Cfi plan apochromat λ 100x

3D-STORM imaging (Huang et al., 2008 (link); Rust et al., 2004 (link)) and epifluorescence imaging were performed on a homebuilt setup based on a Nikon Eclipse Ti-E inverted optical microscope using an oil immersion objective (Nikon CFI Plan Apochromat λ 100x, N/A = 1.45). Lasers at 647nm (MPB Communications) and 560 nm (MPB Communications) were coupled into an optical fiber after an acousto-optic tunable filter and then introduced into the sample through the back focal plane of the microscope. The 560nm laser (~0.2Wcm−2) was used for epifluorescence imaging of Alexa Fluor 555. For STORM of Alexa Fluor 647, the laser beams were shifted towards the edge of the objective so that emerging light reached the sample at incidence angles slightly smaller than the critical angle of the glass–water interface. Continuous illumination of 647-nm laser (~2kWcm⁻²) was used to excite fluorescence from labeled dye molecules and switch them into the dark state. For 3D-STORM imaging, a cylindrical lens was inserted into the imaging path so that images of single molecules were elongated in x and y for molecules on the proximal and distal sides of the focal plane (relative to the objective), respectively (Xu et al., 2013 (link)). Imaging buffer was Tris-HCl (pH 7.5) containing 100mM cysteamine, 5% glucose, 0.8mg/mL glucose oxidase, and 40μg/mL catalase.