4k x 4k ccd camera

Three microliter aliquots of the NV were applied to C-flat holey carbon grids (Protochips, Inc.) and vitrified using a Vitrobot™ Mark IV (FEI Co.) operated at 4°C and with ~90% humidity in the control chamber. The vitrified sample was stored under liquid nitrogen and transferred into a Gatan cryo-holder (Model 626/70) for imaging. The sample was examined using a 4k x 4k CCD camera (Gatan, Inc.) on a Tecnai (FEI Co.) G2 F20-TWIN Transmission Electron Microscope operated at a voltage of 200 kV using low dose conditions (~20 e/Å2). Images were recorded with a defocus of approximately -2µm to improve contrast.

The specimens were visualized on the FEI Tecnai T12 electron microscope operated at 120kv. The tilt series were recorded using the FEI tomography software from –60° to +60° with 2° intervals at a defocus of 6 μm on a 4k x 4k CCD camera (Gatan), with magnification was set to 5,000–25,000x depending on the size of the viral assembly intermediates. The tilt series were aligned using IMOD software package56 (link) and a final 3D reconstruction was computed using the simultaneous iterative reconstruction technique (SIRT) algorithm. For all segmented-tomograms, a Gaussian filter was applied to reduce noise and improve contrast of the tomograms before they were subjected to semi-automated threshold segmentation. Images were rendered using the Chimera software package57 (link).

Sections of retina tissues from each group were cut at a thickness of 400 nm. For each reconstruction, a double-tilt series of images at one-degree tilt increment was collected with an FEI titan hibase electron microscope operated at 300 kV. Images were recorded with a Gatan 4Kx4K CCD camera. The magnification was ×11,000 and the pixel resolution was 0.81 nm. The IMOD package was used for alignment, reconstruction and volume segmentation. Volume segmentation was performed by manual tracing of membranes in the planes of highest resolution with the Drawing Tools and Interpolator plug-ins^{80 (link)}. The mitochondrial reconstructions and surface-rendered volumes were visualized using 3DMOD. Measurements of mitochondrial outer, inner boundary, and cristae membrane surface areas and volumes were made within segmented volumes using IMODinfo. These were used to determine the cristae density, defined as the ratio: sum of the cristae membrane surface areas divided by the mitochondrial outer membrane surface area. Movies of the tomographic volume were made using IMODmovie.