360° rotation of DNA–RNA interaction. Supplemental video to Fig. 5 B.The strength of simultaneous 3D imaging is in the ability to change the perspective within the image volume. Here a subregion of Video 2 showing the DNA (blue) 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 evoking the impression that the mRNA is within the DNA-dense region. If viewed from the side it is obvious that the mRNA sits on top of the DNA-dense region. Because the optical resolution limit along the x axis is close to 1 µm and the signal strength of both DNA and RNA label, using a single plane 2D image would report an incorrect localization of the RNA within the DNA-dense region. 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 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.
