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. 2017 Aug 16;7:8450. doi: 10.1038/s41598-017-08493-x

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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CRISPR-Cas9 mediated PALM is a powerful biological tool for probing protein interactions and live cell imaging. (a) mEos 3.2 tagging of the TubA1B gene allows for convenient multiplexing with high quality antibody probes for multi-colour dSTORM/PALM using the highest performing secondary, Alexa 647. TTLL10, a tubulin ligase is tagged with Alexa 647 and imaged in a Hel 92.1.7 mEos 3.2-TubA1B clone, providing super-resolved imaging of two proteins which are spatially distinct with resolution in the 10 s of nanometers, as indicated by red arrows. (b) The switching properties of mEos 3.2 can be further exploited to enhance sub-pixel radiality needed for high quality SRRF (super-resolved radial fluctuation) imaging, and thus for super-resolved imaging at high temporal resolution with minimal phototoxicity. The lower panel show averaged images from 50 frames of a TIRF image stream and the corresponding SRRF image. (10 μm scale in whole cell images, 1 μm scale in cropped fields).