Super-Resolution Imaging of Single Molecules

dSTORM imaging was performed using an Elyra PS.1 microscope (Carl Zeiss Microscopy) equipped with a Plan-Apochromat 100×/1.46 oil objective and a liquid cooled EMCCD camera (Andor Technology). Imaging was carried out in MEA imaging buffer as previously described (36 (link)). In short, fresh stock solutions (1 M cysteamine in 360 mM HCl, 10% glucose in PBS, 70 mg/ml glucose oxidase in PBS, and 20 mg/ml catalase in PBS) were prepared the day before imaging and stored at 4°C and mixed directly before imaging to final concentrations of 0.124 M cysteamine (Sigma), 44.8 mM HCl, 8.6% glucose, 1.08 mg/ml glucose oxidase from Aspergillus niger (Sigma), and 0.0773 mg/ml catalase from bovine liver (Sigma) in PBS. Imaging was performed in 12.8 × 12.8-μm areas in an inclined total internal reflection fluorescence microscope mode (37 (link)). Single molecule fluorescence detection on the EMCCD camera was acquired with 100 × 100-nm pixel size, 20-ms Exposure time, and 100 Gain. 20,000 image frames were acquired for each channel. Both channels were imaged sequentially in 500 frame sequences and the appropriate filters and lasers for each dye were used (642 nm for Alexa Fluor 647 and 488 nm for Atto 488). The images were analyzed with the ImageJ plugin SMLocalizer (38 (link)).