360° rotation of RNA–NPC interaction. Supplemental video to Fig. 5 C.Here a subregion of Video 2 showing the NPCs (red) and mRNA (green) is rotated around its x axis. In the beginning, middle, and end of the video the perspective is along the z axis. The image volume was recorded in 100 ms, and the 3D rotation was calculated in 1° steps around the x axis. Image processing was done in Fiji. Data were acquired 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). The mRNA is located at the nuclear side of an NPC, only visible in the orthogonal perspective.
