Two-color dSTORM imaging was conducted both for fixed and live cells.
Fixed cells were suspended in a STORM imaging buffer51 (link),52 (link). Live cells were suspended in Imaging buffer (RPMI without phenol red, 10% FBS, 25 mM HEPES.The imaging was performed using a total internal reflection (TIRF) Nikon microscope with a CFI Apo TIRF ×100 oil objective (NA 1.49, WD 0.12 mm). Imaging in TIRF mode served to visualize molecules at the PM of spreading cells in close proximity to the coverslip (up to ~100–200 nm). Fluorophores were activated using a low intensity laser illumination at 405 nm (~0.5% of 30 mW in maximum), and sequentially imaged in a following frame using laser excitation at either 488 nm, or 647 nm (80–100% of 90 mW in 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). For two-color SMLM imaging, we used immunostaining as specified for each experiment.
Fixed cells were suspended in a STORM imaging buffer51 (link),52 (link). Live cells were suspended in Imaging buffer (RPMI without phenol red, 10% FBS, 25 mM HEPES.The imaging was performed using a total internal reflection (TIRF) Nikon microscope with a CFI Apo TIRF ×100 oil objective (NA 1.49, WD 0.12 mm). Imaging in TIRF mode served to visualize molecules at the PM of spreading cells in close proximity to the coverslip (up to ~100–200 nm). Fluorophores were activated using a low intensity laser illumination at 405 nm (~0.5% of 30 mW in maximum), and sequentially imaged in a following frame using laser excitation at either 488 nm, or 647 nm (80–100% of 90 mW in 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). For two-color SMLM imaging, we used immunostaining as specified for each experiment.
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