Plan apo oil immersion objective lens

Immunostaining was carried out as was previously described [36 (link)]. Briefly, the following rabbit monoclonal and polyclonal antibodies against modified histones and DNA were used: anti-dimethyl histone H3 at lysine 4 (1:100; Abcam, cat. no. (ab7766), anti-dimethyl histone H3 at lysine 9 (1:100; Millipore, cat. no. 07–441) and anti-5-methyl-cytosine (1:300, Abcam, cat. no. ab73938). Two secondary antibodies were also applied–Alexa Fluor 488 goat anti-rabbit IgG (Invitrogen, Molecular Probes, cat. no. A-11008) and Alexa Fluor 488 goat anti-mouse IgG (Invitrogen, Molecular Probes, cat. no. A-11001). The fluorescence of DAPI (excitation 405 nm, emission 425–475 nm) and Alexa488 (excitation 488 nm, emission 500–600 nm) was registered using an Olympus FV1000 confocal system (Olympus) equipped with an IX81 inverted microscope, a 60x Plan Apo oil-immersion objective lens (NA 1.35), a 50 mW, 405 nm diode laser and a 100 mW multi-line argon ion laser (Melles Griot BV). An axial series of 2-D fluorescence images of optical sections through the chromosomes (z-stacks) was collected using two separate photomultipliers (R6357, Hamamatsu). Image processing operations were performed with ImageJ (Fiji).

LifeAct-GFP-NLS-expressing HFFs were infected with WT HCMV (MOI of 3) and fixed at RT in 3.7% formaldehyde-DPBS at 24 hpi. 3D-SIM data were collected at the Cell Biology Microscopy Facility at Harvard Medical School using a DeltaVision OMX V4 Blaze system (GE Healthcare) equipped with a 60×/1.42-numerical-aperture (NA) Plan Apo oil immersion objective lens (Olympus), 488 solid state laser, and a pco.edge 5.5 scientific complementary metal oxide semiconductor (sCMOS) camera. z-stacks were acquired with a z-step of 125 nm and with 15 raw images per plane (five phases, three angles). Spherical aberration was minimized using immersion oil matching. Super-resolution images were computationally reconstructed from the raw data sets with a channel-specific measured optical transfer function (OTF) and a Wiener filter constant of 0.001 to 0.002 using softWoRx 6.1.3 (GE Healthcare).