes have been reported, a systematic characterization of the properties of these probes and their impact on super-resolution image quality has been described in only a few cases. Here we quantitatively characterized the switching properties of 26 organic dyes and directly related these properties to the quality of super-resolution images. This analysis provides guidelines for characterization of super-resolution probes and a resource for selecting probes based on performance. Our evaluation identified several photoswitchable dyes with good to excellent performance in four independent spectral ranges, with which we demonstrated low–cross-talk, four-color super-resolution imaging.
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We thank C. Blackstone (US National Institutes of Health) for the myc-ATL1 construct. This work is supported in part by the US National Institutes of Health (to X.Z.). J.C.V. is supported in part by a Burroughs-Wellcome Career Award at the Scientific Interface. K.H.C. acknowledges a National Science Scholarship from the Agency for Science, Technology and Research of Singapore. X.Z. is funded by the Howard Hughes Medical Institute.
The authors declare no competing financial interests.
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Dempsey, G., Vaughan, J., Chen, K. et al. Evaluation of fluorophores for optimal performance in localization-based super-resolution imaging. Nat Methods 8, 1027–1036 (2011). https://doi.org/10.1038/nmeth.1768
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