3D-SIM imaging was performed with a DeltaVision V4 OMX microscope equipped with a ×100/1.4 numerical aperture (NA) Plan Super Apochromat oil immersion objective (Olympus) and electron-multiplying charge-coupled device (EMCCD; Evolve 512B, Photometrics) cameras for a pixel size of 80 nm. Diode lasers at 405, 488, 561 and 647 nm were used with the standard corresponding emission filters. Z-stacks were acquired by scanning the sample in the axial direction (z-step of 125 nm) using five phases and three angles per image plane. Raw images were reconstructed using SoftWorx (version 6.5, GE Healthcare) using channel-specific optical transfer functions (pixel size of reconstructed images = 40 nm). Quality of reconstructed images was assessed using the SIMcheck plugin of ImageJ46 (link). Conventional wide-field images were generated from raw images by averaging angles and phases for each plane.
STED imaging was performed with an Expert line (Aberrior instruments) using an X83 inversed microscope equipped with a ×100/1.4 NA oil immersion objective (Olympus). FISH probes labelled with ATTO-647 were observed by using a 640 nm excitation laser line at 40% of power combined with a depletion completed with a 775 nm laser at 30 % of power. Images were collected with a pixel size of 20 nm and a dwell time of 10 μs with an averaging of 2.
STED imaging was performed with an Expert line (Aberrior instruments) using an X83 inversed microscope equipped with a ×100/1.4 NA oil immersion objective (Olympus). FISH probes labelled with ATTO-647 were observed by using a 640 nm excitation laser line at 40% of power combined with a depletion completed with a 775 nm laser at 30 % of power. Images were collected with a pixel size of 20 nm and a dwell time of 10 μs with an averaging of 2.
