Nis imaging and image analysis software

For SIM analysis, cells were fixed in the desired cell state for 15 min in 4% paraformaldehyde/2% sucrose in PBS solution and washed extensively with PBS. Cells were placed on a glass-bottomed dish as described in the previous section and were kept for the time of imaging in PBS. The samples were imaged on a Nikon N-SIM system equipped with total internal reflection fluorescence Apochromat 100× 1.49 NA oil immersion objective and a single photon–detection electron-multiplying charge-coupled device camera (iXon3 DU-897E; Andor Technology). 488- and 561-nm laser lines were used for excitation of yeGFP and tdTomato/mCherry, respectively, combined with emission band pass filter 520/45 and 610/60. Images were taken sequentially within a small z stack and in consideration to image SPBs close to the coverslip to minimize spherical aberrations. Subsequently, the reconstruction and channel alignment and FWHM measurements were performed with the NIS imaging and image analysis software (Nikon). For the xyz chromatic shift correction, a reference sample with tetraspeck beads was used. Images always show a single stack of the z slices. For the proximity analysis of NPCs close to the mSPB or satellite/dSPB, plot profiles were generated. Signals within a 200-nm diameter around the yeGFP signal peak were counted as colocalizations.

For SIM analysis, cells were fixed on a glass coverslip for 30 min with 4% paraformaldehyde and 2% sucrose in PBS buffer. After several wash steps with PBS, the coverslips were mounted on glass slides with Prolong Glass mounting medium. A Nikon N-SIM microscope system (equipped with total internal reflection fluorescence Apochromat 100 × 1.49 NA oil immersion objective and a single photon-detection, electron-multiplying, charge-coupled device camera [iXon3 DU-897E; Andor Technology]) was used to image the samples. tdTomato-tagged proteins were imaged using a 561 nm laser combined with emission bandpass filter 610/60. A single stack of nucleus cross-sections was imaged, and images were reconstructed using NIS imaging and image analysis software (Nikon).