1.49 na apo tirf objective

The cells were imaged using a Nikon Eclipse Ti2 microscope equipped with a Yokagawa CSU-W1 spinning disk scanner, 100x Nikon 1.49 NA Apo-TIRF objective and an Andor iXon 888 EMCCD camera. The setup was equipped with 405, 488, 561, and 640 nm laser lines and corresponding filter sets. A single confocal plane was selected near the center of the nucleus, and imaging of mCherry-labeled capsids was done with the speed of 10 frames per second for the duration of 40 seconds. Hoechst distribution was recorded by taking one image before and, to verify that no significant movement of chromatin happened during the acquisition, one image after recording the time series of the mCherry channel. The pixel size was 130 nm. Hoechst was excited with a 405 nm diode laser and VP26-mCherry with a 561 nm DPSS laser. Life cell experiments were carried out in a humidified incubation chamber heated to 37°C and 5% CO2 controlled by a gas-mixer with cells were grown on Ibidi 35 mm glass-bottom dishes. The fixed samples were imaged with Leica SP8 X Falcon confocal microscope using HC PL APO 63x 1.4 NA oil immersion objective or with Nikon A1R confocal microscope with CFI Plan Apo VC 60XH 1.4 NA oil immersion objective. The electron microscopy samples were imaged with JEOL JEM -1400 transmission electron microscope at an operating voltage of 80 kV and magnification of 4000–8000.

Spinning-disk microscopy was carried out on a Nikon TI2 (Nikon) based spinning-disk system equipped with a Yokogawa W2, a Nikon 1.49 NA Apo-TIRF objective, and an Andor iXON888 EMCCD (Andor Technology). The resulting pixel size was 130nm, and image acquisition was done with NIS-Elements. Further, the setup was equipped with 405, 488, 561, and 640 laser lines and corresponding filter sets. Live-cell experiments were carried out with a humidified incubation chamber heated to 37°C and 5% CO2 controlled by a gas mixer. For fluorescence microscopy, cells were grown in Ibidi 35mm glass-bottom dishes or Ibidi 8-well glass-bottom chamber slides (Ibidi GmbH), for CLEM in Ibidi 35mm grid polymer bottom dishes. SNAP labeling before live-cell imaging with SNAP-Cell 647-SIR (New England Biolabs GmbH) was done according to the manufacturer’s instructions. Image processing and analysis were performed in ImageJ/FIJI.