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ACS Chem Biol. 2015 Jun 19;10(6):1411-6. doi: 10.1021/acschembio.5b00046. Epub 2015 Apr 1.

A simple method for GFP- and RFP-based dual color single-molecule localization microscopy.

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†Randall Division of Cell and Molecular Biophysics, King's College London, London SE1 1UL, United Kingdom.
§Institute of Biochemistry, ETH Zurich, Otto-Stern-Weg 3, 8093 Zurich, Switzerland.
‡Institute for Chemistry and Biochemistry, Freie Universität Berlin, Thielallee 63, 14195 Berlin, Germany.


The recent development of single-molecule localization-based super-resolution techniques has afforded a resolution in the nanometer range in light microscopy. The ability to resolve biological structures on this scale by multicolor techniques faces significant challenges which have prevented their widespread use. Here, we provide a generic approach for high-quality simultaneous two-color single-molecule localization microscopy imaging of any combination of GFP- and RFP-tagged proteins with the use of nanobodies. Our method addresses a number of common issues related to two-color experiments, including accuracy and density of labeling as well as chromatic aberration and color-crosstalk with only minimal technical requirements. We demonstrate two-color imaging of various nanoscopic structures and show a compound resolution down to the limit routinely achieved only in a single color.

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