1.49 na 100 oil immersion tirf objective

Three-color single-molecule live-cell imaging was carried out as previously described (Schmidt et al., 2016 ), using the CRISPR genome-edited HeLa cell line stably expressing 3xFLAG-HaloTag TERT and mEOS3.2-TRF2 from their respective endogenous loci. Briefly, BFP-coilin was transfected into cells 48 h prior to imaging, followed by a double thymidine block. Imetelstat was added to cells 24 h before imaging, simultaneously with the release from the first thymidine block. Three to 4 hours after release into S-phase, FLAG-HaloTag-TERT was labeled by subjecting cells to a 2-min pulse of 100 nM JF646 HaloTag-ligand (a kind gift from Luke Lavis) in tissue culture medium (Grimm et al., 2015 (link)). BFP-coilin was imaged first for ∼1 s under continuous illumination. 3xFLAG-HaloTag-TERT and mEOS3.2-TRF2 (red state) were imaged simultaneously. Movies were acquired for 15 s on a Nikon N-STORM microscope under highly inclined and laminated optical sheet conditions (Tokunaga et al., 2008 (link)), with a 1.49 NA 100× oil-immersion TIRF objective (Nikon) at 46 frames per second. The two imaging channels were projected onto two iXon Ultra 897 EMCCD cameras (Andor) using TwinCam dual emission image splitter (Cairn). The channels were aligned prior to every imaging session using TetraSpeck microspheres (ThermoFisher).