Super-resolution Imaging of Live and Fixed Cells

Super-resolution imaging experiments were performed on live samples (Fig. 2j, Supplementary Fig. 2c, Supplementary Fig. 4d, Supplementary Fig. 4g) and fixed cells (Fig. 1i, Fig. 2b, Supplementary Fig. 2a, Supplementary Fig. 4a). For live-cell dSTORM imaging the cells were labeled, washed, and imaged directly in DMEM–FBS. For fixed cell preparations, cells were labeled, washed, and fixed in 4% paraformaldehyde (Electron Microscopy Sciences) in PBS buffer (pH = 7.5). The cells were imaged in a sealed cell chamber (Life Technologies) containing nitrogen-degassed redox buffer consisting of PBS supplemented with 50 mM mercaptoethylamine (Sigma–Aldrich), 10% w/v glucose, 0.5 mg/mL glucose oxidase (Sigma–Aldrich), and 28400 U/mL catalase (Sigma–Aldrich). Before imaging, JF₅₄₉ could be efficiently “shelved” in a dark state upon illumination with 2 kW·cm⁻² of excitation light (561 nm), and then activated back to a fluorescent state by blue light (405 nm) with low intensity (~20·W cm⁻²). JF₆₄₆ fluorophores were converted into a predominately dark state using continuous illumination of 637 nm excitation light at 14 kW·cm⁻², after which individual rapidly blinking molecules of JF₆₄₆ fluorophores were observed. These experiments were conducted on the two wide-field microscope systems described above: the Nikon Eclipse Ti epifluorescence microscope (Fig. 1i, Fig. 2j, Supplementary Fig. 2a, Supplementary Fig. 2c, Supplementary Fig. 4g), and the custom-built three-camera microscope with an ASI RAMM frame (Fig. 2b, Supplementary Fig. 4a, Supplementary Fig. 4d).