Fig. 2: Single-molecule and super-resolution confocal microscopy of the same cell.

a A Zeiss LSM880 was extended by an additional beam path for narrow-field single-molecule excitation and high sensitivity detection. This beam path was used for imaging single pre-60S particles labelled by eIF6-HaloTag-JF549 (red). By switching the fully automated microscope to confocal super-resolution mode we were able to image the very same sample region by Airyscan microscopy, which revealed single NPCs lined up at the NE labelled by eGFP-NTF2 (green). For image registration, UV fluorescent microbeads coupled to the cell surface, which were seen in both modes, were used. b Sketch of the measurement principle. Using laser illumination with a wavelength of 561 nm a movie of single eIF6-HaloTag-JF549 (red) inside a cell nucleus was acquired at a high frame rate. After movie acquisition illumination switched from 561 to 405 nm to acquire images of the reference beads. Subsequently, super-resolution imaging of the NPCs labelled by eGFP-NTF2 (green) and of the reference beads was achieved by Airyscan microscopy in the green and UV fluorescence channels, respectively. The EMCCD movie was used to determine pre-60S particle tracks in the nucleus and across the NE. The super-resolution images of the NTF2-labelled NPCs were employed to identify single NPCs and their positions along the NE. The two sets of the reference bead images were used to calculate the transformation matrix that was needed to map the pre-60S trajectories onto the NPC positions. c Hela cells stably expressing NTF2-eGFP (green) and eIF6-HaloTag labelled by JF549 (red) after image registration. Single pre-60S particles could be discerned. Numbers represent time in ms.