HeLa cells were sub-cultured into a glass bottom dish (P35G-1.5-14-C; MatTek, Ashland, MA) using DMEM/F12 (Thermo Fisher) supplemented with 10% fetal bovine serum (Biowest, VWR #S1810-500), 100 U/ml penicillin G (Sigma-Aldrich, St. Louis, MO), and 100 μg/ml streptomycin (Sigma-Aldrich), at 37°C and 5% CO2, 1 day prior to RNA-loading. AF647-labeled RNAs in water were loaded using a bead-loading method (46 (link),47 (link)) as follows: The culturing medium was removed and replaced with a solution containing 2 μM of either AF647-labeled RNA or of the dye itself, together with glass-beads (<106 μm; G4649, Sigma-Aldrich). The glass bottom dish was then gently tapped against the workbench 10 times, after which the glass-bead and RNA solution was removed by washing in PBS three times, before finally adding Opti-MEM I (Thermo Fisher). Measurements were started within minutes after loading and were finished within 2 hours.