As previously described [61 (link)], imaging was carried out with a DeltaVision Elite (Applied Precision, Buckinghamshire, UK) comprising an Olympus IX71 inverted fluorescent microscope, and Olympus UPlanSApo ×100, NA 1.40, oil immersion objective and a CoolSNAP HQ2 camera cooled to −30 °C (Roper Scientific, Sarasota, FL, USA). Cells were adhered to 35 mm glass culture dishes (MatTek, Ashland, MA, USA) precoated with 0.2 mg ml−1 soybean lectin (Calbiochem, Merck Millipore) and immersed in EMM-N media. Culture dishes were placed on the inverted microscope stage in an Environmental Chamber at 28 °C.
For live cell imaging, mCherry, YFP (or GFP) and Cerulean signals were captured with 1.2 s (32% filter), 1.5 s (32% filter) and 0.5 s (32% filter) exposures using Optical Axis Integration, which acquires 3.6 μm of z-axis by a continuous z sweep. This was repeated every 300 s for approximately 12 h. Images were deconvolved and analysed using SoftWoRx 5.5 (Applied Precision). Dead cells observed during the imaging and the subjects moving out of focus were excluded from the study.
For live cell imaging, mCherry, YFP (or GFP) and Cerulean signals were captured with 1.2 s (32% filter), 1.5 s (32% filter) and 0.5 s (32% filter) exposures using Optical Axis Integration, which acquires 3.6 μm of z-axis by a continuous z sweep. This was repeated every 300 s for approximately 12 h. Images were deconvolved and analysed using SoftWoRx 5.5 (Applied Precision). Dead cells observed during the imaging and the subjects moving out of focus were excluded from the study.