Cfi apo tirf 100 oil objective

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.

Imaging was performed on a custom build setup based on an automated Nikon Ti Eclipse microscope equipped with appropriate dichroic and filters (ET dapi/Fitc/cy3 or ZET405/488/561/640m dichroic, ZT405/488/561rpc or ZT405/488/561/640rpc rejection filter, ET610/75 or ET655LP bandpass, all AHF Analysentechnik, Germany), and a CFI Apo TIRF ×100 oil objective (NA 1.49, Nikon). The 488 nm, 561 nm, and 637 nm lasers (Coherent) was modulated via an acousto-optical tunable filter (AOTF) (Gooch and Housego, United States). Fluorescence was detected by an emCCD (iXON Ultra 888; Andor, United Kingdom). The z-focus was controlled by a commercial perfect focus system (Nikon, Germany). The sample was placed on a heating stage and kept at the constant temperature 25°C. Acquisitions were controlled by μManager (Edelstein et al., 2010 (link)).

Imaging was performed on a TIRF microscope (Nikon) 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 nm). Cells were first dropped on the coverslip in imaging buffer and given ∼10 min to spread before imaging commenced. Imaging was conducted for 20–30 cells per experiment at a rate of ∼3 min between cells, hence ∼60–90 min typically passed between the first and last cells imaged. Imaging was conducted at room temperature.
For live cell imaging, cells were illuminated by a 561 nm excitation laser at 80% power for 5,000 frames at 85 fps of an EMCCD Ixon⁺ camera. Fixed cells were further imaged by PALM. For that, they were illuminated using a 405 nm laser for photoactivation of the PAGFP using a changing intensity over the duration of the imaging sequence (typically, using 1–20% power). Illumination with a 488 nm laser at 100% power was used for PAGFP excitation.