C nstrom quad 405 488 561 647 filter

Two-color dSTORM imaging was conducted both for fixed and live cells.
Fixed cells were suspended in a STORM imaging buffer^{51 (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.

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).