Cells (20,000 cells) were seeded on a glass bottom 4 well plate (De Groot—76-D35C4-20). Imaging was started 24 h after the seeding of cells. Images were acquired depending upon the experiment. Cells were cultured, transfected and live cell was performed according to standard protocols19 (link),70 (link). For time-lapse imaging, we used a dual point-scanning Nikon A1R-si microscope equipped with a Piezo stage, using a 60 × PlanApo IR oil objective NA 1.4, 0.3 μm slices, and 0.2–2% laser power (from 65-mW 488-nm laser and 50-mW 561-nm laser) to acquire 3D movies. Images were acquired in resonant-scanning or Galvano-scanning mode. Each Z series was acquired with 0.5- to 1-μm step size and 10–35 steps. For super resolution Structured Illumination Microscopy (SIM) Cells were prepared as described above. Images were acquired using a Nikon nSIM microscope in 2D mode with a 488 nm and 561 nm lasers. A 100 × oil TIRF objective (NA 1.49) was used for the imaging. Prior to imaging the point-spread function was visualized with 100 nm fluorescence beads in order to adjust the correction ring of the objective to the coverslip thickness. The final image was reconstructed using NIS-Elements software (Version 4.1).
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