Inspect3d software

Semithin sections (250 nm thick) were prepared from the same plastic block as was used for the ultrathin sections. After the semithin sections were stained with 2% uranyl acetate and Reynold’s solution, both sides of the sections were carbon coated with the VE-2030 vacuum (Vacuum Device, Ibaraki, Japan). Plasma cleaning was performed with a model 1020 plasma cleaner (Fischione Instruments, Export, PA). Single- or dual-axis images of the semithin sections were acquired with a Tecnai F20 field-emission STEM (FEI Company, Eindhoven, the Netherlands) at 200 kV using an annular dark-field detector (Fischione, Export, PA). The digital images were collected with a 2cosθ° increment over a 60° range with a pixel size ranging from 0.25 to 1 nm. The stack of images was then reconstructed by using the simultaneous iterative reconstruction technique and Inspect3D software (FEI Company, Eindhoven, the Netherlands). For the dual-axis tilt series, the x- and y-axis tilt series were reconstructed by using Inspect3D software (FEI Company, Eindhoven, the Netherlands). The models of the RNP complexes within the virions were created with the Avizo 6.2 image-processing package (Visualization Science Group, Burlington, MA) as previously described (22 (link)).

TEM images were acquired on FEI Tecnai-Spirit (at 120 kV) and Tecnai-Osiris (at 200 kV). HR-TEM, HR-STEM, and tomographic data were acquired on a FEI Titan Themis 60–300 operated at 300 kV. For tomography, a tilt-series of HAADF images (collection angle, 100–200 mrad) were collected over an angular range of −74° to +74° in 2° increments, using an incident beam of 10 mrad convergence semi-angle. The tilt-series images were aligned and a SIRT reconstruction (30 iterations) performed using FEI Inspect 3D software. The tomogram volume rendering and segmentation were done using Avizo software. XPS data were collected on a PHI VersaProbe II scanning XPS microprobe (Physical Instruments AG, Germany) with a monochromatic Al-Kα X-ray source operating at 24.8 W under ultrahigh vacuum conditions. XRD measurements were conducted on a BRUKER D8 Advance instrument with Cu Kα radiation. FTIR analysis was performed on PerkinElmer Spectrum Two.

A set of HSV-1 cryo-ET tilt series was used for the test, and was collected automatically at 1.5° increments over an angular range of [−63°, 60°] on FEI CM120 microscopes. The accelerating voltage was 120 kV, and the total electron doses were approximately 35 electrons/Ų. The undenoised and denoised tilt series were reconstructed using Inspect3D software from FEI, Inc. The results were compared and assessed through visualization.