Cfi apo tirf 100 1.49 na oil immersion objective

STORM data were acquired on Nikon Ti motorized inverted microscope with Perfect Focus System, equipped with Nikon total internal reflection fluorescence (TIRF) illuminator and Prior Proscan II motorized stage, filter wheels, and shutters. For illumination, we used the output of Agilent MLC400B monolithic laser combiner with 405-, 561-, and 647-nm laser lines, reflected by a quad-band dichroic beamsplitter (ZT405/488/561/647rpc; Chroma). Emitted fluorescence was collected by a CFI Apo TIRF 100×/1.49-NA oil-immersion objective (Nikon) and filtered by the dichroic beamsplitter, additional quad-band emission filter (ZET405/488/561/647m; Chroma), and an emission filter (ET600/50m or ET700/75m; Chroma) for CF568 and AF647 emissions, respectively. The filtered emission was projected onto electron-multiplying charge-coupled device camera (Ultra DU-897U-CS0-#BV; iXon). Additional 1.5× intermediate magnification was applied, which resulted in a pixel size of 104.3 nm. NIS-Elements software was used to control the hardware.

Fixed samples were prepared by treatment with 4% platelet-activating factor for 10 min at RT, followed by permeabilization (10 min in PBS and 0.5% Triton X-100) and primary antibody staining for 2 h at RT in PBS and 1% BSA. After several washes in PBT, samples were hybridized with the Alexa Fluor 488– and 568–conjugated secondary antibodies for 1 h at RT. Superresolution imaging on prepared samples was performed with an N-SIM microscope system (Nikon) equipped with a CFI Apo TIRF 100× 1.49 N.A. oil immersion objective and a 897EMCCD Andor camera. 3D-SIM image stacks were acquired with a z-distance of 0.24 mm, covering a range of 5.76 mm (25 stacks/nucleus) using laser power setting 25% and 100-ms exposition time. With this setting, 15 raw images per plane were acquired and computationally reconstructed using the reconstruction slice system from NIS-Elements AR software (version 4.20.01; Nikon). Minimal distance measurements were performed using Volocity (Perkin Elmer) with the find spot function, which allowed us to identify the brightest spots within a 0.05-mm radius in each channel. After the measurement of minimal distance, the results were analyzed by plotting their distribution.