35 mm gridded glass bottom dishes

U2OS cells stably expressing mGFP-Vpr grown on 35 mm gridded glass bottom dishes (MatTek Corporation) were subjected to light microscopy imaging. The resulting cells were fixed with Karnovsky’s fixative, then post-fixed, stained, and resin-embedded as described previously ^{81 (link)}, except that 2% OsO₄ and 1% uranyl acetate were used. In addition, a graded series of Polybed resin (EMS) with ethanol was utilized (2:1 ethanol to resin, 1:1 ethanol to resin, and 1:2 ethanol to resin each for 1 hour). Finally, the cells were incubated at room temperature for approximately 48 hours in resin without activator, followed by a 4-hour incubation in resin with activator BDMA at 32 °C. The cells had a final exchange with degassed resin and were allowed to polymerize for 24 hours at 65 °C. The resin was separated from the glass coverslip by heat shock, and any remaining glass particles were removed by hydrofluoric acid treatment and subsequent washing in ddH₂O. Regions of interest identified by light microscopy imaging were cut out with a jeweler’s saw and glued to resin blocks using super glue. Serial sections were cut en face on an ultramicrotome and collected on formvar-coated slot grids. Grids were post-stained with uranyl acetate and lead citrate according to standard protocols, then were carbon coated before being transferred to the TEM for tomography.

Melanoma cells were seeded at a density of 2 × 10⁵ in 35 mm gridded glass-bottom dishes (MatTek) 24 h before the experiment. Live cells were imaged until NE rupture was spotted, at which point cells were fixed adding 8% (v/v) FA (Taab Laboratory Equipment Ltd, Aldermaston, UK) in 0.2 M phosphate buffer pH 7.4 to the cell culture medium (1:1) for 15 min. Cells were mapped using brightfield light microscopy to determine their position on the grid, and tile scans were generated. Processing details for the Electron Microscopy can be found in Supplementary Information.

Correlative FM-EM allows individual cells to be examined by both FM and EM. Tet-BZLF1/B95-8 cells were cultured on gridded 35-mm glass-bottom dishes (Mat Tek) coated with 1% gelatin in RPMI 1640 medium. To induce lytic EBV replication, doxycycline was added to the medium. After 24 h, cells on the grid were fixed with 4% paraformaldehyde solution in phosphate buffer (PB), stained with specific antibodies, and examined by CLSM. The same specimens were postfixed with 1% osmium tetroxide and 0.5% potassium ferrocyanide in PB for 1 h on ice, washed with distilled water (three times for 1 min each), dehydrated in an ethanol series (50, 70, 90 and 100% for 5 min each), and embedded in Epon-812 (TAAB Laboratories) at 65°C for 48 h. Ultrathin sections of cells were stained with saturated uranyl acetate and Reynold’s lead citrate solution. Electron micrographs were taken using a JEM-1400EX transmission electron microscope (JEOL). For EM, pcDNA-BZLF1 was transfected by electroporation into B95-8 cells to induce the lytic phase. The cells were harvested 24 h after transfection. After fixation, dehydration, and embedding, electron microscopic analysis was performed by using a JEM-1200EX transmission electron microscope at 80kV (JEOL).

Cells were seeded in gridded 35-mm glass bottom dishes (MatTek, Boston, MA) and the media changed to Leibovitz L-15 supplemented with 10% foetal calf serum prior to imaging. Cells were imaged using a 100× 1.4 NA oil objective on a confocal spinning-disk microscope (VOX Ultraview; PerkinElmer, Waltham , MA) with a Hamamatsu ORCA-R2 camera, controlled by Volocity 6.0 (PerkinElmer) running on a Windows 7 64-bit (Microsoft, Redmond, WA) PC (IBM, Armonk, NY). Mitotic cells were first identified using bright-field illumination to minimise phototoxicity. Image stacks (25 z-sections, 0.5 µm apart) were collected every 2 s for 5 min (150 time points per video). Camera pixels were binned 2 × 2, giving an effective pixel size of 138 nm in the lateral direction with a 16-bit per pixel imaging depth. Exposure conditions were set 50 ms per z-slice using a 488-nm laser set to 15% power.