Ixon ultra 897

To reduce the background noise and increase the signal-to-noise ratio for fluorescence detection, we used HILO illumination, which was transmitted in the form of a thin sheet with an incident angle smaller than the critical angle for total reflection. Cells for high-resolution imaging or single-particle tracking were exposed to HILO illumination from a custom-built microscope based on a Nikon microscope body (Ti2e) with 405-nm, 488-nm, 531-nm, and 640-nm excitation lasers (CUBE, Coherent). Sequential images from live cells were acquired through a 100X objective lens of 1.49 numerical aperture (Nikon, Plan Apochromat TIRF 100X oil-type) with an electron-multiplying charge-coupled device (Andor, iXon Ultra 897), and processed using the NIS-Elements software (Nikon). Image analysis was performed using ImageJ scripts and custom-built MATLAB codes.

Cell imaging was performed with highly inclined and laminated optical (HILO) illumination from a Nikon TIRF microscope based on the Nikon Eclipse Ti microscope body. For detections of telomere loci, mCherry was excited with a built-in 561 nm laser in the microscope. Images were acquired through a 100X objective lens of 1.49 numerical aperture (Nikon, Plan Apochromat TIRF 100x oil-type) with the electron multiplying charge coupled device (EMCCD) (Andor, iXon Ultra 897), and processed using the NIS-Elements software (Nikon). For streaming image acquisition, snapshots were taken every 100 ms for 2 min. Image analysis was performed using ImageJ scripts and custom-built MATLAB codes for mean square displacement calculation.