DNA-PAINT Imaging

Super-resolution imaging via DNA-PAINT was performed using an inverted wide-field fluorescence microscope (IX-71; Olympus). For excitation of Cy5 and Cy7, a 641 nm diode laser (Cube 640-100C, Coherent) in combination with a clean-up filter (laser clean-up filter 640/10, Chroma) was used. The laser beam was focused onto the back focal plane of the oil-immersion objective (60×, NA 1.45; Olympus). Emission light was separated from the illumination light using a dichroic mirror (HC 560/659; Semrock) and spectrally filtered by a band-pass filter (FF01-679/41-25, Semrock). Images were recorded with an electron-multiplying CCD camera chip (iXon DU-897; Andor). Pixel size for data analysis was measured to 128 nm. For DNA-PAINT measurement (Figure ​Figure66), 50 000 images with an exposure time of 100 ms (frame rate 10 Hz) and irradiation intensity of 0.5 kW cm^–2 were recorded. For DNA-PAINT measurement (Figure S19), 24 000 images with an exposure time of 20 ms (frame rate 50 Hz) and irradiation intensity of 1.5 kW cm^–2 were recorded. Microtubules were imaged by TIRF microscopy. Experiments were performed in imaging buffer (Figure ​Figure66b and Figure S19b) or imaging buffer supplemented with 10 mM histidine (Figure ​Figure66c and Figure S19a,c) at pH 7.4. All DNA-PAINT results were analyzed with rapidSTORM3.3.⁴⁶ The resolution was estimated via the Fourier ring correlation (FRC)²⁸ plug-in in Fiji with the threshold set to 1/7.⁴⁸