Deltavision omx v4 microscope

Cells grown in YES supplemented with 1 µM oestradiol were synchronized in G1 and released in the presence of 10 µM EdU. After 1 h the analogue and the oestradiol were removed from the medium by washing 3 times with equal volumes of YES. A 25 µg ml⁻¹ CBZ was added before the third mitosis after release and the cells were fixed, processed and EdU detection was performed as described above. The cells were incubated for 5 min with 0.2 µg DAPI in PBS, washed three times with PBS and mounted on 1% agarose slides. Three-dimensional SIM imaging was performed on Deltavision OMX V4 microscope equipped with an Olympus × 60 NA 1.42 objective and three PCO.edge sCMOS cameras and 405 nm, 488 nm, 568 nm and 647 nm laser lines. Cells were illuminated with a grid pattern and for each image plane, 15 raw images (five phases and three rotations) were acquired. Super-resolution images were reconstructed from the raw image files aligned and projected using Softworx software (Applied Precision, GE Healthcare).

Symbiont cell suspensions were applied on high precision coverslips (No. 1.5H, Sigma-Aldrich) coated with 0.01% (wt/vol) of Poly-L-Lysin. After letting the cell dry onto the surface of the coverslip, antifade mounting medium (Vectashield) was applied and the coverslip was sealed to a slide. 3D SIM was performed on a Delta Vision OMX v4 microscope equipped with an Olympus 60X/1.42 Oil Plan Apo N objective or an Olympus 100X/1.42 Oil Plan Apo N objective and 2 sCMOS or EMCCD cameras. The samples were excited with lasers at 488 nm, the emission was detected through emission filters 477/32 nm (Center/Bandpass). The image reconstruction and registration were performed using the SoftWoRx image software running under Linux operating system. For further image analysis of SIM image z stacks, we used Fiji (Schindelin et al., 2012 (link)) Version 2.0.0-rc-54/1.51i. Namely, brightness and contrast were adjusted, stacks were fused to a single image (z projection, maximum intensity), stacks were rotated 90° (resliced) prior z projection for the 90° side view, and videos were created via 3D projection. Regions of interest were cut out and, for uniformity, placed on a black squared background. For clarity a black triangle was placed in the corner of the lower cell in Figure 1C to cover the fluorescence of a neighboring cell. Figures were compiled using Illustrator CC (Adobe Systems Inc. USA).

3D-SIM imaging was carried out with a DeltaVision OMX V4 microscope equipped with an ×100/1.4 numerical aperture (NA) Plan Super Apochromat oil immersion objective (Olympus) and electron-multiplying charge-coupled device (Evolve 512B; Photometrics) camera 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 (z-step of 125 nm) were acquired using 5 phases and 3 angles per image plane. Raw images were reconstructed using SoftWorx v.6.5 (GE Healthcare Systems) using channel-specific optical transfer functions (pixel size of reconstructed images = 40 nm). TetraSpeck beads (200 nm) (T7280, Thermo Fisher Scientific) were used to calibrate alignment parameters between the different channels. The quality of reconstructed images was assessed using the SIMcheck plugin of ImageJ v.1.52i (Ball et al., 2015 (link)).