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Evaluation of fluorophores for optimal performance in localization-based super-resolution imaging

Abstract

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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Figure 1: Principle of single-molecule localization–based super-resolution imaging and modes of switching used for this imaging method.
Figure 2: Quantitative probe characterization for STORM imaging.
Figure 3: Alexa Fluor 647 and Dyomics 654 resolve the hollow structure of immunostained microtubules.
Figure 4: Four-color STORM imaging of in vitro assembled microtubule filaments and cross-talk analysis.
Figure 5: Four-color STORM imaging of cellular structures.

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Acknowledgements

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.

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G.T.D., J.C.V., K.H.C. and X.Z. designed the experiments. G.T.D., J.C.V., K.H.C. and M.B. performed the experiments. G.T.D., J.C.V. and K.H.C. performed the data analysis and interpretation. G.T.D., J.C.V. and X.Z. wrote the manuscript. X.Z. supervised the project.

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Correspondence to Xiaowei Zhuang.

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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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