All microscopy experiments were carried out with a custom-made superresolution setup based on an inverted Zeiss Axiovert 200 body equipped with a Zeiss Apochromat 100×/1.45 NA oil-immersion objective (Zeiss, Oberkochen, Germany). We used a 637 nm Coherent OBIS laser for excitation and an iXon Ultra 897 EM-CCD camera (Andor, Belfast, UK). Fluorescence microscopy measurements were carried out with a freshly prepared dSTORM buffer according to van de Linde et al. [21 (link)]: 500 µL 20% glucose, 200 µL 10× PBS, 230 µL distilled water, 50 µL 1 M cysteamine, 10 µL 50 mg/mL glucose oxidase, 10 µL 12.6 mg/mL catalase. The buffer was added to each chamber immediately before imaging. For each SMLM image, we recorded 40,000 frames in epi-configuration, whereby the first 10,000 were not included in the analysis. The illumination time was 1 ms and the delay between consecutive frames was 7 ms. The raw data was pre-processed with temporal filtering to separate the slowly bleaching background from the rapidly flashing signals [10 (link)], and single-molecule signals were localized using Daostorm [22 (link)]. Finally, images were displayed utilizing a Gaussian kernel density estimator.
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