Two-color dSTORM imaging was conducted both for fixed and live cells. Cells were suspended in a STORM imaging buffer [16 (link),17 (link)]. Imaging cells on coverslips was performed using a Nikon microscope with a CFI Apo TIRF X100 oil objective (NA 1.49, WD 0.12 mm).
Imaging of cells on cells was performed above the coverslip without using the ‘perfect focus’ feature of the microscope. Fluorophores were imaged in a following frame using laser excitation at either 488 nm, or 647 nm (~50% of 90 mW maximum for 488 nm or 200 mW for 647 nm). Laser illumination at all wavelengths covered a circular area with a diameter of 80 µm at the sample. dSTORM acquisition sequence typically took ~2.5 min at 13.4 fps of an EMCCD Ixon+ camera. The pixel size was equivalent to 160 × 160 nm2 at the sample. Excitation and imaging were performed through a quad dichroic (C-NSTROM QUAD 405/488/561/647/FILTER; Nikon, Tokyo, Japan).
Imaging of cells on cells was performed above the coverslip without using the ‘perfect focus’ feature of the microscope. Fluorophores were imaged in a following frame using laser excitation at either 488 nm, or 647 nm (~50% of 90 mW maximum for 488 nm or 200 mW for 647 nm). Laser illumination at all wavelengths covered a circular area with a diameter of 80 µm at the sample. dSTORM acquisition sequence typically took ~2.5 min at 13.4 fps of an EMCCD Ixon+ camera. The pixel size was equivalent to 160 × 160 nm2 at the sample. Excitation and imaging were performed through a quad dichroic (C-NSTROM QUAD 405/488/561/647/FILTER; Nikon, Tokyo, Japan).
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