Iq live cell imaging software

All microscopy analyses except those corresponding to live-microscopy were performed in liquid SC synthetic media using a Nikon Eclipse Ti microscope with a 100x objective. Cell images were captured with a Neo sCMOS Camera (Andor). Images were analysed using ImageJ on 2D-maximum projections from 11-Z-stacks spaced 0.5µ each. For live microscopy, cells were plated in SC synthetic medium on concanavalin A–coated (C2012, Sigma) Lab-Tek chambers (Thermo Fisher Scientific). Imaging was performed using a spinning-disk confocal microscope (Revolution XD; Andor Technology) with a Plan Apochromat 100×, 1.45 NA objective equipped with a dual-mode electron-modifying charge-coupled device camera (iXon 897 E; Andor Technology). Time-lapse series with variable stacks (check figure legends) were acquired every 5 min. iQ Live Cell Imaging software (Andor Technology) was used for image acquisition. Images were analysed on 2D maximum projections and denoised with the Despeckle function in ImageJ 1.46b (National Institutes of Health). Graphs and statistical analysis were performed with Prism or Excel. Fun1 staining analysis was performed with a commercial kit (L-7009 from Molecular Probes) following the standard protocol described in the kit.

All data analysis was done in Matlab 2018b or Prism V8. bedtools was used for preprocessing sequencing data. iQ Live Cell Imaging software (Andor Technology) was used for image acquisition.

For single molecule fluorescence imaging, a custom-built TIRFM system was used as previously described7 (link). The experiment was performed on TIRFM with 100X/1.45NA Plan Apochromat TIR objective (Olympus, Japan) and a 14-bit back-illuminated electron-multiplying charge-coupled device camera (Andor iXon DU-897 BV). Imaging was performed at room temperature. GFP was excited at 488-nm by an argon laser (Melles Griot,Carlsbad, CA, USA) with the power of 1 mW measured after the laser passing through the objective. Movies of 200–300 frames were acquired for each sample at a frame rate of 10 Hz. Sequences of images were stored directly to a computer hard drive for subsequent analysis by IQ live cell imaging software (Andor Technology, BT, UK).