Simultaneous 3D recording of β-actin mRNA mobility inside a living cell. Supplemental video to Fig. 5 A.A stable cell line in which all β-actin mRNAs carry a 24× MS2 stem loop tag expressing MCP-YFP-NLS MS2 coat protein and tdTomato-POM121 labeling the NPC are imaged on a custom-built MFM using a 60×, 1.3 NA, 1.405 RI silicone immersion objective (Olympus); a total magnification of 80× after the MFM unit; and a 1k, back-illuminated EMCCD with 8-µm-pixel size (model 888, built in 2011; Andor Technology). Volumes were recorded every 100 ms. The video shows a total length of the final aligned three-channel data for 50 frames. RNA is green, NPCs are red, and DNA is seen in the blue channel.
