Live Cell Imaging of ER Architecture

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.