Samples were imaged in the presence of a reducing agent to enable reversible photoswitching of the fluorophores. The imaging buffer consisted of 100 mM β-mercaptoethylamine (Sigma-Aldrich, Steinheim, Germany) in PBS (pH 7.4 to 7.8), as previously described (32). Image acquisition was performed on an inverted fluorescence wide-field setup custom-built around an Olympus IX-71 microscope equipped with an oil-immersion objective [APON 60×; numerical aperture (NA), 1.49; Olympus, Tokyo, Japan], a nose-piece stage (IX2-NPS, Olympus) to reduce stage vibration and drift, 514-nm (500 mW) and 639-nm (1000 mW) solid-state lasers (OPSL, Genesis MX STM-Series, Coherent, Santa Clara, CA, USA), and a suitable dichroic mirror (R442/514/635, Chroma, Bellows Falls, Vermont, USA). The fluorescence emission of Alexa Fluor 647 and Alexa Fluor 532 dyes was acquired sequentially and projected on two separate electron-multiplying charge-coupled device (EMCCD) cameras (iXon Ultra 897, Andor, Belfast, Northern Ireland) by means of a dichroic mirror (630 DCXR customized, Chroma) and two bandpass filters (582/75 and 679/41 BrightLine series, Semrock, Rochester, NY, USA). The excitation intensity ranged from 1 to 5 kW/cm2, depending on the fluorophore and labeling density. For each color, 40,000 frames were acquired at 60 Hz. This single-molecule localization data were analyzed using the open source software rapidSTORM (version 3.3). The fitting process and the reconstruction of super-resolved images were performed as previously described (60). Localization precision was estimated on the basis of a nearest neighborhood analysis approach (see Supplementary Results for details) (61).

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