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.
Cfi apo tirf 100 oil objective
The CFI Apo TIRF ×100 oil objective is a high-numerical aperture objective designed for total internal reflection fluorescence (TIRF) microscopy. It provides a magnification of 100x and is optimized for use with oil immersion.
4 protocols using cfi apo tirf 100 oil objective
dSTORM Imaging of 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.
Automated Multicolor Fluorescence Microscopy
Live-cell and PALM Imaging of Protein Dynamics
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.
Automated super-resolution microscopy
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