dSTORM Imaging

Two-color dSTORM images were acquired on an inverted wide-field fluorescence microscope (Olympus, IX-71) with a 639 nm diode laser (Coherent, Genesis MX639-1000; Chroma) and a 561 nm diode laser (Coherent, Genesis MX561-500) for excitation of Cy5 and CF568, respectively. The lasers were spectrally cleaned (Chroma, Laser Clean-up filter 640/10 and 561/14) overlaid by a LaserMUX filter (Semrock, LM01-552), widened, and focused on the back-focal plane of an oil immersion objective (Olympus, APON 60× NA 1.45) by a lens assembly of a 25 mm (Qioptiq, G322284000) and a 120 mm (Qioptiq, G322303000) focal achromatic lens. Emission light was separated from excitation light by a polychromatic mirror (Chroma, FF410/504/582/669). Cy5 and CF568 emission was split and filtered by a dichroic beam splitter (Chroma, 630dcxr) and two bandpass filters (Shemrock, HC679/41 and BP607/70) mounted in a dual-camera adaptor (Andor, TuCam). For detection, two EMCCD cameras (Andor, iXon Ultra 897) were used with a measured pixel size of 128 nm for the Cy5 channel and 132 nm for the CF568 channel. Prior to two-color dSTORM imaging both cameras were aligned by centering fluorescent beads in the FOV of both cameras. Imaging was performed in 100 mM MEA (Sigma-Aldrich, #M6500) in PBS at a pH of 7.5–7.7. For both channels 25.000 frames were captured at 20 ms exposure time in epifluorescence at an illumination intensity of approximately 3 kW/cm². To avoid bleaching of Cy5 by 561 nm light the Cy5 channel was captured first. Image reconstruction was carried out by analyzing the image series with rapidSTORM 3.3.²² To correct for chromatic aberration between Cy5 and CF568 channels five to ten series of 100 frames of fluorescent microspheres (Thermo Fisher Scientific, #T7279) with 100 ms exposure time were captured after dSTORM imaging and reconstructed with rapidSTORM 3.3. Elastic transformations were then created using the ImageJ plugin bUnwarpJ²³ and applied to the dSTORM images. Localization data was binned to 10 nm (pixelsize), and bit-depth was scale from 0–10⁶ ADC (analog-to-digital converter) counts.