Plan apochromat 100x 1.4 psf oil immersion objective lens

SIM imaging was carried out on a DeltaVision OMX v4 Blaze microscope (GE Healthcare) equipped with 488 nm 568 nm and 647 nm lasers and a BGR-FR filter drawer (emission wavelengths 528/48 for Alexa 488, 609/37 for Alexa 568 and 683/40 for Alexa 647), a Plan Apochromat 100x/1.4 PSF oil immersion objective lens (Olympus) using an oil of 1.514 refractive index, and individual Evolve liquid-cooled EM-CCD cameras (Photometrics). A total of 15 images were acquired per section per channel (comprising 3 rotations and 5 phase shifts of the structured illumination pattern) at a z-spacing of 0.125 μm^{38 (link),39 (link)}. Structured illumination reconstruction and alignment were carried out using the SoftWoRx (GE Healthcare) program. For processing three colour 3D SIM cross-sectional view image processing Bitplane Imaris software (Oxford Instruments Company) was used.

To image the ER in living cells, MRC5 cells were grown on Mattek 35 mm dishes and transiently transfected with EGFP-ER^{6 (link)} 18 hours before imaging. A mixture of 70 ng EGFP-ER, 140 ng mCherry Rab5 and 2.59 µg of pBluescript (as carrier of DNA^{48 (link)}) were combined with 8 µl FUGENE in 280 µl OptiMEM and added to each 35 mm dish. The following day, the cells were incubated in 200 nM SiR-tubulin^{49 (link)} (Spirochrome; Stein-am-Rhein, Switzerland)) diluted in fresh EMEM/FBS media (see above) for an hour at 37 °C, 5% CO₂. Cells were then imaged using a DeltaVision OMX microscope (GE Healthcare) in wide-field mode, at 37 °C and 5% CO₂. All channels were acquired simultaneously using three independent Evolve liquid cooled EMCCD cameras (Photometrics), but only the ER images were used here. EGFP was imaged with a 488 nm band of excitiation light from a solid state illuminator together with the BGR-FR filter drawer containing a quad-band dichroic cascade and 528/48 nm emission filter. An Olympus Plan Apochromat 100x/1.4 PSF oil immersion objective lens was used (1.514 refractive index immersion oil). A sequence of 400, 16 bit, 512 × 512 pixel images were collected at a frame interval of 115.15 ms, at 12.983 pixels/µm. Image sequences after acquisition were processed in Fiji.